The Regulation of Starch Biosynthesis in Barley

The Regulation of Starch Biosynthesis in Barley (Hordeum vulgare)
by
Zaheer Ahmed
A Thesis
presented to
The University of Guelph
In partial fulfillment of requirements
for the degree of
Doctor of Philosophy
in
Molecular and Cellular Biology
Guelph, Ontario, Canada
© Zaheer Ahmed, January, 2014
ABSTRACT
THE REGULATION OF STARCH BIOSYNTHESIS IN BARLEY (Hordeum
vulgare)
Zaheer Ahmed
University of Guelph, 2014
Advisor: Dr. Michael J. Emes
Starch has enormous uses in the food and non-food industries in different forms.
An important form of starch in the food industry is resistant starch (RS). Resistant
starches (RS) have potential benefits for human health as a source of low-glycemic
carbohydrate and as a prebiotic for the large colon. Such starches are often referred to as
high-amylose though this may reflect changes in amylopectin as well the proportion of
amylose in the starch granule. This study investigated the relationship between resistant
starch (RS) and physical properties of the starch which may contribute to increased RS.
The role of protein phosphorylation, and protein complex formation between enzymes of
starch synthesis, was also studied in a range of barley genotypes exhibiting high, low and
normal amylose phenotypes. In the final part of the thesis, the relationship between
variations in starch physiochemical properties and starch granule proteome from
genotypes with no known mutations in starch biosynthetic enzymes was investigated. The
results indicate that increased RS is positively associated with increased amylose and Bgranule content. Detailed biochemical analysis of barley mutants, with alterations in the
starch biosynthesis pathway, revealed that formation of phosphorylation-dependent
multi-enzyme complexes among isoforms of starch synthases (SS) and branching
enzymes (BE) are potentially important mechanisms of regulating RS biosynthesis.
Barley lines down regulated with SBEIIa or SBEIIb, demonstrated distinct patterns of
protein-protein interactions compared with a reference genotype, suggesting functional
complementation for the loss of either isoform by SBEI and SP (starch phosphorylase). In
an ssiia mutant no protein complexes were formed indicating that SSIIa plays an
important role in recruiting different components into protein complexes. Detailed
biochemical analysis revealed that in some of the mutant lines, different protein
complexes are involved in the synthesis of A- and B-granules. These variations in protein
ii
complexes are reflected in the complement of starch synthesizing enzymes detected in
starch granules (A- and B-) of different genotypes. The results reinforced the hypothesis
that the multi-enzyme complexes play a functional role in biosynthesis of A- and Bgranules. Finally, studies of the physiochemical properties of seed and starch in cultivars
with no known mutations in starch biosynthetic enzymes, suggest that significant
component of variation lie elsewhere, and are independent of the starch granule
proteome.
iii
ACKNOWLEDGEMENTS
In the name of ALLAH, the merciful and compassionate, Who bestowed upon
me the will and ability to complete this task. Thousands of blessings and clemencies of
ALLAH are upon the Holy Prophet Muhammad (Peace Be upon Him), the pride of
humanity, who took the responsibility to take the humanity out from the swamp of
ignorance and showed us the righteousness.
I feel highly privileged in taking opportunity to express my profound gratitude
and sense of devotion to my supervisors, Dr. Michael J. Emes and Dr. Ian J. Tetlow for
their continuous supports and supervision throughout this study project, particularly for
their extremely scrupulous reading of this thesis through its various stages. It was only
because of their inspiring guidance, consistent encouragement, sympathetic attitude and
dynamic supervision during the entire study program that I could prepare this thesis.
Thanks are extended to my committee member Dr. Duane E. Falk (Plant
Agriculture, University of Guelph), for his technical and constructive guidance and for
providing barley material for this study. I would like to thank Dr. Matthew Morell and
Dr. Regina Ahmed (CSIRO, Australia) for various barley mutant lines and wheat
antibodies. I am thankful to Dr. Qiang Liu’s laboratory (AAFC) for starch analysis. I
would like to thank Dr. Koushik Seetharaman’s laboratory (Food Science, University of
Guelph) for starch analysis. I would also like to thank Michael Mucci and Tannis
Slimmon for green house, phytotron facility.
I also like to acknowledge my heartful indebtedness and sincere thanks to past
and present members of the Emes/Tetlow lab for their assistance with experiments, but
more importantly for their support and friendship. I am especially thankful to Dr. Amina
Makhmoudova, Dr. Fushan Liu, Dr. Nadya Romanova, Dr. Wendy Allen, Dr. Renuka
Subasinghe, Dr. Mark Burrell, Usha Rayirath, John Hollingshead, Adam Harris, Qianru
Zhao (Ruby), Lily Nasanovsky, Sarah Massey, Jenelle Patterson and John ( a high
school student), for their help and support. I am thankful to my friend, Kashif Mahmood,
who helped me a lot with his valuable suggestions all the time and he was always there to
encourage me. I am also thankful to my other friends, Fazal Abbas, Shezad Rauf,
iv
Muhammad Uzair, Attiq rehman, Farukh Iftikhar Ali, Khurram Shahzad and Madhulika
Sareen for their support and company. Special thanks to colleagues and staff in the
Science Complex Fourth Floor West Wing for their company and for sharing in mutual
frustrations and celebrations.
Words cannot express the feeling of my love, devotion, thanks and gratitude to
my sweet parents. My success is really the fruit of sincerest prayers of my mother, father,
brother, sisters and other members of the family. What so ever, I am today is only by the
grace of ALLAH Almighty and due to the prayers of my parents they are always very
kind to me. Tributes are due for my family, by whom I was always inspired and
encouraged for my studies. My deep appreciations are due to my affectionate and loving
parents, brother and sisters, who are always praying for my success and brilliant career. I
submit my earnest thanks to all of them for their encouragement and moral support which
made this possible.
At the end I would like to thank, Government of Pakistan, University of
Agriculture Faisalabad (UAF) Pakistan and Higher Education Commission of Pakistan
(HEC) for allowing me and providing financial support for this study.
v
TABLE OF CONTENTS
ABSTRACT ............................................................................................... ii
ACKNOWLEDGEMENTS ...................................................................... iv
TABLE OF CONTENTS.......................................................................... vi
LIST OF TABLES .................................................................................. xiii
LIST OF FIGURES ................................................................................ xiv
ABBREVIATIONS ................................................................................. xvi
Chapter 1: General Introduction .......................................................... - 1 1.1 Overview ........................................................................................ - 2 1.2 Endosperm Development.............................................................. - 3 v Double fertilization ................................................................................ - 5 v Syncytium formation ............................................................................. - 5 v Cellularization ........................................................................................ - 6 v Differentiation ........................................................................................ - 7 v Embryo surrounding region (ESR) cells ............................................... - 7 v Transfer cells (TCs) ............................................................................... - 7 v Aleurone layer ........................................................................................ - 9 v Starchy endosperm .............................................................................. - 10 v Endoreduplication................................................................................ - 12 v Maturation ........................................................................................... - 13 v Programmed cell death ........................................................................ - 13 v Desiccation............................................................................................ - 13 -
1.3 Starch and its Composition ........................................................ - 15 1.3.1 Molecular structure of starch granule ........................................ - 18 1.3.2 Variations in physiochemical properties of A- and B-granules . - 23 1.3.2.1 Uses of starch granules with different properties............................ - 25 1.3.2.1.1 Uses of starch in food industry .................................................. - 25 1.3.2.1.1.1 Digestion of food ................................................................... - 26 1.3.2.1.1.1.1 What is RS and its health benefits? .................................. - 26 1.3.2.1.1.1.1.1 Types of RS .............................................................. - 29 1.3.2.1.1.1.1.1.1 Factors affecting resistant starch ......................... - 30 -
1.4 Starch Biosynthesis .................................................................... - 33 vi
1.4.1 Enzymes involved in starch biosynthesis .................................. - 33 1.4.1.2 Adenosine 5’ Disphosphate Glucose Pyrophosphorylase (AGPase, EC
2.7.7.27)......................................................................................................... - 33 1.4.1.3 Starch Synthases (SS, EC 2.4.1.21) .................................................. - 38 1.4.1.3.1 Granule-bound starch synthase (GBSS) ................................... - 42 1.4.1.3.2 Starch synthase (SSI) ................................................................ - 43 1.4.1.3.3 Starch synthase (SSII) .............................................................. - 44 1.4.1.3.4 Starch synthase (SSIII) ............................................................. - 45 1.4.1.3.5 Starch synthase (SSIV) ............................................................. - 48 1.4.1.4 Starch Branching Enzymes (SBEs, EC 2.4.1.18) .......................... - 48 1.4.1.4.1 Starch branching enzyme I (SBEI) ......................................... - 49 1.4.1.4.2 Starch branching enzyme II (SBEII)......................................... - 51 1.4.1.5 Debranching enzymes (DBEs, EC 3.2.1.41 and EC 3.2.1.68) ...... - 52 1.4.1.6 Starch Phosphorylase (SP, EC 2.4.1.1) ....................................... - 56 1.4.1.7 Disproportionating enzyme (D-enzyme, E, C. 2.4.1.25) .................. - 58 1.4.2 Post-translational modification of starch synthesizing proteins - 59 1.4.3 Starch granule-bound proteins ................................................... - 63 -
1.5 Experimental Material................................................................ - 67 1.6 Hypotheses ................................................................................... - 67 1.7 Aims and Objectives of the Study .............................................. - 68 Chapter 2: Evaluation of physical characteristics of resistant starch
from (a range of) barley genotypes ..................................................... - 71 2.1 Introduction ................................................................................ - 72 2.2 Material and Methods ................................................................ - 75 2.2.1 Selection of genotypes.................................................................. - 75 2.2.1.1 Selection of genotypes for further detailed analyses ....................... - 75 2.2.2 Isolation of starch granules ......................................................... - 75 2.2.3 Measurement of amylose and RS contents ................................. - 76 2.2.4 Amylose content determination by gel permeation chromatography
.............................................................................................................. - 76 2.2.5 Measurement of amylose by iodine binding ............................... - 77 2.2.6 Granule size distribution of different genotypes ........................ - 78 2.2.7 Granule morphology ................................................................... - 78 2.2.8 Seed characteristics ..................................................................... - 79 vii
2.2.8.1 Thousand grain weight (TGW) ........................................................ - 79 2.2.8.2 Starch content ................................................................................... - 79 2.2.9 High-performance anion exchange chromatography ................ - 79 2.2.10 Statistical analysis ..................................................................... - 79 -
2.3 Results.......................................................................................... - 80 2.3.1 Amylose content .......................................................................... - 80 2.3.2 Resistant Starch (RS) Content .................................................... - 82 2.3.3 Comparison of different methods for determination of amylose
content .................................................................................................. - 84 2.3.4 Physical characteristics of seed ................................................... - 84 2.3.4.1 Seed characteristics .......................................................................... - 84 2.3.4.2 Thousand grain weight (TGW) ........................................................ - 84 2.3.4.3 Starch content ................................................................................... - 86 2.3.5 Granule size, number and surface area distributions of different
genotypes .............................................................................................. - 86 2.3.6 Contribution of A-, B- and C-granules in total mass of starch .. - 87 2.3.7 Granule morphology ................................................................... - 90 2.3.8 Amylopectin chain length distribution ....................................... - 92 2.3.9 Principal component analysis (PCA) .......................................... - 97 -
2.4 Discussion .................................................................................. - 100 2.5 Conclusion ................................................................................. - 104 Chapter 3: Post-translational modification, protein-protein interactions
among enzymes of starch biosynthesis and their affect on physical
properties of starch in high-amylose barley genotypes .................... - 106 3.1 Introduction .............................................................................. - 107 3.2 Materials and Methods ............................................................. - 110 3.2.1 Plant material ............................................................................ - 110 3.2.2 Isolation of starch granules ....................................................... - 110 3.2.3 Separation of A- and B-type starch granules ........................... - 111 3.2.4 Extraction of amyloplasts and preparation of endosperm whole cell
extracts ............................................................................................... - 112 3.2.5 Isolation of starch granule-bound proteins .............................. - 112 3.2.6 SDS-PAGE and immunoblotting of total starch, A and B granules
......................................................................................................... ...- 113 3.2.7 Co-immunoprecipitation ........................................................... - 113 viii
3.2.8 Detection of SS and SBE activity following non-denaturing gel
electrophoresis.................................................................................... - 114 3.2.9 Detection of SP activity ............................................................. - 115 3.2.10 Sliver staining .......................................................................... - 116 3.2.11 Pro-Q Diamond phospho-protein staining ............................. - 116 3.2.12 Starch gelatinization ................................................................ - 117 3.2.13 Estimation of amylose and RS content ................................... - 118 3.2.14 Protein content ........................................................................ - 118 3.2.15 Mass spectrometry .................................................................. - 118 3.2.16 Statistical analysis ................................................................... - 118 -
3.3 Results........................................................................................ - 119 3.3.1 Physiochemical properties of mutant starches in barley ......... - 119 3.3.1.1 Starch, amylose and RS content..................................................... - 119 3.3.1.2 Starch gelatinization ....................................................................... - 119 3.3.2 Biochemical characterization of different mutants .................. - 121 3.3.2.1 Amyloplast stromal proteins .......................................................... - 121 3.3.2.2 Detection and estimation of SS activity ......................................... - 123 3.3.2.3 Detection and estimation of SBE activity ...................................... - 126 3.3.2.4 Detection and estimation of starch phosphorylase (SP) activity ... - 126 3.3.3 Protein-protein interactions among different proteins of starch
biosynthesis ........................................................................................ - 129 3.3.3.1 Phosphorylation dependent protein-protein interactions ............. - 130 3.3.4 Analysis of starch granule-associated proteins ........................ - 133 3.3.4.1 Phosphorylation state of the starch granule proteome ................. - 134 -
3.4 Discussion .................................................................................. - 139 3.5 Conclusion ................................................................................. - 145 Chapter 4: Physiochemical and biochemical properties of starch and its
relationship to granule size distribution in barley genotypes from
diverse genetic back grounds ............................................................ - 148 4.1 Introduction .............................................................................. - 149 4.2 Material and Methods .............................................................. - 154 4.2.1 Plant material ............................................................................ - 154 4.2.2 Isolation of starch granules ....................................................... - 154 4.2.2.1 Separation of A- and B-type starch granules ................................ - 155 ix
4.2.3 Isolation of starch granule-bound proteins .............................. - 156 4.2.4 SDS-PAGE and immunodetection of granule-bound proteins - 157 4.2.5 Silver staining ............................................................................ - 157 4.2.6 Estimation of protein phosphorylation by Pro-Q diamond staining
.......................................................................................................... ..- 158 4.2.7 Measurement of total granule-bound protein content ............. - 159 4.2.8 In-gel protein quantification ..................................................... - 159 4.2.9 Starch gelatinization.................................................................. - 159 4.2.10 High-Performance Anion Exchange Chromatography (HPAEC)
......................................................................................................... ...- 160 4.2.11 Granule size distribution of different genotypes .................... - 161 4.2.12 Granule morphology ............................................................... - 161 4.2.13 Thousand grain weight (TGW) ............................................... - 162 4.2.14 Starch content.......................................................................... - 162 4.2.15 RS Content............................................................................... - 162 4.2.16 Amylose content ...................................................................... - 162 4.2.17 Mass spectrometric analysis .................................................... - 162 4.2.18 Statistical analysis ................................................................... - 163 -
4.3 Results........................................................................................ - 164 4.3.1 Physical characteristics of seed ................................................. - 164 4.3.1.1 Seed morphology characteristics ................................................... - 164 4.3.1.2 Thousand grain weight (TGW) ...................................................... - 164 4.3.1.3 Starch content ................................................................................. - 164 4.3.1.4 Internal seed structures and starch packing within endosperm ... - 164 4.3.2 Physiochemical properties of starch ......................................... - 166 4.3.2.1 Amylose and resistant starch (RS) contents .................................. - 166 4.3.2.2 Granule Size, number and surface area distributions of different
genotypes .................................................................................................... - 166 4.3.2.3 Contribution of A-, B- and C-granules in total mass of starch ..... - 168 4.3.2.4 Granule surface morphology ......................................................... - 173 4.3.3 Starch gelatinization (Thermal properties) .............................. - 173 4.3.4 Amylopectin chain length distribution (CLD).......................... - 178 4.3.5 Biochemical characterization of barley genotypes ................... - 184 4.3.5.1 Amyloplast stromal proteins (soluble proteins) ............................ - 184 4.3.5.2 Analysis of starch granule-associated proteins.............................. - 184 x
4.3.5.2.1 Total amount of starch granule-bound protein ...................... - 184 4.3.5.2.2 Individual isoforms of starch granule-associated proteins ..... - 185 4.3.5.2.3 Quantitation of individual granule-bound proteins ................ - 189 4.3.5.2.4 Proteomic analysis of C-granules ............................................ - 192 4.3.5.3 Phosphorylation of proteins in starch granule .............................. - 192 -
4.4 Discussion .................................................................................. - 196 4.5 Conclusion ................................................................................. - 203 Chapter 5: General discussion and future work .............................. - 205 5.1 General Discussion .................................................................... - 206 5.1.1 Analysis of the physiochemical properties of RS as determinant of
increased RS genotypes...................................................................... - 206 5.1.2 Regulation of starch biosynthesis in the barley endosperm is
governed by the formation of multi-enzyme protein complexes ...... - 207 5.1.3 Formation of multi-enzyme complexes containing starch
biosynthetic enzymes in barley endosperm is regulated by protein
phosphorylation ................................................................................. - 210 5.1.4 Physiochemical properties of seed and starch do not correlate with
starch granule proteome .................................................................... - 210 -
5.2 Future Work ............................................................................. - 212 REFERENCES .................................................................................. - 213 Appendix 1: Description of genotypes used in the study. ................. - 250 Appendix 2: Comparison of three methods used for amylose
determination. .................................................................................... - 251 Appendix 3: PCA analysis shows association of three methods used for
amylose determination. ...................................................................... - 252 Appendix 4: Determination of amylose with 6CL-B column. .......... - 253 Appendix 5: Iodine-staining of barley grains (cross section). .......... - 254 Appendix 6: DSC data of different genotypes. .................................. - 255 Appendix 7: Immunological characterization of endosperm amyloplast
lysates from different barley mutants. .............................................. - 256 Appendix 8: Detection of SS activity and protein. ............................ - 257 Appendix 9: Detection of SBE activity and protein. ......................... - 258 Appendix 10: Detection of SP protein and activity in different genotypes.
............................................................................................................ - 259 -
xi
Appendix 11: Co-immunoprecipitation of stromal proteins from
amyloplasts of different genotypes with SSI, SBEIIa and SBEIIb
antibodies. .......................................................................................... - 260 Appendix 12: Summary of novel protein–protein interactions formed
between amylopectin-synthesizing enzymes in barley endosperm
following either loss of single gene (SSIIa, SBEIIa and SBEIIb) or
alteration in single gene (ssiii-, amo1 mutant). .................................. - 263 Appendix 13: Starch, A- and B-granules bound proteins. ............... - 265 Appendix 14: Granule-bound phospho-proteome analysis and starch, Aand B-granules bound proteins detected by silver staining. ............. - 268 Appendix 15: Analysis of starch composition and granule morphology by
light microscope (A, B & C) and electron microscope (D & E). ....... - 269 Appendix 16: Single nucleotide polymorphisms (SNPs) of barley ssIIIa
genomic DNAs from different barley genotypes. .............................. - 270 Appendix 17: SNPs of cDNA sequences of barley ssIIIa genomic DNAs
from different barley genotypes. ....................................................... - 274 Appendix 18: Changes of polypeptide sequences of barley SSIIIa protein
from different barley genotypes. ....................................................... - 276 -
xii
LIST OF TABLES
Table 2.1: Physical characteristics of seed………………………………………....-85Table 2.2: Degree of polymerization (%) of amylopectin from barley genotypes.-93Table 3.2: Physiochemical properties of starch……………………………............-119Table 3.3: DSC data of different genotypes…………………………………..........-121Table 4.4: Physiochemical properties of seed and starch of barley genotypes......-164Table 4.2: Thermal properties of starch……………………………………….......-176Table 4.5: Amylopectin chain length in different barley genotypes………...........-179Table 4.4: Amount of total starch granule-bound protein (µg/ 50 mg of starch)
…………………………………………………………………………………...........-185Table 4.5: Correlation between different physiochemical properties of starch
determined by principal component analysis………………………………...........-196-
xiii
LIST OF FIGURES
Figure 1.1: Sucrose metabolism and starch biosynthesis in different cells of plants.
................................................................................................................................ ...- 4 Figure 1.2: Diagrammatic presentation of endosperm cellularization process. ..... - 8 Figure 1.3: Presentation of different phases of maize endosperm development. . - 14 Figure 1.4: Starch granules morphologies from different cereals. ....................... - 17 Figure 1.5: Structural differences between amylose and amylopectin. ................ - 19 Figure 1.6: Schematic representation of higher order molecular structure. ........ - 21 Figure 1.7: Presentation of different types of crystallinity in starch originated from
different sources...................................................................................................... - 22 Figure 1.8: Digestion and absorption of different food ingredients in the human
digestive tract. ......................................................................................................... - 27 Figure 1.9: Schematic presentation of starch biosynthesis in the cereal endosperm.
.............................................................................................................................. ...- 35 Figure 1.10: Comparison of cereal SS domains. .................................................... - 41 Figure 1.11: Diagrammatic representation of the coordinated actions of different
enzyme classes in the synthesis of amylopectin. .................................................. - 57 Figure 1.12: Phosphorylation dependent functional complex formation and
entrapment of proteins within starch granule in different maize mutants........... - 66 Figure 2.1: % amylose of barley genotypes. .......................................................... - 81 Figure 2.2: % RS of barley genotypes.................................................................... - 83 Figure (2.3A & B): Granule size distribution. ....................................................... - 88 Figure 2.4: Amount of A-, B- and C-granules in total mass of starch. ................. - 89 Figure 2.5: Morphology of starch granules from normal and high RS/amylose
genotypes observed by SEM. .................................................................................. - 91 Figure 2.6: Difference plot of (different genotypes versus wild-type) of amylopectin
chain length distribution. ....................................................................................... - 95 Figure 2.7: Difference plot of (Different genotypes versus waxy) of amylopectin
chain length distribution. ....................................................................................... - 96 Figure 2.8: PCA analysis shows association of different characters. .................... - 98 Figure 2.9(A & B): Linear correlation between A- and B-granules with % amylose...
................................................................................................................................. - 99 Initial Heating Summary ...................................................................................... - 122 Figure 3.1: Immunological characterization of endosperm amyloplast lysates from
different barley mutants. ...................................................................................... - 124 Figure 3.2. Detection of SS activity. ..................................................................... - 125 Figure 3.3: Detection of SBE activity and protein. .............................................. - 127 Figure 3.4: Detection of SP protein and activity in different genotypes. ............ - 128 Figure 3.5: Co-immunoprecipitation of stromal proteins from amyloplasts of
different barley genotypes with SSI, SBEIIa and SBEIIb antibodies................. - 132 Figure 3.6: Starch, A- and B-granules bound proteins. ...................................... - 136 Figure 3.7: Starch granule bound proteome phospho-proteome. ....................... - 138 Figure 3.8: Protein–protein interactions formed between amylopectin-synthesizing
enzymes in barley endosperm. ............................................................................. - 147 Figure 4.1: Iodine-staining of barley grains (cross section). ............................... - 167 xiv
Figure 4.2 (A & B): Starch granule size distribution of different genotypes...... - 169 Figure 4.3 (A & B): Starch granule number distribution of different genotypes.
................................................................................................................................- 170 Figure 4.4 (A & B): Starch granule surface area distribution of different genotypes.
............................................................................................................................... - 171 Figure 4.5: Starch granule distribution into (A-, B-, and C-) classes. ................ - 172 Figure 4.6: Surface morphology of starch granules from different genotypes
observed by SEM. ................................................................................................. - 174 Figure 4.7: Difference plot of (other genotypes versus wild-type) for amylopectin
chains with different DP. ...................................................................................... - 181 Figure 4.8: Difference plot of (other genotypes versus waxy) for amylopectin chains
with different DP. ................................................................................................. - 182 Figure 4.9: Difference plot of (other genotypes versus low amylose) for amylopectin
chains with different DP. ...................................................................................... - 183 Figure 4.10: Immunodetection of granule-bound proteins in A- and B-starch
granules. ................................................................................................................ - 188 Figure 4.11: Quantitation of individual granules bound proteins. ..................... - 191 Figure 4.12: Granule-bound proteins (starch, A- and C-granules) visualized by
silver staining. ....................................................................................................... - 193 Figure 4.13: Analysis of starch composition and granule morphology by light
microscope (A, B & C) and electron microscope (D & E). .................................. - 194 Figure 4.14: Phosphorylation of starch granule associated proteins. ................. - 195 -
xv
ABBREVIATIONS
AGPase
Adenosine diphosphate glucose pyrophosphorylase
APase
Alkaline phosphatase
AgNO3
Silver nitrate
AMG
Amyloglucosidase
Ae
Amylose extender
CaCl2.2H2O
Calcium chloride
CLD
Chain length distribution
CSIRO
Commonwealth scientific and industrial research organisation
Con A
Concanavalin A
DAP
Days after pollination
DMSO
Dimethyl sulphoxide
DP
Degree of polymerization
DTT
Dithiothreitol
ELC
Extra-long unit chains
GPC
Gel Permeation Chromatography
GOPOD
Glucose oxidase/peroxidise reagent
GBSS
Granule-bound starch synthase
HAG
High amylose glacier
HPAEC
High-Performance anion exchange chromatography
HPAEC-PAD
High performance anion exchange chromatography with pulsed
amperometric detection
HPLC
High performance liquid chromatography
xvi
I2-KI
Iodine and potassium iodide
ISA
Isoamylases
KOH
Potassium hydro oxide
MgCl2
Magnesium chloride
MnCl2.4H2O
Manganese chloride
Na2-EDTA
Ethylene diamine tetra acetic acid disodium salt
Na2S2O3
Sodium thiosulfate
NaCl
Sodium chloride
NaOH
Sodium hydroxide
PCA
Principal component analysis
RDS
Rapidly digestible starch
RS
Resistant starch
Rpm
Revolution per minute
RSI
Resistant starch I
RSII
Resistant starch II
RSIII
Resistant starch III
RSIV
Resistant starch IV
SBEIIa
Starch branching enzyme IIa
SBEIIb
Starch branching enzyme IIb
SEM
Scanning electron microscopy
SCFA
Short chain fatty acids
SL
Seed length
SDS
Slowly digestible starch
SDS
Sodium dodecyl sulphate
xvii
ST
Seed thickness
SBEI
Starch branching enzymes
DBE
Debranching enzymes
SS
Starch synthases
SW
Seed width
TGW
Thousand grain weight
Tricine-KOH
Tricine-potassium hydroxide
V/V
Volume/volume
W/V
Weight/volume
xviii
Chapter 1: General Introduction
-1-
1. General Introduction
1.1 Overview
Starch is an important and widely distributed carbohydrate in plants. It is the most
significant form of carbon reserve in the majority of species. It is important in terms of
the amount made, its distribution among many different plant species and its economic
importance. It is an important dietary source of energy for humans and represents 7080% of the average daily caloric intake (Tetlow, 2006) and also serves as a source of
feed, fiber, biofuels and a raw material in many industrial applications. Cereal grains,
pulses and tubers are the major sources of starch (Tauberger et al., 2000). As a principal
storage compound, starch plays important roles in the life cycle of the plant. In plants,
starch is synthesized in plastids, which are derived from proplastids. There are two
particularly important types of plastids. Chloroplasts which are present in photosynthetic
tissues like leaves, and are responsible for transient starch synthesis: and amyloplasts
which are present in non-photosynthetic storage tissues like seeds, tubers and roots and
which are responsible for storage starch synthesis (Tetlow, 2006, 2011).
The type of plastid and the plant tissue from which starches are derived are
important in determining the functionalities of a particular starch. All the starches which
are synthesized in different parts of the plant are degraded at some stage, For example,
during the day time transient starches are synthesized in leaves and degraded at night to
provide carbon for non-photosynthetic metabolism (Streb and Zeeman, 2012). This
supply of carbon from degradation of starch at night is important for normal growth of
the plant. By comparison storage starches in developing seeds serve as a long term
carbon store for the next generation (Gerard et al., 2001; Tetlow, 2006).
-2-
The starch synthesized in chloroplasts during the day time is degraded to maltose
during the dark period. The majority of derived hexoses are converted to triose phosphate
and transported to the cytosol for sucrose synthesis. To support plant growth this sucrose
can readily be transported to non-photosynthetic tissues and then carbon is transported to
amyloplasts for starch synthesis. The starting point for the chloroplast pathway of starch
synthesis is fructose-6-phosphate, a product of photosynthetic carbon fixation. The
starting point for the amyloplast pathway is glucose-1-phosphate, a product of sucrose
degradation. In potato, pea, and maize glucose-6-phosphate, in addition to glucose-1phosphate, can be imported into the amyloplast and can serve as the starting point for
starch biosynthesis (Tauberger et al., 2000). Figure, 1.1 shows details of the starch
biosynthetic pathway of transient and storage starches.
Starch produced in the cereals endosperm is an abundant and renewable source
and has many commercial uses (Hannah and James, 2008; Zhang et al., 2008). In the
food industry starch is used in cereals and snacks, flavours and beverages. Starch is also
utilized in many non-food industries includes textiles, explosives, cosmetic and
pharmaceutical, paper, mining and construction industries (Lauro, 1999). The physical
and chemical properties of starch granules are important in determining their end use
(Lindeboom et al., 2004).
1.2 Endosperm Development
Starch is synthesized in the cereals seeds during endosperm development.
Endosperm is the nutritive tissue that surrounds the embryo within seeds of flowering
plants. The nutritive endosperm initiated by double fertilization is a unique feature of
angiosperms. The endosperm is essential for reproduction because it provides nutrition to
-3-
Figure 1.1: Sucrose metabolism and starch biosynthesis in different cells of plants.
TP, triose phosphate; G1P, glucose 1-phosphate; G6P, glucose 6-phosphate; AGPase,
adenosine 5' diphosphate glucose pyrophosphorylase; ADPG, adenosine diphosphate
glucose; Glc, glucose; Mal, maltose; HXP, hexose phosphate; Open and closed circles, TP
and ADPG transporter; Open hexagon, hoxose and maltose transporter; closed hexagon,
HXP transporter. Figure 1.1 shows that triose phosphate and hexoses are transported to
the cytosol of the source via transporters and are converted into hexose phosphate, and
then converted into sucrose. This sucrose is transported to different cells. In the cereal
endosperm, sucrose is utilized to produce ADP-glucose by cytosolic and plastidial
AGPase via some intermediate products (G6P & G1P). ADP-glucose is used by starch
synthesizing enzymes to produce starch. (This picture has been transformed from
Mitsui et al., 2010).
-4-
the germinating embryo. In cereal endosperm, different storage products accumulate
during development. Among storage products, starch and proteins are prominent. The
great economic and nutritional value of cereals like maize, wheat, rice and barley is due
to their starch-enriched grains. In cereals, such as maize, starch represents approximately
75 % of the mature seed weight and proteins represent 10 % of the total grain weight
(Keeling and Myers, 2010). The process of endosperm development in cereals has been
divided into several distinct phases, which overlap considerably. These phases are: early
development, differentiation and maturation. Early development involves double
fertilization, syncytium formation and cellularization.
Double fertilization, all angiosperms including maize, wheat and barley (Kiesselbach,
1949; Bennett et al., 1975) show double fertilization in which the fusion of one sperm
nucleus with the egg nucleus forms a diploid embryo, and fusion between a sperm
nucleus and two polar nuclei giving rise to a triploid endosperm, occur simultaneously.
After double fertilization, the triploid endosperm shows a very fast mitotic activity
compared to the embryo. The underlying mechanism of this process is not yet known in
cereals (Sabelli and Larkin, 2009a).
Syncytium formation, the newly fertilized nucleus of the endosperm undergoes a
repeated cycle of synchronous divisions in the absence of cell wall formation and
cytokinesis, which gives rise to a multi-nucleate structure called a syncytium as shown in
Figure (1.2B) (Lopes and Larkins, 1993). Cell division is slower in the zygote, as by the
time the zygote has divided for the first time, there are four to eight endosperm nuclei in
maize, and an increase up to 512 nuclei occurs within three days after pollination (DAP)
(Randolph, 1936). These nuclei arrange themselves at the periphery of the endosperm cell
-5-
as shown in Figure (1.2B). Proliferation of embryonic cells is less compared to
endosperm because endosperm nuclei do not involve the synthesis of cytoplasm, cell
membranes and cell walls.
Cellularization, the formation of the internuclear radial microtubule system (RMS), and
an open-ended alveolation process that starts from the periphery of the endosperm and
proceeds towards the vacuole, is responsible for the cellularization of the coenocytic
endosperm in cereals. Microtubules radiating from the nuclear surface (Figure 1.2C)
define the nuclear-cytoplasmic domains, which result in the equal distance lining of the
nuclei with the cell wall (Olsen, 2004). Through the deposition of adventitious
phragmoplast and the repolarization of microtubules, initiation of cell wall formation
takes place anticlinally. This event is followed by the centripetal extension of the cell
wall (Figure 1.2D) forming alveoli (open tubular structures in which the inner periclinal
cell wall that surrounds each nucleus is absent). After centripetal extension of the cell
wall, synchronous and periclinal division of the nuclei occur, immediately followed by
cytokinesis. With an overlaying layer of residual syncytial cytoplasm, the alveoli layer
displaces farther towards the central cavity (Figure 1.2E). The process of cellularization
continues centripetally until the central cavity is filled with cells (Figure 1.2F). In cereals
it takes 3-6 DAP. Cellularization is mediated due to interaction between the cell cycle
progression machinery and the microtubular cytoskeleton (Olsen, 2001). Cellularization
is important because these are the cells from where further differentiation will take place.
Cellularization is a metabolically active process and requires energy. During the early
phase of development (syncytium formation and cellularization) certain changes are very
prominent. These changes include maximum expression of proteins related to actin,
-6-
tubulin and cell organization. Similarly, proteins involved in respiration metabolism
(glycolysis and Krebs cycle) and protection against reactive oxygen species also have
high expression (Prioul et al., 2008). These changes coincide with early development
where, due to cell division and cellularization, high metabolic rates exist, requiring more
energy, which results in an increase in respiration.
Differentiation, during this phase of development, endosperm cells differentiate into four
major cell types including embryo surrounding region (ESR) cells, transfer cells (TCs),
aleurone layer and starchy endosperm cells, which are the main constituents of the
endosperm.
Embryo surrounding region (ESR) cells, ESR comprises several cell layers, which
completely cover the young embryo at around 4 DAP. With the growth of the embryo,
ESR shrinks, and around 12 DAP there are only vestigial remnants of the ESR present at
the base of the endosperm (Kiesselbach, 1949). In maize, these cells have dense
cytoplasmic contents. Their function is to supply the embryo with sugars through an
apoplastic route (Cossegal et al., 2007). An additional function is to protect the embryo
against pathogens. These cells are also metabolically active. The mechanism which
specifies the fate of ESR is not known.
Transfer cells (TCs), in cereal endosperm, several cell layers near the placenta
differentiate into TCs before cellularization is complete. These cells are found at the base
of the endosperm, but their position within the caryopsis varies among species (Rost et
al., 1984). In maize they are located over the chalazal pad. These cells are characterized
by prominent secondary cell wall ingrowths. The plasma membrane of TCs grows,
clinging to the cell wall ingrowths, which results in an increase in its superficial area, and
-7-
Figure 1.2: Diagrammatic presentation of endosperm cellularization process.
(A), the fertilized triploid nucleus (orange), vacuole (yellow).
(B), multinucleate cell with a large central vacuole.
(C), cellularization, formation of RMS, at the surface of the endosperm nuclei.
(D), cell wall formation, each nucleus surrounded by a tube-like wall structure
(alveolus).
(E), continued growth of alveoli.
(F), mitotic division.
This picture has been adapted from Olsen, (2004).
-8-
magnification of the surface-to-volume ratio of the protoplast. The morphology of TCs is
optimal for absorbing and secreting substances, which facilitate nutrient uptake by the
endosperm (Zheng and Wang, 2010). These cells have dense cytoplasm, enriched with
spherical mitochondria. The presence of an enormous amount of mitochondria shows that
during differentiation of TCs higher metabolic activities are required. TCs produce
antimicrobial- resembling proteins, which show that these cells also protect the kernel
from potential pathogenic invaders (Magnard et al., 2003).
Aleurone layer, aleurone cells form a sheet around the endosperm, except in the transfer
cell region. The number of layers of cells in the sheet varies with the species, for
example, one in maize and wheat, three in barley and several in rice (Sabelli and Larkins,
2009b). In maize their differentiation occurs between, 6-10 DAP from the outer layers of
the endosperm cells. Due to accumulation of spherosomes and protein bodies these cells
become cuboidal. Differentiation of aleurone cells depends upon the species (Brown et
al., 1994). The presence of numerous small vacuoles with inclusion bodies, termed
“aleurone grains”, makes the cytoplasm of aleurone cells dense and granular. These
inclusion bodies store protein, lipids and other molecules. These cells possess a welldeveloped endoplasmic reticulum and a large number of mitochondria. The different
range of colors observed in the maize kernels is due to the presence of anthocyanins in
mature aleurone cells. This is the only live tissue in endosperm at maturity due to the
presence of a specific developmental program that protects it from desiccation (Hoecker
et al., 1995). Positional information stored in the periphery of the endosperm, close to the
former central cell wall is required for aleurone cell fate specification. The function of the
aleurone cells is to synthesize the proteolytic and hydrolytic enzymes, which digest the
-9-
endosperm cell wall and remobilize the starch and protein stored in the endosperm, to
provide sugars and amino acids for the growing embryo. Production of these enzymes in
aleurone cells is stimulated by gibberellic acid (GA) from the imbibed endosperm
(Ritchie et al., 2000).
Starchy endosperm, the transition from the cell division to the storage phase of
endosperm depends upon different signals. One important factor is the glucose/sucrose
ratio in the caryopsis. A high glucose/sucrose ratio leads to endosperm cell proliferation,
while low glucose/sucrose sucrose ratio will lead to the starch accumulation phase
(Sabelli and Larkins, 2009a). Cereal endosperm is one of the most important food sources
because it has about 70 % starch by weight. This starch provides more than 80% of daily
caloric intake for humans (Keeling and Myers, 2010). Initiation of starch deposition in
endosperm varies among species. In wheat it starts soon after cellularization, while in
maize it begins around 10 DAP. Starch synthesis is controlled by a series of enzymes
which are discussed in this thesis.
The grain filling rate and seed weight are directly related to the number of starch
granules in the endosperm. In turn, starch granule number is directly related to the
number of cells. This observation shows that initiation and duration of the cell division
phase are important in endosperm development and grain yield (Commuri and Jones,
1999). High levels of ATP and high energy states are associated with starch accumulation
and granule size, which shows that high metabolic activity is associated with cell
expansion and starch accumulation, and these reactions are energy limited (Rolletschek et
al., 2005). During endosperm development, cereals store carbohydrates, protein, oils and
other compounds. These stored compounds provide nutrition and other necessary
- 10 -
elements for the germination of the embryo. The stored proteins serve as a source of
nitrogen and sulphur for the growing seedling. The major stored proteins in cereals are
prolamins and globulins. Prolamins are highly hydrophobic and are soluble in organic
solvents or denaturing solvents while globulins are soluble in saline solution. Additional
minor proteins are also stored in the endosperm (Coleman and Larkins, 1999).
In wheat and barley, two forms of prolamins are present which are closely related.
In wheat, the monomeric form is termed gliadin and the polymeric form glutenin, while
in barley the polymeric form is hordein (kreis et al., 1985). In maize, prolamins are called
zeins (Coleman and Larkins, 1999). Prolamins are rich in certain amino acids like proline
and glutamine, and are deficient in lysine and tryptophan. Prolamins represent 50-60 %
of total endosperm stored protein in maize, barley and wheat but in rice accounts for only
5-10 % (Laudencia-Chingcuanco et al., 2007). In the lumen of rough endoplasmic
reticulum (RER) both prolamins and globulins form insoluble accretions called protein
bodies. In maize and rice, the protein bodies are retained within the RER, the mechanism
of which is unknown. In wheat, these accretions are trafficked to vacuoles (Herman and
Larkins, 1999; Holding et al., 2007). Prolamins accumulate during middle and late
periods of endosperm development. Nucleic acids are also present in starchy endosperm.
These are not specific storage molecules but they are present in the starchy cells and
remain there in the dead cells. After imbibition, nucleases released by the aleurone
degrade starchy endosperm nucleic acid contents (Brown and Ho, 1986). After
cellularization, a phase of mitotic cell division occurs that generates the final population
of endosperm cells. In central endosperm this phase lasts until 8-12 DAP but in aleurone
and subaleurone layers it continues until 20-25 DAP. The mitotic cell division index is
- 11 -
maximum around 8-10 DAP, and after that it declines. Endosperm grows rapidly from 810 DAP to fill the entire seed cavity. This growth is correlated with cell division, cell
enlargement and endoreduplication (Knowles and Philips, 1988). Different phases of
endosperm development have been presented in the Figure (1.3).
Endoreduplication, maize endosperm cells around 8-10 DAP switch from a mitotic to
an endoreduplication cycle. In this cycle, without chromatin condensation, sister
chromatid segregation and cytokinesis, completed and reiterated rounds of DNA
synthesis take place (Sabelli and Larkins, 2008). As a result, a gradient of nuclear size is
observed in the tissue due to the presence of small nuclei at the periphery and
increasingly larger nuclei in the inner central region of the endosperm (Knowles and
Philips, 1988). Endoreduplication is also a metabolically active process which requires
energy. Endoreduplication seems to be correlated with rapid caryopsis growth, and the
synthesis and accumulation of starch and storage proteins. The precise function of
endoreduplication is not yet known (Sabelli and Larkins, 2009b). During this phase of
development (differentiation) certain changes are prominent, which include the presence
of a large amount of proteins which are involved in proteolysis. This turnover in proteins
is consistent with a switch from growth and differentiation to storage. During this phase
of growth, enzymes related to metabolism are also increased because of endosperm
storage filling. The prominent proteins which are expressed at a maximum rate during
this stage are chaperones and proteins involve in degradation of metabolites. The
presence of the maximum amount of proteins involved in protein folding (chaperone)
during this stage is associated with storage protein accumulation in the endosperm. One
interesting change at this stage is the decrease in the amount of enzymes involved in the
- 12 -
Kreb’s cycle relative to the glycolytic enzymes. This condition is in agreement with the
recent demonstration that starch accumulation in the endosperm takes place under
hypoxic conditions (Rolletschek et al., 2005). Due to lack of oxygen, Kreb’s cycle
enzymes are not functioning, which influences corresponding enzyme expression. ATP
needed for starch synthesis from sucrose is produced in the absence of oxygen by
glycolysis and the sucrose synthase UGPase-AGPase cycle. During this stage
accumulation of other proteins like those involved in starch synthesis is also increased.
Maturation, maturation is comprised of programmed cell death (PCD), dormancy and
desiccation (Oslen, 2001). These final steps permit seed dispersal, long term storage and
tolerance to harsh environments.
Programmed cell death, programmed cell death is an important mechanism in cereal
endosperm development which facilitates nutrient hydrolysis and uptake by the embryo
at germination (Naguyen et al., 2007). In maize, endosperm PCD starts in two different
regions, the central starch endosperm cells and apical cells near the silk scar at around 16
DAP. In this way, the top half of the endosperm becomes dead at around 28 DAP
(Young and Gallie, 2000a), while aleurone cells undergo PCD at around 30 DAP (Young
and Gallie, 2000b). It is thought that certain proteases and hormones (ethylene) are
involved in the progression of PCD (Naguyen et al., 2007). It is thought that abscisic acid
(ABA) is also involved in PCD (Young and Gallie, 2000a).
Desiccation, desiccation is also important in PCD, because this makes the aleurone
responsive to GA. An interesting change which occurs around 21 DAP involves, sudden
onset of Pyruvate-Phosphate Dikinase (PPDK). This PPDK plays an important role in the
accumulation of storage products. This protein is believed to be involved in regulation of
- 13 -
Figure 1.3: Presentation of different phases of maize endosperm development.
(A) shows, double fertilization, syncytium formation, and cellularization (3 DAP). The
pollen tube and sperm nuclei (yellow), polar nuclei and endosperm (red), The egg cell
nucleus and embryo nuclei (green). Endosperm and embryo (red and green outline).
(B) represents 4-20 DAP, endosperm mitotic division and cell proliferation, followed by
endoreduplication (from 8-10 DAP). Programmed cell death (PCD) starts around 16
DAP). The graphs with different C values represent increase in genetic material.
(C) is presenting parameters of endosperm development, fresh weight, nuclei number,
mitotic index, and average DNA content (red, blue, brown and green lines respectively).
Abbreviations: Aleurone (Al); central starchy endosperm (CSEn); embryo (Em);
endosperm (En); nucellus (Nu); pericarp (Pe); placentochalaza (Pl); subaleurone layer
(SAl); transfer cells (TC). This picture has been adopted from Sabelli and Larkins,
(2009a).
- 14 -
storage product synthesis in different ways (Prioul et al., 2008). Other changes at this
stage of development are gradual decrease in metabolic activity, which is consistent with
PCD.
1.3 Starch and its Composition
Normal starch exists as semi-crystalline insoluble granules varying in size (0.5100µm) and shape (spherical, elliptical, or polyhedral). The starch granule is composed
of two polymers, amylose and amylopectin (Martin and Smith, 1995). Granule size,
number and morphology are the characteristics of the organ and species in which they are
produced (Jane et al., 1994; Shapter et al., 2008). Based on the size distribution of starch
granules, different species have either unimodal (maize) or bi-or trimodal (wheat and
barley) granule size distributions (French, 1984). For example, in wheat and barley, bi- or
tri-modal granule size distribution is differentiated as (A-, B- and C-) type granules. A- ,
B- and C-type granules can be distinguished based on size, shape, relative number and
the timing of their initiation during seed development. A-granules are lenticellular,
varying in size from 10-50 µm in diameter and make upto 70-80 % of the volume but
only 10 % of the total number of the starch granules (Hughes and Briarty, 1976;
Langeveld et al., 2000). B-granules are spherical, varying in size from 5-10 µm in
diameter and represent < 30 % of the volume and > 90 % of the total number of starch
granules. Starch granules of size less than 5 µm in diameter are termed as C-granules
(Bechtel and Wilson, 2003; Wilson et al., 2006). The small size of C-granules makes it
difficult to isolate and quantify them which sometimes lead to their classification as Bgranules.
Timing and location of granule synthesis, the initiation of A-, B- and C-granules is
- 15 -
developmentally regulated. A-granules are synthesized when endosperm is still actively
dividing i.e. between 4-14 days postanthesis (DPA), B-granules are initiated between 1016 DPA in the evaginations of A-granule-containing plastids (Langeveld et al., 2000;
Bechtel and Wilson, 2003) and C-granules start to appear at about 21 DPA (Bechtel and
Wilson, 2003). Morphology and size of A- and B-granules from different cereals are
shown in Figure (1.4).
Starch is synthesized in plastids in different tissues within the same plant. In
developing endosperm these plastids are called amyloplasts. Rice and oats have
compound granules in which many granules are formed in a single amyloplast. These
granules are small at maturity and are polyhedral in shape due to pressure from the
surrounding granules. These polyhedral granules become compressed together to form
compound granules which at low magnification appears as a single granule. In
Panicoideae, typically a simple, single granule is formed in the individual amyloplasts.
However, Triticeae have a unique endosperm bimodal granule size distribution,
consisting of large lenticellular A- and small spherical or ovoid B-granules. The Agranules are synthesized in the body of amyloplasts while B-granules are formed
independently in the outgrowths of the same cell without appearing as compound
granules (Rahman et al., 2000). However, Li et al. (2001) reported the existence of
compound granules in certain barley genotypes, which represented clusters of a few
granules but with the appearance of a single granule.
There are number of factors involved in the differentiation of the starch granule.
These include multiple and complex genetic control and biochemistry which regulate the
size and number of plastids, and also environmental conditions during seed development
- 16 -
(Shapter et al., 2008).
Figure 1.4: Starch granules morphologies from different cereals.
This figure illustrates starch granules from the endosperm of cereal seeds using light
microscopy and scanning electron microscopy, the methods of which are described in
later part of this thesis. (A), light micrograph presenting compound starch granules
within amyloplasts of an oat endosperm. Each compound granule is composed of many
smaller granules which look like a single granule.
(B), scanning electron micrograph is presenting tightly packed A- and B-granules within
the barley endosperm cells.
(C), scanning electron micrograph of maize endosperm starch.
(D), scanning electron micrograph of wheat endosperm starch. Both large A-granules
(the arrows indicate equatorial grooves) and small B-granules are clearly visible (Smith,
2010).
- 17 -
1.3.1 Molecular structure of starch granule
Starch is made up of amylose and amylopectin. Amylose and amylopectin are
made up of the same basic glucan polymers but with different length and degree of
branching (Figure 1.5 A & B). Amylose is essentially a linear molecule with a molecular
weight varying between (105-106 Daltons), in which glucose residues are joined via α-1, 4
linkages with very few α-1, 6 linkages and makes upto 20-30% of the starch (Figure, 1.5
A). While amylopectin with a molecular weight of (107-109 Daltons) contributes 70-80 %
share in total starch and contains linear chains of various lengths. Almost 5 % of the
glucose units in amylopectin are joined by α-1, 6 linkages, which introduce branches in
the amylopectin (Davis et al., 2003) (Figure, 1.5 B). In amylose the degree of
polymerization of glucose is species dependent (Morrison and Karkalas, 1990). In
amylopectin branching of glucan chains exhibits regular periodicity and its length and
pattern play a critical role in the proper formation of the granule (Stamova et al., 2009).
Intermolecular attraction and association of neighbouring amylose molecules result in
unstable aqueous solutions of amylose leading to increase in viscosity, retrogradation
and, under specific conditions, precipitation of amylose particles (Hedley, 2002). The
characteristics of aqueous solutions of amylopectin include high viscosity, clarity,
stability, and resistance to gelling. These properties are important in terms of starch use.
For example rehydration of starch depends upon both viscosity and granule size.
Similarly, resistance to gelling makes starch stable at high temperature (Hedley, 2002). In
the native form of starch, amylose and amylopectin are organised in granules as
alternating semi-crystalline and amorphous layers. The ordered regions constituting the
semi-crystalline layers are composed of double helices formed by short amylopectin
- 18 -
Figure 1.5: Structural differences between amylose and amylopectin.
The starch granule is composed of two types of glucan polymers; amylose and
amylopectin.
(A), Amylose is a relatively less branched polymer with longer chains containing
predominant α(1→4) bonds.
(B), Amylopectin is a highly branched and complex glucan polymer with α(1→4) linked
chains in which branches are introduced by α(1→6) linkages.
- 19 -
branches, most of which are further ordered into crystalline structures (Hedley, 2002).
Amylopectin has a polymodal glucan chain distribution, which allows the condensing of
shorter glucan chains and the subsequent development of efficiently packed parallel
double helices. These helices coupled with regular branch point clustering give rise to the
basis of the organized semi-crystalline nature of the starch granule matrix as shown in
Figure (1.6c) (French, 1984; Hizukuri, 1986; Tetlow, 2006). This conserved architecture
of amylopectin is responsible for the semi-crystalline, water insoluble starch granule.
Granule formation is regulated by the semi-crystalline properties of amylopectin, which
are determined by clustering, the length of the linear chains of amylopectin, and the
frequency of a-1, 6 linkages (French, 1984; Hizukuri, 1986; Myers et al., 2000). In
granules of different sizes the molecular structure of the amylose and amylopectin
fractions varies. For barley, it was found that with decreasing granule size, the average
degree of polymerization of amylopectin decreased. On the other hand granule size did
not affect amylose polymerization (Tang et al., 2001). In amylopectin with large
granules, long amylopectin B chains are present in greater number as compared to small
granules from the same cultivar (Naka et al., 1985). The crystallites composed of double
helices may be densely packed forming an orthogonal pattern, as in cereal starches, and
are termed as A-type, or in a less densely packed hexagonal pattern, as in potato starch,
when they are termed B-type as shown in (not to be confused with A- and B-starches
based on size). The type of crystallinity in cereal and potato is shown in Figure (1.7).
The amount and mobility of the structural water contained in both types of crystallites is
greater in B type crystallites. Hence, cereal starches are termed A- and potato starches Btype. There are some other species, for example pea, which contain both A- and B-type
- 20 -
A
B
C
Figure 1.6: Schematic representation of higher order molecular structure.
(1.6 A), illustrates amorphous and semi-crystalline zones with in starch granule.
(1.6 B & C), Enlargement of semi-crystalline growth rings, showing the arrangement of
the alternating crystalline and amorphous lamellae, the crystalline lamella is about 6 nm
and amorphous lamella is about 3 nm. In a starch granule both these regions repeat
many times to give rise to a well-organized starch granule (Tetlow, 2006).
- 21 -
A
B
Figure 1.7: Presentation of different types of crystallinity in starch originated from
different sources.
(A), shows A type crystallinity in cereals, when crystallites composed of double helices
are densely packed forming an orthogonal pattern.
(B), shows B type crystallinity in potato, when crystallites composed of double helices
are less densely packed forming hexagonal pattern (Ratnayake and Jackson, 2008).
- 22 -
crystallites confined to specific regions of the granule (Hedly, 2002).
1.3.2 Variations in physiochemical properties of A- and B-granules
Previous studies have shown that starches from different barley genotypes vary
widely in structure, composition and properties (Kang et al., 1985; Morrison et al., 1993;
Lorenz & Collins, 1995; Song and Jane, 2000; Yoshimoto et al., 2000). Besides
variations in morphology, size, and origin, wheat and barley large and small starch
granules also have differences in characteristics and properties with regard to chemical
composition. For example amylose, amylose-lipid complex and phosphorus contents
(Raeker et al., 1998; Shinde et al., 2003; Geera et al., 2006; Ao and Jane, 2007),
molecular structure (Sahlstrom et al., 2003), resistance to α-amylase digestion (Bertoft
and Kulp, 1986),
relative granule crystallinity and gelatinization temperatures
(Vermeylen et al., 2005; Ao and Jane, 2007) gelatinization temperature and
retrogradation (Peng et al., 1999; Singh and Kaur, 2004), granule swelling (Van Hung
and Morita, 2005), reactivity to modifying agents (Van Hung and Morita, 2005) and
pasting behavior (Geera et al., 2006; Ao and Jane, 2007) may also be different in large
and small granules. The isolated and purified B-granules of barley exhibit more
susceptibility to cereal α-amylases digestion and acid hydrolysis than the A-granules.
This is primarily due to the larger surface area of small granules as compared to large
granules (Bertoft and Kulp, 1986; Vasanthan and Bhatty, 1996). Large A-granules of
wheat show an increased enthalpy of gelatinization, lower gelatinization temperatures,
increased retrogradation (Peng et al., 1999; Singh and Kaur, 2004) and soft textured
flours compared to smaller B-granules (Gaines et al., 2000). Similarly, Sandhu et al.,
(2004) reported that varieties of maize with small starch granules exhibits lowest swelling
- 23 -
power, amylose content, solubility and retrogradation. A study with waxy maize and
millet and a very low amylose species, amaranth, showed that the small granule size of
amaranth was associated with its slower retrogradation (Choi et al., 2004). Wheat
starches possessing different granule sizes exhibited different degrees of susceptibility to
enzymatic hydrolysis, as well as thermal and pasting properties. Along with granule size,
difference in amylose content, protein content, and branch chain length of amylopectin in
A- and B-type starch granules, are also major factors responsible for differences in
digestibility and other functional properties of starch (Liu et al., 2007). Wheat starch from
five different genotypes, four possessing the same amylose content and one lacking
amylose (waxy) was separated into A-, B- and unfractionated starches. It was found that
A-granules had a smaller proportion of short chains of 6-12 DP and a higher proportion
of intermediate chains of 25-36 DP than B-granules. The lamellar repeat distance in Agranules was larger than that of B-granules. And the lamellar distances of both A- and Bgranules from the waxy genotype were smaller than those of non-waxy starches.
However, no differences were observed in the crystallinity of either A-, B- or
unfractionated starch. In vitro digestion kinetics with α-amylase of A- and B-granules
demonstrated differences. Initially B-granules were digested to a greater extent than Agranules. But after 4 h of incubation, A-granules showed more digestibility than that of
B-granules, while waxy starch showed similar in vitro digestibility of unfractionated and
fractionated granules (Salman et al., 2009). Investigation of chemical composition
showed that A-granules had lower lipid and higher amylose contents than B-granules.
Similarly A-granules also had the highest gelatinization enthalpy and peak and final
viscosity, while B-granules had the highest gelatinization temperature and amylose-lipid
- 24 -
complex enthalpy (Dengate and Meredith, 1984; Soulaka and Morrison, 1985; Peng et
al., 1999; Sahlstrom et al., 2003; Shinde et al., 2003; Vermeylen et al., 2005).
1.3.2.1 Uses of starch granules with different properties
Because of differences described above, these two types of starch granules are
utilized differently, both in food and non-food industries. Along with the above
mentioned properties, granule morphology also has an important impact on starch
physiochemical properties (Da Silva et al., 1997; Lindeboom et al., 2004) and granule
size determines many of the potential food and industrial applications of starch (Ji et al.,
2004). For example, small starch granules are suitable as a fat substitute, a carrier
material in cosmetics and in paper coating, while large granules are used in the
manufacturing of biodegradable plastic film and carbonless copy paper (Lindeboom et al.
2004). Similarly cereal cultivars with different proportion of large and small granules
would be very useful to different food and non-food industries (Wei et al., 2010).
1.3.2.1.1 Uses of starch in food industry
As indicated, starch has many important uses in the food industry. Factors
such as the ratio of amylose to amylopectin affect its physicochemical properties and end
use (Izdorczyk et al., 2000; Hang et al., 2007). For example waxy (amylose free) starch
has wide application in the food industry for conferring properties like uniformity,
stability, texture, and better freeze-thaw ability (Chibbar and Chakraborty, 2005).
Depending upon the % amylose content, barley starch can be classified into normal (2527 %), waxy (<5 %), and high amylose (>35 %) (Bhatty and Rossnagel, 1992; Bhatty et
al., 1998; Izdorczyk et al., 2000; Zheng et al., 1998). These differences in starch
composition and structure can be utilized in food applications to reduce the risk of
- 25 -
diabetes and/or digestive tract related diseases like colon cancer which are associated
with increased use of starch rich food.
1.3.2.1.1.1 Digestion of food
Digestion of food containing starch is a complex process which is influenced by
rate of digestion and absorption in the digestive tract. This process is affected by many
factors including source, components, physical nature and processing methods of food,
and the presence of enzyme inhibitors (O’Dea et al., 1980; Goni et al., 1997). Due to the
high viscosity created by fibre enriched food, the rate of carbohydrate absorption
decreases in the upper digestive tract (Englyst and Kingman, 1990; O‘Dea et al., 1991). It
is thought that the fiber component of food excludes the enzymes responsible for
carbohydrate hydrolysis hence lowering the rate of starch hydrolysis (O’Dea et al., 1980).
Starch has been divided into three categories: rapidly digestible starch (RDS), slowly
digestible starch (SDS), and resistant starch (RS). Digestion of food in the human
digestive tract is shown in detail in Figure (1.8).
1.3.2.1.1.1.1 What is RS and its health benefits?
RS was first identified in 1982 and categorized as part of dietary fibre (Englyst et
al., 1982). Increasing dietary fibre uptake is related to lower rates of obesity,
cardiovascular disease, diabetes and certain cancers (National Health and Medical
Research Council, 2006). Similarly RS resists α-amylase digestion in the small intestine,
and is fermented by the bacteria in the large intestine, producing a variety of end
products, the most important of which are the short chain fatty acids (SCFA) (Englyst et
al., 1996). SCFA primarily consists of butyrate, propionate and acetate and are the
preferred sources of energy for the colonocytes (cells lining the colon). Other beneficial
- 26 -
Figure 1.8: Digestion and absorption of different food ingredients in the human
digestive tract.
Food digestion occurs in a series of phases. Starch which has been consumed after
gelatinization (cooked), which disrupts the molecular structure of starch and makes
glucose chains accessible, is more easily digested by α-amylases. Almost all of the starch
in food is digested and absorbed in the small intestine. However some starch escapes
this digestion process and reached in the large intestine, this starch is called resistant
starch (RS). This RS is utilized by the bacteria of the large intestine and different
beneficial components including short chain fatty acids are produced. Use of RS and
ultimately its consumption in the large intestine has many health-associated benefits,
including decreasing the incidence of colorectal cancer.
- 27 -
effects of SCFA include increased colonic blood flow, improvement in the mineral
bioavailability, reduction in the growth of pathogenic bacteria by lowering pH in the
lumen, and prevention of abnormal colonic cell development (Topping and Clifton,
2001). SCFA, particularly butyrate, also been shown to facilitate other important
physiological changes such as, an ability to reverse neoplastic changes in vitro (Ferguson
et al., 2000), positive and nutritive effects on the colonic epithelium, and induction of
apoptosis (programmed cell death) of damaged cells (Mentschel and Claus, 2003). SCFA
help in maintaining healthy viscera (Wei et al., 2010). RS is assumed to be one of the best
substrates for butyrate production because RS fermentation produces butyrate at twice the
rate of wheat fibre and four times that of pectin (Champ, 2004).
In recent years, the glycemic index (GI) has not only been used as a potentially
diet planning tool for diabetic patients, but also as a measure to prevent diabetes,
dyslipidemia, cardiovascular disease, and even certain cancers as well (Jenkins et al.,
1981). The glycemic index provides a measure of how quickly blood sugar (glucose)
levels rise after eating a particular type of food. Glycemic response of an individual is
greatly influenced by the digestion of starch present in the food (Liu et al., 2007).
The Commonwealth Scientific and Industrial Research Organisation (CSIRO),
has recommended that around 20 g of RS should be consumed per day, this amount is
almost four times greater than a typical western diet is currently providing (Baghurst et
al., 1996). Studies on a particular type of RS2 (Hi-maize®) showed that 17g/day or more
RS is required in the diet for a positive impact on one or more of the accepted parameters
of digestive health, (Muir et al., 1995; Phillips et al., 1995; Birkett et al., 1996; Noakes et
al., 1996; Muir et al., 2004). Because of the prebiotic properties of RS, it has a symbiotic
- 28 -
effect where it can provide protection to beneficial bifid bacteria in vivo during their
travel through the upper gastrointestinal tract (Wang et al., 1999). It has been shown that
during diarrhoea and cholera, use of oral rehydration solutions containing RS reduce
fecal fluid loss and shorten the duration of disease. This finding can provide further
insight into production of oral rehydration solutions which can be used for different
purposes. Recently it was shown that fermentation of resistant starch is associated with
elevated levels of gut hormones (PYY and GLP-1) which have a role in satiety and
potentially long-term energy balance (Keenan et al., 2006). Although this research is still
preliminary, the possible link between fermentation products of RS and gene expression
of hormones, related to reduced energy intake, is of considerable importance (Higgins,
2004).
1.3.2.1.1.1.1.1 Types of RS
There are four different types of RS, designated RSI, RSII, RSIII and RSIV. RSI
is physically inaccessible starch, as found in partial or intact cereal seeds. α-amylases do
not have access to starch as the gastrointestinal tract does not possess enzymes which
degrade cellulose, hemicelluloses, lignins, and other constituents of plant cell walls. RSII
represents raw (uncooked) starch in its granular form of some plant species, e.g. potato
and banana. The phenomenon of raw starch resistance to digestion varies from species to
species and depends upon different factors (Leszczyñski, 2004). For example in potato,
due to large size of granules, limited area is available to enzymes (Ring et al., 1988).
Other factors responsible for raw starch resistance include enzyme adsorption on the
surface of starch granule (Leloup et al., 1992a), shape, structure of granule, pore size on
the surface of granule and crystallinity (Leszczyñski, 2004), amylopectin chain length,
- 29 -
occurrence of large blocklets (Kossman and Lloyed, 2000), and amylose contents
(Gallant et al., 1997). RS III is retrograded starch. When starch-containing foods are
cooked and cooled, a water-insoluble semi-crystalline structure precipitates from starch
paste which is resistant to digestion. Retrogradation results in the formation of more
thermo-stable structures by amylose than by amylopectin. The amount of RSIII produced
by retrogradation is directly proportional to the amylose content of starch (Leloup et al.,
1992b; Colquhoun et al., 1995; Leszczyñski, 2004; Rahman et al., 2007). RS IV is
chemically or physically modified starch in industrially processed food. RSIV includes
hydroxypropyl distarch phosphate and acetylated distarch phosphate. The former has two
times lower susceptibility to the activity of amylases than native starch (Östergård et al.,
1988; Hoover & Zhou, 2003).
1.3.2.1.1.1.1.1.1 Factors affecting resistant starch
Starch is hydrolyzed by α-amylases, the actions of which are influenced by
various physical and structural properties including granule size, phosphorus content,
amylose: lipid complexes (Crowe et al., 2000). The molecular structure of starch is also
important in determining resistance to digestion; association of amylopectin chains, and
the degree of helix formation in amylose and amylopectin (Haralampu, 2000; Miao et al.,
2009), occurrence and perfection of crystalline region in both amorphous and crystalline
lamellae of the granule, (Eerlingen and Delcour, 1995) and starch crystallinity and
packing (Jane et al., 1997) are of significant importance. Other properties, such as hilum
and surface channels/pores connection (Kim and Huber, 2008), porosity, degree of
integrity and structural inhomogeneity (Copeland et al., 2009), and the presence of αamylases inhibitors like maltose and maltotriose (Colonna et al., 1988) also contribute in
- 30 -
resistance to α-amylases digestion. Previously, presence of pore or pin holes on the
surface of starch granules (Hall and Sayre, 1970; Fannon and BeMIller, 1992) and
variation in their distribution within and among different individual species (Fannon et
al., 2004) have also been reported to be important factors in determining starch
digestibility. In species like sorghum, maize and millet, the presence of pores on the
surface of the granule is related to increased rate of enzymatic digestion of the granule.
Pores connect the outer surface of the granule to the inner cavities (Huber and BeMiller,
2001; Benmoussa et al., 2006). The presence of such features is important in some
cereals like sorghum which has limitations in its end uses because of inherently poor
digestibility (Shapter et al., 2008). When starch is heated above a certain temperature, it
is gelatinized, which results in the leaching of amylose molecules in the form of coiled
polymers from the swollen starch granules. Upon cooling, these coiled polymers
associate as double helices and form hexagonal networks (Jane and Robyt, 1984;
Haralampu, 2000). In waxy starch, aggregate formation between amylopectin molecules
occurs, which is more susceptible to amylases for hydrolysis (Miao et al., 2009). Those
factors which make starch inaccessible to amylases contribute to overall resistant starch
content. Amylopectin chain length distribution (CLD) and packing play an important role
in determining starch digestibility (Asare et al., 2011). For example, the RS content of
maize ae-mutant lines was positively correlated with the apparent and the absolute
amylose content and the larger proportion of longer glucan chains of amylopectin (Li et
al., 2008). The ae-mutant lacks SBEIIb enzyme which is responsible for the addition of
branches in the growing amylopectin molecule. Due to absence of SBEIIb, branch
frequency decreases significantly, which results in the amylopectin with longer glucan
- 31 -
chains. Because of this structural change, the resultant amylopectin resembles amylose
hence this starch is termed as “high amylose”. Analysis of starch from different botanical
sources revealed no significant correlation between functional and physical properties
such as granule size, shape and apparent amylose content of starch but there was a strong
association with chain length distribution of amylopectin (Zhang et al., 2006). The
presence of lipids and proteins is also important in determining the digestibility of starch
and hence affects RS content (Zhang et al., 2006).
Rapidly digestible starch (RDS) is negatively correlated with amylose content and
variations in RS content are significantly influenced by amylose content in meal and pure
starch samples. RS is also associated with B-type granules ranging from (5-15 μm) in
size and the amylopectin fraction with 19-36 DP (Asare et al., 2011). A study conducted
with amylose extender (ae-), waxy (wx-) and wx ae- mutants showed that ae- endosperm
starch accumulated an increased amylose content plus long chain amylopectin. There
were no significant differences in the unit chain-length distribution of amylopectin or
starch granule morphology in ae- and wx/ae-starches. While wx/ae- starch had a higher
pasting temperature, higher peak viscosity and higher gelatinization peak temperatures
than that of the wild-type starch. The primary structure of the rice wx ae-amylopectin
with high proportion of long chains changes the granular and crystal structure of the
starch and increases resistance to in vitro or in vivo digestion by amylases (Kubo et al.,
2010). Thus starches from different origins have different physiochemical properties and
hence have different applications in food and non-food industries. The factors responsible
for variations in starch structure from different biological sources include: amylose to
amylopectin ratio, glucan chain length distribution, degree of branching, and granule
- 32 -
size. These are in turn controlled by the activities of different enzymes involved in the
synthesis of starch, the subject of which will now be discussed.
1.4 Starch Biosynthesis
1.4.1 Enzymes involved in starch biosynthesis
It is generally accepted that the complex process of starch biosynthesis is
catalyzed by a series of biosynthetic enzymes including, ADP-glucose pyrophosphorylase
(AGPase), starch synthase (SS), starch branching enzyme (SBE), and starch debranching
enzyme (DBE) (Smith et al., 1997; Myers et al., 2000; James et al., 2003; Grimaud et al.,
2008; Radchuk et al., 2009). The other enzymes which are also important in starch
synthesis are starch phosphorylase (SP) and disproportionating enzyme (D-enzyme) (Li
et al., 2003; Leterrier et al., 2008). Across species, highly-conserved families of genes
encode these enzymes (Ball & Morell, 2003; Ball & Deschamps, 2009). By studying
genetic modifications of starch synthesizing enzymes in numerous plant species, it has
been suggested that each enzyme class has a uniquely conserved role in the process of
starch synthesis (Peter et al., 2010). A schematic presentation of different enzymes
involved in starch biosynthesis is shown in Figure (1.9).
1.4.1.2 Adenosine 5’ Disphosphate Glucose Pyrophosphorylase (AGPase, EC
2.7.7.27)
In higher plants, adenosine 5' diphosphate glucose pyrophosphorylase (AGPase)
is responsible for the catalysis of the first committed step in starch biosynthesis. It
controls the synthesis of the nucleotide diphosphate sugar ADP-glucose (ADP-Glc) from
glucose-1-posphate and ATP. This ADP-glucose (ADP-Glc) serves as the soluble
precursor and substrate for starch synthases (Tetlow, 2006; Bowsher et al., 2007). The
- 33 -
reaction catalyzed by the AGPase is often considered as the rate-limiting step in starch
biosynthesis (Tetlow et al., 2003b). AGPase has a single, highly conserved N-terminal
catalytic region and a C-terminal domain made up of a parallel beta helix structure which
is involved in allosteric regulation and subunit oligomerization (Jin et al., 2005; McCoy
et al., 2007). The AGPase reaction in the majority of plant cells exclusively takes place in
plastids. However, an extra-plastidial, cytosolic form of AGPase is also present in the
endosperms of cereals and other graminaceous plants (Beckles et al., 2001). A number of
studies have revealed that the extra-plastidial form of AGPase is responsible for the
majority of AGPase activity in maize, barley, rice, and wheat (Denyer et al., 1996;
Thorbjornsen et al., 1996; Sikka et al., 2001; Tetlow et al., 2003b). The enzyme is largely
extraplastidial in cereal endosperm in contrast to other cereal tissues and non cereal
plants (Giroux et al., 1994; Denyer et al., 1996; Shannon et al., 1996; Thorbjørnsen et al.,
1996; Beckles et al., 2001; Comparot-Moss & Denyer, 2009). In wheat, cytosolic
AGPase accounts for about 65 %-95 % of the total activity (Tetlow et al., 2003a), and in
maize endosperm cytosolic AGPase activity was found to be > 95 % of the total (Denyer
et al., 1996). In barley the cytosolic AGPase activity accounts for 85 % of activity
(Denyer et al., 1996; Thorbjornsen et al., 1996; Johnson et al., 2003). Barley mutants
lacking cytosolic AGPase (Johnson et al., 2003) had reduced starch synthesis (44 %)
even though plastidial AGPase activity was unaffected (Tester et al., 1993).
In cereal endosperm, ADP-glucose synthesized in the cytosol is transported into
plastids in exchange for ADP via a small inner envelope protein encoded by the Brittle1
gene (Sullivan, 1995; Mohlmann et al., 1997; Shannon et al., 1998; Emes et al., 2003). It
was shown that the maize mutant brittle1 (bt1) accumulated >13% ADP-glucose more
- 34 -
SSIV
Figure 1.9: Schematic presentation of starch biosynthesis in the cereal endosperm.
In cereal endosperm ADP-glucose (ADPG) is generated by cytosolic and/or plastidial
AGPase, and used as a substrate for starch biosynthesis. GBBS is the only enzyme which
is involved in amylose biosynthesis. Synthesis of amylopectin is a complex process which
is regulated by number of enzyme classes including (SS, SBE, DBE and SP). SSs add linear
chains of various lengths in growing amylopectin molecule and branches are introduced
by SBEs. Inappropriately attached branches are trimmed by DBE. The exact role of SP in
starch biosynthesis is yet not known (Rahman et al., 2007).
- 35 -
than normal, but had reduced starch content (60 % of wild-type) even though activities of
starch synthases and starch branching enzymes were unaffected (Shannon et al., 1996).
Bowsher et al., (2007) showed that in wheat endosperm, the import of ADP-glucose into
amyloplasts is dependent on counter-exchange with the adenylates ADP and AMP.
Further, the rate of ADP exported from the amyloplasts was equal to the rate of ADPglucose utilized by starch synthases, suggesting that ADP is the most likely to be the
form of adenylate which exchanges with ADP-Glucose. AGPase is present in all starchsynthesizing tissues of higher plants and involved in the biosynthesis of both transient
starch in chloroplasts or chromoplasts and storage starch in amyloplasts. In higher plants,
AGPase is heterotetrameric, composed of two large (AGP-L) and two small (AGP-S)
catalytic subunits which are highly homologous in both sequence and structure, however
each is encoded by different genes (Ballicora et al., 2004; Hannah and James, 2008). In
maize, the large and small subunits of AGPase are encoded by shrunken2 (sh2) and
brittle2 (bt2) genes respectively (Bhave et al., 1990; Bae et al., 1990). While large and
small subunits of the maize plastidial AGPase are encoded by the Agp1 and Agp2,
respectively, but no mutants are available (Rosti & Denyer, 2007). Multiple genes
encoding the AGPase subunits are differentially expressed in different plant organs
which results in variable AGPase subunit composition in different parts of the same
plant such as potato (La Cognata et al., 1995), rice (Nakamura and Kawaguchi, 1992),
and barley (Villand et al., 1992). The sequences of small subunit of AGPase from
various eudicots and monocots vary in exon1 (Hannah et al., 2001).
Different mechanisms have been reported to regulate AGPase activity. First,
AGPase is subjected to transcriptional regulation, with elevated (sugar) sucrose causing
- 36 -
an increase in expression (Salanoubat and Belliard, 1989; Sokolov et al., 1998; MullerRober et al., 1990) while nitrate and phosphate shown a decrease (Nielsen et al., 1998;
Scheible et al., 1997). A second mechanism involves the allosteric regulation of AGPase,
being activated by glycerate-3-phosphate (3PGA) and inhibited by inorganic phosphate
(Pi) in leaf chloroplasts (Neuhaus and Stitt, 1990), in amyloplasts in cereal endosperm
(Tetlow et al., 2003b), and in storage tubers (Tiessen et al., 2003). The level of
sensitivity of AGPase to these allosteric effectors seems to be dependent upon tissue,
plastid type, the subcellular localization of the enzyme, and the ratios of the allosteric
effectors in different species. For example, in wheat endosperm the plastidial AGPase
activity is much less sensitive to 3-PGA activation and Pi inhibition compared to potato
tubers (Hylton and Smith, 1992; Gomez-Casati and Iglesias, 2002; Tetlow et al., 2003a).
Similarly chloroplast AGPase, is highly sensitive to concentrations of allosteric
effectors, being activated by micromolar concentrations of 3-PGA and inhibited by Pi
(Ghosh and Preiss, 1966).
Fu et al. (1998) also proposed a post-translational mechanism of modification of
AGPase involving thioredoxin. They exposed recombinant potato AGPase to oxidized
thioredoxin and observed the subsequent formation of disulfide bonds between the Ntermini of the small AGPase subunit. In leaves of different plants, starch synthesis is
controlled by post-translational regulation of AGPase in response to light and sugar
levels. When isolated chloroplasts are illuminated, or sucrose is supplied to leaves in the
dark through the petiole, the small subunit of AGPase is rapidly converted from a dimer
to a monomer. The reverse happens when pre-illuminated leaves are darkened (Hendriks
et al., 2003). AGPase of potato tuber is also subjected to redox-dependent post-
- 37 -
translational regulation, in which an intermolecular cystene (Cys) bridge is formed
between the two catalytic small subunits (Tiessen et al., 2002).
The cytosolic localization of AGPase in cereal endosperm may provide an
advantage where large amounts of carbon are partitioned to starch when there is a
plentiful sucrose available.
1.4.1.3 Starch Synthases (SS, EC 2.4.1.21)
Starch synthases (SSs) produce α-1,4-glucan linkages by transferring sugar
moieties from an activated donor molecule (ADP-glucose) to a specific acceptor
molecule (growing glucan chain) in a distributive mechanism in which the enzyme
dissociates from its substrate during each catalytic cycle (Denyer et al., 1999). Among
starch biosynthesis enzymes, SSs has the highest number of isoforms (Fujita et al., 2011).
These enzymes are found in the starch granules but are also present in the plastid stroma,
leading to their classification as soluble starch synthases, with the exception of granulebound starch synthase (GBSS), which is exclusively found within starch granules. Five
classes of starch synthases are consistently present in higher plants which can be divided
into granule-bound starch synthase (GBSS), and soluble starch synthases (SSI, SSII,
SSIII and SSIV). These different classes may have multiple isoforms e.g. GBSSI and
GBSSII, three SSII isoforms (SSIIa, SSIIb and SSIIc [also defined as, SSII-3, SSII-2 and
SSII-1], respectively), two SSIII isoforms (SSIIIa and SSIIIb [SSIII-2 and SSIII-1,
respectively]), and two SSIV isoforms (SSIVa and SSIVb [SSIV-1 and SSIV-2,
respectively]) (Hirose and Terao, 2004; Fujita et al., 2007). Phylogenetic analyses
separate the GBSS, SSI and SSII from SSIII and SSIV classes (Ball and Morell, 2003;
Patron and Keeling, 2005; Leterrier et al., 2008).
- 38 -
Sequence alignment comparisons of different isoforms of SSs show that all
isoforms of SSs in higher plants and green algae contain a highly conserved core, or
catalytic region, of approximately 60 kDa, with a C-terminus similar to that of glycogen
synthases (GSs) (Tetlow, 2011). The K–X–G–G–L motif is thought to be responsible for
substrate (ADP-glucose) binding in higher plant SSs and in prokaryotic (GSs) (Furukawa
et al., 1990, 1993; Busi et al., 2008), and is only present in the C-terminus of higher
plants and green algal SSs (Nichols et al., 2000). However, K-X-G-G-L domains are
distributed across the GSs protein sequence in prokaryotes (Fukukawa et al., 1990).
Glucan primer preference is determined by the presence of lysine in the K–X–G–G–L
domain (Gao et al., 2004). Further, in maize SSs, the glutamate and aspartate were found
as important residues for catalytic activity and substrate binding (Nichols et al., 2000). In
contrast to GS and GBSS all SSs (SSI, SSII, SSIII, and SSIV) have an additional
sequence located at N-terminal to the catalytic region; which is sometimes referred as the
N-terminal extension. Considerable variation has been found within this region upstream
of the catalytic core, and this extension can vary greatly in length from 2.2 kDa in GBSSI
to approximately 135 kDa in maize SSIII (Gao et al., 1998). Studies with SSs truncated at
N-terminal extension showed that this extension is not required for catalysis (ImparlRadosevich et al., 1998; Edwards et al., 1999) or glucan affinity (Commuri & Keeling,
2001), however it is involved in determining chain-length specificities of the enzymes or
possibly for protein-protein interactions (Hennen-Bierwagen et al., 2008). On the basis of
predicted amino acid sequence, the phylogenetic and sequence analyses of plants SSs
(Arabidopsis thaliana, wheat and rice) and algal SS and prokaryotic GS isoforms
suggests that SSI, SSIIs and GBSSIs have distinct evolutionary origins as compared to
- 39 -
SSIIIs and SSIVs (Leterrier et al., 2008). In particular, the valine residue present within
the highly conserved K-X-G-G-L motif seems to have faced strong evolutionary selection
in SSIIIs and SSIVs and in these SSs it may affect primer/substrate binding compared to
SSI, SSIIs and GBSSIs (Leterrier et al., 2008). SSIII and SSIV also have another
prominent difference from other SSs, which is the presence of a highly conserved, G-X-G
motif near the nucleotide-binding cleft (Leterrier et al., 2008). Sequence comparison of
different SS isoforms is shown in Figure (1.10).
Apart from GBSS, other enzyme classes are also found within the starch granule.
The partitioning of starch synthases between the soluble plastid stroma and starch
granules varies among plant species, plastids type and developmental stage of the plant
(Ball and Morell, 2003). In higher plants, a specific group of starch synthesizing enzymes
(SSI, SSIIa, and isoforms of SBEII) are consistently found within starch granules as well
as the stroma, whereas other enzyme classes (SSIV, SBEI, SP, isoforms of isoamylase,
pullulanase-type DBE and D- enzyme) which also play an important role in amylopectin
biosynthesis are either absent from the granules or are present in small amounts (Tetlow,
2011). The relative activities of different SSs isoforms are species-, organ- and
developmental stage-dependent. For example in maize endosperm, 60 % of the soluble
starch synthase activity is contributed by SSI (Cao et al., 1999), in pea embryos SSIIa
makes 60 % of the total activity (Denyer and Smith, 1992) and in potato tubers 80 % of
soluble starch activity is shared by SSIII (Marshall et al., 1996). These variations may
contribute to differences in starch structure in different organs and species. Genetic and
biochemical evidence suggest that each SS isoform has different properties and a distinct
role in the biosynthesis of amylopectin. This will be discussed below.
- 40 -
Figure 1.10: Comparison of cereal SS domains.
The five known isoforms of SS with their constituent number of amino acids are shown
with the name of the corresponding mutant in maize (in parentheses). The Figure shows
comparison of SS domain sequences in which C- terminal catalytic domain (including
ADP- glucose binding domain) is presented as black.
The different SSs have N-terminal domains of varying lengths which is shown as hatched
bars. In SSIII particularly a unique N-terminal extension is present which is thought to
be involved in controlling protein–protein interactions (Tetlow, 2011).
- 41 -
1.4.1.3.1 Granule-bound starch synthase (GBSS)
Granule-bound starch synthase (GBSS) is different from other SSs because it is
exclusively associated with starch granules. There are two isoforms encoded by the waxy
locus. GBSS I is confined to storage tissues while GBSS II is encoded by a separate gene
and is responsible for transient starch biosynthesis in leaves and other non-storage tissues
(Fujita and Taira, 1998; Nakamura et al., 1998; Vrinten and Nakamura, 2000). Studies
with different GBSS mutants (amylose free) showed that this is the only enzyme
responsible for amylose biosynthesis in maize and other plant species (Denyer et al.,
2001). In leaves and endosperm of rice, and presumably other plant species, highly
similar GBSS isoforms produced from different genes are also present (Vrinten &
Nakamura, 2000). In potato, GBSS also has a significant influence on the granule
structure (Fulton et al., 2002). Mutation in the waxy gene leads to loss of GBSS activity,
which results in amylose-free (waxy) starches. These waxy starches are still able to form a
granule and maintain its semi-crystalline property, which indicates that insoluble granule
synthesis does not require amylose (Denyer et al., 1999).
One hypothesis regarding the mechanism, by which GBSSI synthesizes amylose,
indicates that GBSSI is stimulated by malto-oligosaccharides (MOS). MOS diffuse into
the granule matrix where amylose synthesis by GBSSI takes place by elongating the
MOS primers (Denyer et al., 2001; Denyer et al., 1996). It can also elongate the glucan
chain within amylopectin. GBSS does not disassociate from the growing glucan chain
after addition of glucose units but it remains associated to add new glucose units (Yeh et
al., 1981; Hizukuri et al., 1989; Reddy et al., 1993; Maddelein et al., 1994). GBSS
absence does not affect granule size distribution significantly (Fujita et al., 1998;
- 42 -
Mangalika et al., 2003), however its activity is related to formation of growth rings in
starch granules (Pilling and Smith, 2003). In the early stage of grain filling, soluble SSs
could be the predominant enzyme responsible for starch granule size distribution while
GBSS may play an important role in the ratio of large to small granules, especially in the
late grain filling stage (Chuanhui et al., 2010).
1.4.1.3.2 Starch synthase (SSI)
SSI is responsible for the synthesis of the shortest glucan chains,
approximately 10 glucosyl units or less (Commuri and Keeling, 2001), while other SS
isoforms are responsible for the further extension of glucan chains. The hypothesis that
SSI is involved in the synthesis of short chains comes from the study of Arabidopsis
(Dauvillée et al., 2005) and rice (Fujita et al., 2006) mutants lacking SSI which show
deficiencies in shorter (DP 6–12) glucan chain lengths. In potato, SSI mutants exhibit no
detectable changes in starch structure, which suggests that this isoform has only a
minor activity in potato tubers (Kossmann et al., 1999). Similarly the SSI mutation did
not affect starch contents, size and shape of developing seeds and starch granules
significantly (Fujita et al., 2006), but alteration in amylopectin structure has been found
where the proportion of chain length of DP 6-12 decreased, however the proportion of
chain length of DP 16-19 was increased. Similar results have been reported in
Arabidopsis transient starch with the SSI mutation (Dauvillée et al., 2005). The catalytic
activity of SSI was significantly reduced when longer glucan chains were used as
substrate and most of the ADP-glucose was incorporated into shorter chains with DP <10
(Jeon et al., 2010). Thus, smaller glucan chains are extended by SSI up to a certain
critical length and then SSI becomes bound to longer amylopectin chains and entrapped
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there as an inactive protein within the starch granule. Further extension of glucan chains
must be taken over by other SSs for continued amylopectin biosynthesis (Jeon et al.,
2010). In cereals, the complete absence of SSI has no effect on the size and shape of
seeds, starch granules and the crystallinity of endosperm starch, which indicates that
other SS enzymes are able to compensate for SSI function (Fujita et al., 2006). In contrast
to this, in barley, soluble SSs are thought to have a role in determination of granule size.
A mutation at the barley shx locus results in reduced SSI activity which leads to reduction
in the size of A-granules and transforms the normal bimodal granule size distribution to
unimodal (Schulman and Ahokas, 1990; Tyynela and Schulman, 1993; Tyynela et al.,
1995). However, Chuanhui et al. (2010) reported that starch granule size distribution in
wheat is associated with activities of starch synthases and not specifically SSI. These
variations may suggest that impact of SSI on starch synthesis is species dependent.
1.4.1.3.3 Starch synthase (SSII)
Two genes classes (SSIIa and SSIIb) are present in monocots encoding SSII.
SSIIa is present in cereal endosperm, while SSIIb is expressed in photosynthetic tissues.
In monocots and green algae, SSII is involved in the synthesis of intermediate glucan
chains of DP 12-24 by elongating short chains of DP ≤ 10 (Fontaine et al., 1993; ImparlRadosevich et al., 2003; Morell et al., 2003). SSIIa mutation causes a decrease in the
proportion of intermediate chains of DP 12-25 and an increase in short chains of DP 6-10
in amylopectin. The gene encoding SSIIa in the endosperm has been found in many crop
species, including maize, wheat, rice, barley and pea (Campbell et al., 1994; Craig et al.,
1998; Yamamori, 2000; Umemoto et al., 2002; Morell et al., 2003; Zhang et al., 2004).
Although the contribution of SSIIa in the total measureable activity of SSs in the cereal
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endosperm is minor, loss/down regulation of this protein has a major impact on the
amount and composition of starch (Tetlow, 2011). In Arabidopsis, loss of SSII has no
affect on growth rate or starch quantity, but causes an increase in amylose content and a
decrease in amylopectin (Zhang et al., 2008). In monocots, loss of SSIIa results in
reduced starch content, change in granule morphology reduction in amylopectin chainlength distribution, and decreased crystallinity. SSIIa mutants of barley and wheat
possess low seed starch content, reduced chain length distribution of amylopectin and
crystallinity. Altered granule morphology and amount of amylose in barley starch
granules has been significantly increased up to 70 % (Morell et al., 2003; Kosar-Hashemi
et al., 2007). The relevant SSIIa mutation in maize is sugary2, which results in more
short chains of DP 6-10, fewer chains of DP 12-30 and increased levels of amylose up to
40 % (Zhang et al., 2004). This mutation has important applications in the food industry
due to changes in the functional properties of starch (Harn et al., 1998). Similar effects
have been observed in potato tubers (Edwards et al., 1999; Lloyd et al., 1999) and in
Arabidopsis leaves (Zhang et al., 2008). In the rice endosperm, abundant transcripts of
SSIIa and SSIIIa were found during grain filling, suggesting a crucial role of SSIIa and
SSIIIa during starch biosynthesis (Jeon et al., 2010).
1.4.1.3.4 Starch synthase (SSIII)
SSIII is involved in the synthesis of longer glucan chains of DP 25-35 or greater
(Tomlinson and Denyer, 2003; Zhang et al., 2004, 2005, 2008). SSIII mutants lack longer
chains in amylopectin which results in adverse alteration in molecular architecture
(Gerard et al., 2009). In rice, SSIII is encoded by two genes, SSIIIa in the endosperm and
SSIIIb in leaves (Hirose and Terao, 2004; Dian et al., 2005). After SSI, the second
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highest catalytic activity in cereal endosperm (maize and rice) is exhibited by SSIII (Cao
et al., 1999; Fujita et al., 2006). SSIII has a primary role in amylopectin synthesis, but
the impact of SSIII loss is different within different genetic backgrounds. In an
in vitro experiment when glycogen was used as the primer, a SSIIIa purified fraction
from rice endosperm generated long chains from DP ≤ 11 chains (Fujita et al., 2006). In
ssIIIa mutants of rice, glucan chains with DP 6-8, DP 16-20, and DP ≥ 30 were reduced,
whereas glucan chains with DP 9-15 and DP 22-29 were increased (Fujita et al., 2007;
Ryoo et al., 2007). This shows that in the rice endosperm SSIIIa contributes to the
synthesis of amylopectin chains with DP ≥ 30 in vivo. Interestingly endogenous SSI
activity has been seen to increase due to loss of SSIIIa, thereby enhancing the synthesis
of glucan chains with DP 9-15 and DP 22-29, respectively (Fujita et al., 2007). Similar
observations have been made with maize, where the SSI activity in the SSIIa mutant was
higher than wild-type (Cao et al., 1999). In potato, mutation in SSIII causes alteration in
glucan chain-length distribution (Abel et al., 1996), whereas in maize, mutation in SSIII
produces less significant phenotypic effects, which are detectable only in waxy mutants
(Gao et al., 1998). The (SSIII) du1 mutants of maize and rice show altered granule
morphology and crystallinity and reduced proportion of longer glucan chains of DP ≥ 30,
which suggests the role of SSIII in the elongation of these chains (Inouchi et al., 1983;
Fujita et al., 2007; Ryoo et al., 2007) white-core, floury endosperm. Similarly, maize
ssIIIa mutants show a dull phenotype with a glassy and tarnished endosperm (Gao et al.,
1998). In addition to alteration in starch structure and physical properties, in maize
endosperm loss of SSIII is associated with pleiotropic effects on other SS and SBE,
causing increased activities of SSs (Cao et al., 1999) and reduction in the activity of
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SBEIIa (Boyer and Preiss, 1981). But expression of the genes coding different enzymes
involved in starch biosynthesis in (ssiii-) amo1 appears more or less unaffected at both the
transcript and protein levels (Borén et al., 2008) However qualitative analysis with
zymogram suggested that the branching enzyme (SBEs) activity differed between amo1
and normal type and starch granule protein content of amo1 was higher than the normal
type (Boren et al., 2008). Amo1 is ssiii- mutant in barley, the same mutation in maize is
called dull. Amo1 mutant does not lack SSIII protein, however it has a leucine to arginine
residue substitution in a conserved domain compared to the wild-type protein. This
substitution results in reduction in activity of SSIII protein compared to wild-type (Li et
al., 2011). Observation with mutants shows that, in addition to catalytic role of SSIII, it
also possesses regulatory properties with respect to control over the starch biosynthetic
pathway. Several studies have revealed that SSIII is a negative regulator of starch
synthesis and mutation in this gene leads to elevated levels of amylose (Li et al., 2011) In
Chlamydomonas, mutations in SSIII is associated with increased amount of GBSSI
protein and transcript giving rise to more long chains in amylopectin (Ral et al., 2006).
Studies with potato single and double mutants for SSII and SSIII showed that these
enzymes make distinct contributions towards amylopectin biosynthesis, and they act
synergistically, rather than independently, during amylopectin synthesis. Single mutants
for both the SSII and SSIII produced minor effects on starch granules, whereas double
mutant for SSII and SSIII showed remarkable alteration in starch phenotype (Edwards
et al., 1999; Lloyd et al., 1999; Zhang et al., 2008). In Arabidopsis, loss of both SSII
and SSIII results in slower plant growth, dramatically reduced starch content and change
in amylopectin structure, and their function cannot be substituted by any other conserved
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SS, specifically SSI, GBSSI and SSIV (Zhang et al., 2005, 2008) Thus the synergistic
effects of the loss of both SSII and SSIII are more severe than the loss of individual
isoform, which suggest, partial redundancy with respect to the function these two
isoforms in amylopectin biosynthesis (Tetlow, 2011).
1.4.1.3.5 Starch synthase (SSIV)
SSIV is the most recently discovered form of SSs from higher plants (Dian et al.,
2005). The precise role of SSIV in starch biosynthesis is yet not well elucidated, but the
SSIV mutants of Arabidopsis showed defective granule initiation and it is expressed in
the grain during development (Hirose and Terao, 2004; Dian et al., 2005; Roldan et al.,
2007). In plants there are two isoforms of SSIV which are differentially expressed in the
endosperm (SSIVa) and in leaves (SSIVb) (Leterrier et al., 2008). SSIV mutants of
Arabidopsis show a decrease in granule number but an increase in the granule size
(Roldan et al., 2007; Zhang et al., 2008). SSIV protein is different from other SS isoforms
in the sense that it has an N-terminal extension with two coiled-coil domain and a
putative 14-3-3 protein binding site (Leterrier et al., 2008). These features may enable
SSIV to interact with other proteins and thus contribute in the granule initiation. In the
absence of SSIV, SSIII may be responsible for initiation of single granules since it has a
glucosyl transferase domain closely related to SSIV. Double mutants of SSIII and SSIV
lack starch in their leaves, although 60 % of the measureable soluble starch synthase
activity exists from the remaining isoforms (Szydlowski et al., 2011).
1.4.1.4 Starch Branching Enzymes (SBEs, EC 2.4.1.18)
SBEs catalyze the hydrolysis of an existing α-1,4 linked glucan chain, with
subsequent transfer of the cleaved portion of glucan chain with six or more glucose units
- 48 -
to the C6 portion of the same glucan chain (intra chain transfer) or an adjoining glucan
chain (inter chain transfer) via an α-1,6 linkage to form the branched structure of
amylopectin. There are two major classes of highly conserved SBEs across plant species
(SBEI and SBEII) sometimes also referred to as SBEB and SBEA, respectively, and they
have a primary role in amylopectin synthesis, although they may also have a role in
amylose synthesis which is lightly branched (Keeling and Myers, 2010). There are two
isoforms of SBEII namely (SBEIIa and SBEIIb). The functional SBE enzymes have
multiple highly conserved regions incorporating two carbohydrate binding sites and an αamylase domain (Abad et al., 2002). The N-terminal region of SBEs appears to be
important for catalysis and structural stability (Guan et al., 1997; Hamada et al., 2007),
whereas substrate preference and chain length transfer are determined by both the N- and
C-terminus (Kuriki et al., 1997).
1.4.1.4.1 Starch branching enzyme I (SBEI)
The two SBEs classes differ primarily in terms of length of glucan chain
transferred in vitro and substrate specificities. For example, SBEII transfers shorter
glucan chains and has a higher affinity for amylopectin than SBEI which shows higher
rates of branching with amylose (Guan and Preiss, 1993; Takeda et al.,1993; Rydberg et
al., 2001). SBEI has a lower km for amylose and tends to produce shorter constituent
chains, compared to SBEIIa or SBEIIb (Gao et al., 1996). Some of these isoforms are
tissue, or developmental-stage specific, in their expression patterns (Yamanouchi and
Nakamura, 1992; Gao et al., 1997; Sun et al., 1998; Regina et al., 2005). For example, in
maize, SBEI is expressed moderately during middle stages (12–20 DAA) of kernel
development and more strongly during the later stages (22–43 DAA), but is only
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moderately expressed in vegetative tissues (Kim et al., 1998). The roles of SBEI and
SBEIIa are less clear because SBEI mutants of rice have reduced intermediate and long
chains (Nakamura, 2002), whereas in maize the chain lengths are unaffected in mutants
of SBEI and SBEIIa (Blauth et al., 2001, 2002). SBEI mutants do not show a significant
effect on starch structure, but in maize, double mutants of SBEI and SBEIIb same
noticeable alterations in amylopectin branching pattern which suggests that SBEI has
some role in storage starch biosynthesis (Yao et al., 2004). In monocots or dicots, downor up-regulation of SBEI has minimum effects on starch synthesis and composition in
storage and transient starch synthesis (Blauth et al., 2002; Satoh et al., 2003). It was
found that loss of enzyme activity was only detected in SBEIIa or SBEIIb mutants
(Blauth et al., 2002), suggesting either that the lack of SBEI was compensated by other
two SBE isoforms or that SBEI does not have a significant role in determining starch
quantity or quality in leaves or endosperm (Blauth et al., 2002). Although the precise role
of SBEI is not clear, in cereals like wheat and barley, an isoform of SBEI, termed SBEIc,
is only found within the large A-granules (Peng et al., 2000). However, in contrast,
SBEIc was found in both A- and B-granules of wheat (Bancel et al., 2010). Expression of
three functional SBE genes of maize in a yeast strain lacking yeast glucan polymer
branching enzymes showed that SBEI was inactive in the absence of either SBEIIa or
SBEIIb, and that SBEII acts prior to SBEI on precursor polymers (Seo et al., 2002). In
plants, SBEI is highly conserved and has been shown to physically interact with other
starch biosynthetic enzymes (Liu et al., 2009; Tetlow et al., 2004) which suggest that
SBEI plays some function in regulating the starch biosynthetic process. Similarly loss of
SBEI in SBEIIb-deficient back ground resulted in increased branching, which also
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suggests a regulatory role for SBEI in influencing other branching enzymes (Yao et al.,
2004).
1.4.1.4.2 Starch branching enzyme II (SBEII)
In monocots, two SBEII gene products (SBEIIa and SBEIIb) are closely related
(Rahman et al., 2001). The affect of loss of SBEII on starch phenotype is more
pronounced compared to SBEI. Both SBEIIa and SBEIIb are expressed in the developing
endosperm of barley and are partitioned between soluble and granule-bound fractions of
amyloplasts (Morell et al., 1997; Sun et al., 1998; Rahman et al., 2001). But the
expression pattern of both enzymes is different in barley endosperm from other cereals
like maize and wheat. In maize, SBEIIa is present 50 times less than SBEIIb. In contrast
SBEIIb is present at much lower levels than SBEIIa in wheat endosperm (Gao et al.,
1997; Morell et al., 1997; Regina et al., 2005). However, in barley endosperm both
proteins are expressed at approximately equal levels (Sun et al., 1998). In maize and rice,
loss of SBEIIa produced a clear phenotype of transient starch but no apparent effects on
storage starch biosynthesis in the endosperm (Blauth et al., 2001). There was no
significant change in kernel phenotype, starch content, starch structure and chain length
distribution in the endosperm. This suggest that SBEIIa has a primary role in transient
starch biosynthesis but no critical role in storage starch biosynthesis, or that its role is
easily compensated by other SBEs (Blauth, et al., 2001; Nakamura, 2002).Based on
biochemical studies of the isolated enzyme (Guan et al.,1997; Takeda & Preiss, 1993)
and structural analysis of the maize and rice mutants (Nishi et al., 2001; Takeda et al.,
1993; Stinard et al., 1993), it was determined that SBEIIb is primarily responsible for
transferring longer chains of amylopectin. The maize gene encoding for SBEIIb is
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amylose extender (ae), the mutation of which leads to severe alteration in the structure of
amylopectin with fewer branches and increased level of apparent amylose up to 50 %,
(Garwood et al., 1976; Klucinec & Thompson, 1998). The primary reason of increase in
measureable amylose is the synthesis of modified amylopectin, where longer glucan
chains are formed with few branches. The resultant amylopectin “resembles” amylose
which can be detected by iodine-binding, and hence the resultant phenotype is often
termed as “high amylose” although this is misleading. In barley, elimination of either
SBEIIb or SBEIIa does not result in a significant alteration in the number of branches in
amylopectin or an increase in apparent amylose content although the branch frequency of
amylose was increased in sbeiib- (Regina et al., 2010). In wheat, suppression of both
genes SBEIIa and SBEIIb by RNAi- is required to produced starch with amylose contents
> 70 % (Regina et al., 2006). A double mutant (GBSSI/ SBEIIb) of rice produced starch
with much longer chains and few chains with DP ≤ 17, with the greatest decrease in chain
length between DP 8-12 (Nishi et al., 2001). This same trend has been observed in a rice
ae single mutant. This suggests that SBEIIb plays an important role in the synthesis of
amylopectin A chains. In the mutant, SBEI and SBEIIa levels remained unaffected which
shows that the extent of change in the chain length profile was related to decreased
SBEIIb activity (Tanaka et al., 2004). In an RNAi- generated sbeiib- mutant of barley,
increased branching frequency in amylose has been found (Regina et al., 2010).
1.4.1.5 Debranching enzymes (DBEs, EC 3.2.1.41 and EC 3.2.1.68)
Debranching enzymes, also termed isoamylases, are involved in cleaving
inappropriately attached glucan chains and contribute to the organized crystalline
structure of amylopectin (Ball et al., 1996; Zeeman et al., 1998). This important role of
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DBEs has been suggested by observing variations in the amylopectin structure of mutants
lacking some of the DBEs. Thus, in addition to SSs and SBEs, isoamylases play an
important role in the development of crystalline amylopectin. DBEs are of two types:
isoamylase-type and pullulanase-type. The isoamylases hydrolyze a-1,6 linkages in
amylopectin and the pullulanases hydrolyze a-1,6 linkages in pullulan, a fungal polymer
of malto-triose. The genes encoding three evolutionarily conserved isoamylase-type
DBEs and one pullulanase-type DBE have been identified (James et al., 1995; Dinges et
al., 2003). The different classes of isoamylases are distinguishable by their amino acid
sequence and substrate specificities (Zeeman et al., 2010). The ISO type DBE has three
classes (ISO1, ISO2 and ISO3). ISO1 and ISO2 are strongly involved in amylopectin
synthesis while ISO3 and pullulanase are primarily involved in starch degradation. In
Arabidopsis leaves and potato tuber, ISO1 forms a heteromultimeric enzyme complex
with ISO2 (Hussain et al., 2003; Delatte et al., 2005; Wattebled et al., 2005). Whereas, in
the endosperm of rice and in other cereals, ISO1 is found as a homomultimeric and as a
heteromultimer with ISO2 (Utsumi and Nakamura, 2006). ISO1 has more affinity
towards substrate with longer external chains such as solubilised amylopectin, while
ISO3 and LDA are more active on substrates with short external chains such as β-limit
dextrin. ISO2 appears to be catalytically inactive, and may be a regulatory subunit to
ISO1 rather than contributing directly to catalytic activity (Hussain et al., 2003).
Transgenic plants or mutants lacking ISO1 or ISO2 type DBEs have reduced amylopectin
content, and in its place accumulate large amounts of the water-soluble polysaccharide
(WSP), phytoglycogen (Zeeman et al., 1998; Bustos et al., 2004; Wattebled et al., 2005).
Genetic alteration in ISO1 activity in maize and rice resulted in significant changes in
- 53 -
starch granule structure (Jane et al., 1994; Kubo et al., 1999) while a barley ISOI mutant
produced compound instead of simple starch granules (Burton et al., 2002). In double
ISO1 and ISO2 antisense mutants, potato accumulated a large number of small granules
in tubers (Burton et al., 2002; Bustos et al., 2004). These observations and studies with
barley mutants and transgenic rice show that isoamylase-type DBE activity plays a
crucial role in the initiation of starch granules (Burton et al., 2002; Bustos et al., 2004;
Kawagoe et al., 2005). The expression of an isoamylase-type DBE (ISO1) depends upon
the stage of development, as it is highest in developing endosperm and undetectable in
mature grains (Sun et al., 1991). In maize, a severe phenotype has been observed in a
ISO1 mutant known as sugary1 (su1-) where reduction in crystalline starch and
accumulation of a (WSP) pytoglycogen increased (Dinges et al., 2001; Burton et al.,
2002; Fujita et al., 2003; Bustos et al., 2004). These observations suggest that DBEs
function in starch synthesis by the selective cleavage of inappropriate branch linkages
before crystallization of the molecule (Ball et al., 1996; Myers et al., 2000). Reduction in
PUL activity did not produce pleiotropic effects on the other starch synthesizing enzymes
in rice (Fujita et al., 2009), However, studies with starch mutants such as rice floury-2
and su1 mutants do induce pleiotropic effects on PUL activity (Kawasaki et al., 1996). A
maize PUL mutant zpul-204 was isolated by gene tagging method (Dinges et al., 2003).
The structure and composition of endosperm starch from maize zpul-204 were not
different to that wild-type; however amylopectin of transient starch contained
significantly fewer chains with DP 8-15 than wild-type because of the pleiotropic effect
of SBEIIa activity. Similarly, developing endosperm of zpul-204 accumulated more
branched malto-oligosaccharides which were not found in the wild-type (Dinges et al.,
- 54 -
2003). In ISO1 (sug1) deficient back ground zpul-204 showed more accumulation of
pytoglycogen in the seeds which was not seen in the wild-type. This indicates that PUL
partially compensates for ISO1 deficiency and is important during starch synthesis and
degradation (Dinges et al., 2003). PUL function partially overlaps with ISO1, though
effects of deficiency in PUL1 are much smaller on amylopectin biosynthesis than that of
ISO1, and variations of su1 phenotype are not significantly dependent upon activities of
PUL (Fujita et al., 2009). In transgenic rice generated by transforming a wheat ISO1 gene
into su1 rice, phytoglycogen synthesis in the endosperm was substantially replaced by
starch synthesis (Kubo et al., 2005) These observations suggest that in maize and rice,
su1 mutations are caused by deficiency of ISO1 and its activity plays a crucial role in
normal amylopectin biosynthesis (Fujita et al., 2009).
Two models have been proposed for the function of DBEs in starch synthesis and
phytoglycogen accumulation. The glucan trimming model explains the function of DBEs
as removing any branches that would inhibit crystallization and aggregation of
amylopectin into an insoluble granular structure (Ball et al., 1996; Myers et al., 2000), as
inappropriately attached branches on the surface of the growing starch granules prevent
crystallization. Another model suggests a ‘‘clearing role’’ of DBEs and proposes that
they are involved in removing soluble glucan not attached to the granule from the stroma
(Zeeman et al., 1998). This concept is based on the theory that SSs and SBEs will
continue to synthesize glucan polymers if sufficient substrate is present and there will be
random synthesis of glucan polymers which could cause accumulation of phytoglycogen,
ultimately leading to a reduction in the rate of starch synthesis.
Contribution of different enzyme classes and their isoforms in the synthesis of
- 55 -
organized starch granule is shown in Figure (1.11).
1.4.1.6 Starch Phosphorylase (SP, EC 2.4.1.1)
Starch phosphorylase, a tetramer, is responsible for the reversible transfer of
glucosyl units from glucose 1-phosphate to the non-reducing end of a-1, 4-linked glucan
chains. Depending upon the concentration of the soluble substrates, SP may work
either in a synthetic or a degradative direction. Plastidial SP referred to as Pho1 (or
the L-form) has higher affinity towards amylopectin than glycogen (Mu et al., 2001).
Based upon properties or timing of expression, SP from maize endosperm can be
separated into different forms (Tsai and Nelson, 1969). SPI (Pho1) is present during all
stages of endosperm development and at germination as well. However, SPII and SPIII
are present during the period of rapid starch synthesis and are absent during germination
and can initiate glucan chain synthesis without a primer. SP consists of N-terminal and Cterminal, where C-terminal domain shares significant similarity with the nucleotidebinding domain of SS (Buschiazzo et al., 2004). In developing rice endosperm, 96 % of
the total phosphorylase activity is controlled by Pho1 (Satoh et al., 2008). In potato SP is
classified as high (SP-H) and low (SP-L) isozymes, according to their affinity for glucans
(Mori et al., 1993). An extra 80–amino acid insertion is present in the plastidic form,
which is absent from cytosolic Pho2. The plastidic form has a high affinity towards low
molecular weight malto-oligosaccharides (MOS) and amylose, while the cytosolic
isoform (Pho2 or Pho-H) has a high affinity towards glycogen (Yu et al., 2001). Studies
with Arabidopsis mutants clearly indicate that SP is not required for starch degradation
(Zeeman et al., 2004). Activity of the plastidial isozyme (L-form) in sweet potato roots
is regulated by proteolysis of a 78-amino acid peptide. Digestion of this peptide by
- 56 -
Figure 1.11: Diagrammatic representation of the coordinated actions of different
enzyme classes in the synthesis of amylopectin.
The Figure demonstrates that in growing amylopectin molecules, linear chains are
added by the SS. When these chains are extended to a particular length, SBE cleaves
additional glucan chains and adds branches to the growing amylopectin molecule.
During this process some branches are inappropriately attached which can hinder the
formation of proper crystalline structure. Thus these inappropriately attached
branches are trimmed by DBE, which facilitates proper crystallization of the molecule.
- 57 -
endogenous protease results in an increase in catalytic activity of SP in the
phosphorolytic direction (Chen et al., 2002). In wheat, three variable phosphorylase
activity forms (P1, P2, P3) of SP have been identified. P1 and P2 are cytosolic in younger
leaves, whereas mature leaves have only the plastidic form (P3) (Schupp, 2004). SSIV
mutants of Arabidopsis showed increased activity of SP, but starch structure or the
amylose/ amylopectin ratio remained unaffected in these mutants. However, in the dark,
starch accumulation was decreased in two eco-types as compared to mutant leaves with a
significant influence on granule size (Roldan et al., 2007).
1.4.1.7 Disproportionating enzyme (D-enzyme, E, C. 2.4.1.25)
D-enzyme or disproportionating enzyme is present in the soluble fraction of
plastids in different starch synthesizing organs of plants (Takaha et al., 1993; Lin et al.,
1988). D-enzyme catalyses transfer of two glucose units from malto-triose to a longer
glucan chain, making them available for β-amylosis and the remaining glucose monomer
becomes available for export from the plastids. An Arabidopsis mutant of D-enzyme
showed reduced rates of nocturnal starch degradation which indicates role of D-enzyme
in the pathway of chloroplast starch degradation (Critchley et al., 2001). Some studies
suggested that D-enzymes may work together with SP, contributing to starch
synthesis by a phosphorolytic SP reaction (Takaha et al., 1998). This model, is based
on the ‘‘glucan-trimming’’ model. According to this model, short-chain maltooligosacharides (MOS) liberated by the trimming action of DBEs are converted to
longer-chain glucans by D-enzyme, which in turn become substrates for phosphorolysis
by SP, liberating G-1-P, being used to synthesize ADP-glucose by plastidial AGPase
(Takaha et al., 1998). It was shown by Colleoni et al. (1999) that phosphorolytic SP
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reaction in Chlamydomonas reinhardtii, can be stimulated by the presence of Denzyme.
1.4.2 Post-translational modification of starch synthesizing proteins
Some of the enzymes involved in starch biosynthesis are subjected to posttranslational modification involving protein phosphorylation, allosteric and redox
modification. Similarly there is increasing evidence that starch synthesis does not take
place in a simple, linear fashion, as described in previous sections, but involves proteinprotein interactions, i.e. formation of protein complexes coordinates the multiple
actions of different proteins involved in the synthesis of starch polymer. Thus,
synthesis of highly organized crystalline starch granule is due to the coordinated action of
starch synthases (SSs), starch branching enzymes (SBEs), together with starch
debranching enzymes (DBEs) (Ball and Morell, 2003). Allosteric and redox modification
of certain proteins to control their activities have already been described in the previous
section (see above). However, other important mechanisms of post-translational
modification involve phosphorylation of certain proteins as described by Tetlow et al.
(2004). Activities of different stromal isoforms of SBEII and SSIIa and the association of
SBEI with SBEIIb and SP were found to be regulated by phosphorylation (Tetlow et al.,
2004). When intact plastids from wheat were incubated with g-32P-ATP, three isoforms
of the SBE’s (SBEI, SBEIIa, and SBEIIb) were found to be phosphorylated on serine
residues (Tetlow at al., 2004). This suggests that phosphorylation is also directly involved
in the regulation of enzyme activity and in the formation of protein complexes (Tetlow
et al., 2004, 2008; Liu et al., 2009). Evidence that protein phosphorylation is directly
involved in protein complex formation came from experiments with wheat endosperm
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amyloplasts, in which SP and SBEIIb could be co-immunoprecipitated with SBEI when
phosphorylated within the soluble protein fraction (Tetlow et al., 2004). Conversely,
dephosphorylation with alkaline phosphatase caused disassembly of this protein complex.
Based on the analysis of enzyme kinetics and mutations in the starch biosynthetic
pathway there is evidence which suggests that enzymes involved in starch biosynthesis
have physical and functional coordination (Yao et al., 2002, 2004; Colleoni et al., 2003;
Seo et al., 2002; Dinges et al., 2001, 2003; Nishi et al., 2001; Beatty et al., 1999; Gao et
al., 1998; Boyer and Preiss, 1979, 1981; Hawker et al., 1974). Most recently, biochemical
analysis of endosperm soluble extracts provided direct evidence of protein-protein
interactions among enzymes involved in starch synthesis (Liu et al., 2009; HennenBierwagen et al., 2008, 2009; Tetlow et al., 2004, 2008). The idea of heteromeric enzyme
complex formation is further supported by the observation of numerous pleiotropic
effects on SSs, SBEs, and AGPase resulting from mutations in genes encoding specific
enzymes of the starch biosynthesis pathway (Singletary et al., 1997; Boyer & Preiss,
1981). In maize, mutations which affect both a pullulanase-type DBE (zpu1-204) and
an isoamylase-type DBE (ISO1), results in loss of SBEIIa activity, although the
amount of SBEIIa protein is unchanged (Dinges et al., 2001; Dinges et al., 2003;
James et al., 1995). However this effect was not observed when catalytically inactive
SU1 was present, which suggests that SU1 has both enzymatic and non-enzymatic effects
and different enzymes involved in starch biosynthesis may form protein complexes
(James et al., 2003). Similarly in the maize sugary2 (su2-) mutant, loss of SSIIa
caused decreased association of SSI, BEIIb, and SSIII within the starch granule, and
SSI levels increased in the amyloplast lysate (Grimaud et al., 2008). In an ae- mutant
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of maize lacking SBEIIb, binding of SBEI to starch granules was significantly increased,
which suggests that loss of SBEIIb might unmask a binding site for SBEI in a
multisubunit complex (Grimaud et al., 2008; Hennen-Bierwagen et al., 2008). In
developing endosperm of sex6 mutant of barley, which lacks SSIIa, the mutation also
loses the binding of SSI, SBElla and SBEIIb to the starch granules, despite the fact that
there is no detectable alteration in their expression levels in the soluble fraction (Morell et
al., 2003). Similarly, in wheat and rice endosperms, loss of SSIIa results in reduction of
amylopectin synthesis and loses the presence of SSI, SBEIIa, and SBEIIb within the
starch granules (Yamamori, 2000; Umemoto and Aoki, 2005). These pleiotropic effects
are consistent with a central role for SSIIa in mediating protein-protein interactions. In
maize, the interdependency of starch synthase IIa (SSIIa), SSIII, starch branching enzyme
IIb (SBEIIb), and SBEIIa was tested by assessing assembly into multi-subunit
complexes. It was found that mutations that eliminated one of these proteins also
prevented the other proteins from assembling into a high molecular complex of almost
670 kDa. Similarly, in high molecular weight fractions of developing maize and wheat
endosperm, different enzymes complexes, containing SSI, SSIIa, SSIII, SBEIIa, and/or
SBEIIb, in various combinations have been reported (Hennen-Bierwagen et al., 2008,
Tetlow et al., 2008). In maize kernel extracts, SSI activity was stimulated by the addition
of purified SBEI or SBEII (Boyer and Preiss, 1979; Seo et al., 2002) suggesting some
interaction. In barley SSI, SSII, and SBEIIa are able to bind with amyloplast 14-3-3
proteins in a phosphorylation-dependent manner. This may suggest a potential
mechanism for assembly of the SS and SBEII proteins via protein phosphorylation and
plastidial 14-3-3 proteins (Alexander and Morris, 2006). In maize, SSIII is involved in
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protein-protein interactions with other enzymes (Hennen-Bierwagen et al., 2008, 2009)
and, in addition, possesses sequences that confer a glucan binding function (Palopoli et
al., 2006; Senoura et al., 2007; Valdez et al., 2008). Hennen-Bierwagen et al. (2009)
reported a novel protein complex in maize endosperm containing SSIII, SSIIa, SBEIIa,
SBEIIb, large and small subunits of AGPase, and Pyruvate Phosphate Dikinase (PPDK).
Existence of these novel protein complexes could have a broader metabolic significance
because proteomic analyses of maize endosperm have given rise to the hypothesis that
PPDK is a key factor in regulating the partioning of carbon flux between starch and
protein (Mechin et al., 2007). The concept that different proteins involved in the same
metabolic pathway are interacting with each other in stable complexes suggests that these
interactions are of significant physiological importance. Recently Liu et al. (2009) also
reported that in ae- mutant of maize due to loss of SBEIIb, a novel protein complex
containing (SSI, SSIIa, SBEI, SBEIIa and SP) forms, as opposed to wild-type in which a
protein complex containing only SSI, SSIIa and SBEIIb was observed. The formation of
these protein complexes was found to be phosphorylation-dependent because following
dephosphorylation by alkaline phosphatase, no protein complex was observed in both
genotypes. However, re-phosphorylation with ATP resulted in the restoration of protein
complex formation in both genotypes. Similarly, Liu et al. (2009) also reported that
SBEIIb present in the wild-type complex and SBEI and SP present in the ae- mutant
complex were phosphorylated. Formation of phosphorylation-dependent complex and
phosphorylation of individual proteins are shown in Figure (1.12).
Although existence of protein complexes among starch biosynthetic enzymes has
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been reported in different plant species, it is not clear whether different starch
synthesizing organs in same the plant have similar protein complexes because protein
complexes found in the amyloplasts of wheat endosperm were not found in the
chloroplasts of starch synthesizing leaves of wheat (Tetlow et al., 2004).
1.4.3 Starch granule-bound proteins
The starch biosynthetic enzymes described earlier are distributed between the
soluble fraction of the plastids and the insoluble starch granules to varying degrees (Ball
and Morell, 2003; Tetlow et al., 2004, 2011; Grimaud et al., 2008; Dai et al., 2009). Thus,
other than GBSS, proteins are also found within the starch granule. The proteins
entrapped within the starch granules are involved in the synthesis of glucan chains of
short to intermediate length which form clusters, resulting in semicrystalline lamellae.
The amylopectin biosynthesis model proposed by French, 1984 and Hizukuri, 1986 states
that gradual and periodic synthesis of amylopectin clusters joined by amorphous lamellae
result in entrapment of proteins involved in starch biosynthesis with in starch granules.
However, detailed kinetic analysis of some of SSs isoforms suggest that increased
affinity of these proteins for longer glucan chains during catalysis may be a cause for
their association with the starch granules (Commuri and Keeling, 2001). But the
mechanism by which these proteins become granule-bound is not well understood.
However, Liu et al. (2009) reported that different enzymes of starch synthesis form
functional protein complexes (described earlier) and these complexes become entrapped
in the starch granule. Similarly it was also reported that different components of a protein
complex are phosphorylated. Thus, from these observations it can be speculated that
these phosphorylated proteins are coordinated as a protein complex and become
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entrapped within the starch granule. This idea is further supported by evidence which
shows that some of the proteins found in the starch granules are phosphorylated. In
wheat, GBSS, SSI and SSII were phosphorylated in their starch granule-bound state
(Bancel et al., 2010). Genetic analyses of the maize mutant lacking BEIIb, the ae mutant
shows significant increases in BEI, BEIIa, SSIII, and SP in the granule, without affecting
SSI or SSIIa (Grimaud et al., 2008). Staining the internal granule-associated proteins with
phospho-protein-specific dye showed that at least three proteins, GBSS, SBEIIb, and SP,
are phosphorylated in the starch granules (Grimaud et al., 2008; Liu et al., 2009). The
idea that starch biosynthetic enzymes form functional protein complexes and these
complexes become entrapped within the starch granule is further supported by two
independent studies by Liu et al. (2012a, b). In a maize mutant, ae 1.2, which was
expressing SBEIIb enzyme lacking glucan binding ability due to single nucleotide
mutation. However, this protein was found within starch granule because this protein was
found as part of a protein complex which was trafficked to the starch granules. Similarly,
in a su2 mutant of maize which was expressing a catalytically inactive SSIIa, no protein
was observed in the granule.
Apart from these proteins, other proteins which are not involved directly in starch
biosynthesis or degradation, and do not have a assigned function, have also been found
within the starch granules, such as plastid-localized starch granule-associated glycogen
synthase kinase (GSK-3)-like protein kinase in Medicago and Arabidopsis. Overexpression of GSK-3-like kinase results in accumulation of starch in leaves under saline
conditions (Kempa et al., 2007).
Similarly granule associated phosphatases have also been found which play an
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important role in starch catabolism by priming the polymer for degradation via
phosphorylation of glucose units (Comparot-Moss et al., 2010; Kotting et al., 2009; Kerk
et al., 2006; Niittyla et al., 2006; Sokolov et al., 2006; Fordham-Skelton et al., 2002).
Although substantial progress has been made in understanding the function of
different starch synthesizing enzymes, it is still not clear how these enzymes (SSs, SBEs
and DBEs) are coordinated and how different structures of amylopectin arise. There is
increasing evidence that heteromeric protein complexes act as functional assemblies to
improve polymer construction efficiency (Tetlow, 2006).
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Figure 1.12: Phosphorylation dependent functional complex formation and
entrapment of proteins within starch granule in different maize mutants.
(A), shows WT amyloplast stroma in which protein complex formation is shown to be
phosphorylation dependent and this phosphorylated protein complex becomes
entrapped with in starch granule.
(B), shows that in ae- mutant of lacking SBEIIb, phosphorylation dependent novel protein
complex containing (SSI, SSIIa, SBEI, SBEIIa and SP) is formed in which SBEI and SP are
phosphorylated and this protein complex also becomes entrapped within starch
granule.
(C), Su2- mutant expressing a catalytically inactive SSIIa protein due to a mutation which
resulted in the loss of SSIIa glucan binding ability. This mutation did not affect ability of
SSIIa to coordinate with SSI and SBEIIb in a trimeric protein complex. Thus due to loss of
glucan binding ability of SSIIa, other proteins (SSI and SBEIIb) were not able to bind to
starch granule. This Figure also explains that SSIIa plays a major role in trafficking other
proteins into to the starch granule (Liu et al., 2009, 2012).
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1.5 Experimental Material
In this study, barley (Hordeum vulgare) was used as the experimental material.
Barley is among the most widely cultivated and utilized cereals after maize, rice and
wheat, with the ability to provide valuable nutrients necessary for humans and domestic
animals (Zeeman et al., 2010). But this cereal is seldom for human consumption and
hence referred as poor man’s food in underdeveloped countries (Asare et al., 2011).
However, in the recent past there is renewed interest in barley as a food because of the
potential health benefits associated with constituents of its grain, including dietary fibre
(β-glucans), phenolic compounds and vitamins (Jadhav et al., 1998). Barley grain
constitutes of carbohydrates (60-80 %), water (10-15 %), protein (9-13 %), crude fibre
(5-6 %), ash (2-2.5 %), fat (1-2 %) other (5-6 %) (Chibbar et al., 2004). Although starch
is the major carbohydrate in barley grain ranging from (62-77 %) of the grain dry weight
(Bhatty and Rossnagel, 1998), it has not been studied to the same extent as in other
cereals such as corn, wheat and rice. As experimental material, barley has other
attractions because of the large collections of genetic variants available, and because of
its diploid nature, it is easier to study relative to wheat.
1.6 Hypotheses
Starch is a renewable biopolymer which has numerous food and industrial
applications which mostly depend upon physiochemical properties of starch. The diverse
physiochemical properties of starch are either controlled by complex genetic variations or
by a process which is regulated by the coordinated interaction of different enzymes and
their isoforms involved in starch biosynthesis. There is increasing evidence that these
enzymes co-operate physically, forming functional proteins complexes. Thus mutations in
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these enzymes can also alter other starch synthesizing enzymes pleiotropically. The
present study is based on an investigation of the following hypotheses:
I.
Increased RS is associated with distinctly altered physiochemical properties of
starch, which could be used as a diagnostic tool to determine high RS genotypes.
II.
The relationship between the granule proteome and starch properties from barley
mutants with defined mutations can be related to variation in protein-protein
interactions in the stroma.
III.
Variation in the granule proteome of A- and B-granules can be used to understand
the relationship between granule type, its proteome and mechanisms underpinning
granule size and morphology.
IV.
Some variations in starch physiochemical properties do not always occur due to
defined mutations in starch biosynthetic enzymes, but are result of complex
interaction between genetic back ground and biochemical processes.
1.7 Aims and Objectives of the Study
This research project focused on the following objectives,
I.
The first objective of the study was to analyze starch physical properties including
(amylose and RS contents, granule size, number and surface area distributions,
granule morphology, amylopectin chain length distribution and thermal properties
etc.) in a wide range of barley cultivars to determine characteristics of resistant starch
genotypes (Chapter 2).
II. The second objective of the study was to analyze soluble and starch granule
associated proteins from different high RS/amylose genotypes to determine
relationships among partitioning of enzymes between the stroma and granule and RS
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content (Chapter 3).
III. The third objective of this study was to investigate the composition of different
soluble protein complexes, the mechanisms involved in heteromeric complex
formation in different mutants, and binding of different proteins to starch granule and
also to determine whether different protein complexes contribute to the formation of
A- and B- type granules in barley (Chapter 3).
IV. The fourth objective of this study was to investigate barley genotypes with known
variations in the physiochemical properties of starch but for which there is no
characterized mutation, and to relate variations in starch biosynthetic enzymes, and
the granule proteome to the resulting starch phenotype (Chapter 4).
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Chapter 2 is prepared as a research article to be submitted for publication
- 70 -
Chapter 2: Evaluation of physical characteristics of resistant
starch from (a range of) barley genotypes
- 71 -
2.1 Introduction
Starch is the most important and widely distributed storage polysaccharide in
plants and the major storage biopolymer in cereal endosperms providing more than 75 %
of daily caloric intake and is a major source of feed, fiber, biofuels and a raw material in
many industries (Stamova et al., 2009). It occurs in a well-organized granular form
composed of linear and highly branched glucan polymers called, respectively, amylose
and amylopectin. Amylose and amylopectin are found in semicrystalline, water insoluble
lamellae within the granule. Amylose is essentially a linear molecule with predominantly
α-1,4 linked glucose units with a low frequency (< 1 %) of α-1,6 linked branches,
whereas, amylopectin is a much larger and highly branched molecule with a high
frequency (~ 5 %) of α-1,6 linkages (Myers et al., 2000). In amylopectin, branching of
glucan chains exhibits regular periodicity (Davis et al., 2003), and its length and pattern
play a critical role in the proper formation and organized structure of the starch granule
and the resisting physiochemical properties (Liu, 2005). The size, shape and number of
starch granules vary within and among species (Shapter et al., 2008).
In Festucoid grasses, including wheat and barley, starch granules can be
distinguished as A-, B- and C-granules, based on size, shape, relative number and the
timing of their initiation during seed development (Wei et al., 2010). A-granules are lens
shaped, varying in size from 10-50 µm in diameter and make 70-80 % of the volume and
normally account for approximately 10 % of the total number of the starch granules
(Langeveld et al., 2000; Hughes and Briarty, 1976). B-granules are spherical, varying in
size from 5-15 µm in diameter and represent < 30 % of the volume and > 90 % of the
total number of starch granules. The starch granules of size < 5 µm in diameter are
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termed C-granules (Wilson et al., 2006; Bechtel and Wilson, 2003). Wheat and barley
starches with different granule sizes show different degrees of amylolysis and thermal
and pasting properties (Liu et al., 2007). Along with granule size, differences in amylose
content, protein content, and branch chain length of amylopectin in A- and B- type starch
granules, could be important factors responsible for differences in digestibility and
determining the end use of starch (Liu et al., 2007).
Starch has many uses in food industries and, depending upon nutritional
classification, starch has been divided into three categories: rapidly digestible starch
(RDS), slowly digestible starch (SDS), and resistant starch (RS). RS was first identified
in 1982 by Englyst et al. (1982), and defined as the starch that resists α-amylase digestion
in the small intestine and is fermented by the bacteria in the large intestine, producing a
variety of end products, the most important of which are short chain fatty acids (SCFA)
(Leszczynski, 2004). RS can be classified into different types: RSI is physically
inaccessible starch as found in partial or intact cereal seeds. RSII is the raw starch in its
granular form in some plant species, e.g. potato and banana. RSIII is retrograded starch,
which occurs when starch-containing foods are cooked and cooled and RSIV is
chemically or physically modified starch, or processed food (Leloup et al., 1992a;
Colquhoun et al., 1995; Leszczynski, 2004; Rahman et al., 2007).
The Commonwealth Scientific and Industrial Research Organisation (CSIRO) in
Australia, has recommended that around 20 g of RS should be consumed per day, but the
consumption level of RS in a typical western diet is only about 25 % of the recommended
amount (Baghurst et al., 1996). To meet this deficiency, the best approach is to develop
crop genotypes high in RS and barley is one of the best candidates for this purpose.
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Barley (Hordeum vulgare) is the fourth most widely cultivated cereal after maize, rice
and wheat. Interest in the use of barley as a food component with the ability to provide
valuable nutrients necessary for humans and domestic animals has increased because of
the potential health benefits associated with constituents of its grain, including dietary
fiber, phenolic compounds and vitamins (Jadhav et al., 1998; You and Izydorczyk, 2007).
Barley grain is composed of carbohydrates (60-80 %), water (10-15 %), protein (9-13 %),
crude fibre (5-6 %), ash (minerals) (2-2.5 %), fat (1-2 %) and other (5-6 %) (Fahrenholz,
1998; Chibbar et al., 2004). Starch is the major carbohydrate in barley grain ranging from
(62-77 %) of the grain dry weight (Bhatty and Rossnagel, 1998). Although starch is the
major component of barley grain, it has not been studied to the same extent for human
consumption as the starch of corn, wheat and rice. Barley has many advantages for use as
a model plant which includes its relative simple genome compared to wheat and corn,
and the large number of morphological and biochemical mutants available. This study
was conducted using barley and tested the hypothesis that increased RS is associated with
distinct physical properties of starch, which could be used as a diagnostic tool to
determine high RS genotypes. Comprehensive analyses were used to investigate which
physical parameters of barley starch can be used to improve the selection of high RS
genotypes.
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2.2 Material and Methods
2.2.1 Selection of genotypes
In this study 33 genotypes of barley have been used. These genotypes were
selected based on diverse agronomic and physiological characteristics from different
genetic back grounds and different geographical origins. These genotypes include
covered and hulless, two and six rowed varieties or advance lines in different
combinations. Similarly, different mutants with known mutations in starch biosynthetic
enzymes such as, granule bound starch synthase (GBSS), starch synthase (SSIII or
amo1), starch branching enzymes down regulated lines (sbeiia- and sbeiib-) and lines
derived from amo1 are also included. Detailed description of the genotypes used in this
study has been given in the Appendix 1.
2.2.1.1 Selection of genotypes for further detailed analyses
From the initial 33 screened genotypes, 12 genotypes either high in amylose or
RS were selected for further detailed study. The rationale for selecting these genotypes
from the wider pool, were based on the following, the reason behind increased
amylose/RS content in different genotypes may be different. It is may be due to mutation
in specific genes, different genetic background or geographical origin of different
genotypes. All these variations can give a better understanding that how different
physical parameters under different conditions are important in producing starch of the
desired RS phenotype. Along with these genotypes, one normal genotype was also used
as reference.
2.2.2 Isolation of starch granules
Mature dry barley grains (≈ 55 g) were completely ground to flour with a
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Retsch® MM301 homogenizer in liquid nitrogen. Equal amounts of flour (≈ 50 g) were
suspended in 150 ml of buffer containing 100mM Tricine-KOH, pH 7.8, 1mM Na2EDTA, 1mM DTT and 5mM MgCl2 at 4oC (Liu et al., 2009). The suspension was mixed
thoroughly by vortexing for 5-10 minutes and then was left for 5-10 minutes on ice. The
suspension was filtered through six layers of cheese cloth to remove debris and bran.
Buffer solution was used to rinse traces of starch from the cheese cloth. The sieved milky
suspension (≈ 450-500) ml was centrifuged at 16000 g for 15 min. The supernant was
discarded and pellet was washed in ≈ 100 ml buffer containing 50 mM TRIS-acetate, pH
7.5, 1 mM Na2-EDTA, and 1 mM DTT. The pellet was suspended in the same washing
buffer and was centrifuged at 5000 g for 5 min. This washing step was repeated 5-7 times
until only a thick yellow layer of debris was left on the top of starch. The yellow layer
containing very small pieces of debris was completely removed with spatula attempting
to avoid any starch losses. The starch was again washed twice in buffer and centrifuged at
5000 g for 5 min. Purified starch was washed three times with acetone followed by three
washes with 2% (w/v) sodium dodecyl sulphate (SDS), and three wahes with water and
then dried with speed vacuum (Eppendorf, VacufugeTM) at 25 oC for 3 h.
2.2.3 Measurement of amylose and RS contents
The apparent amylose and RS from raw starch were measured with Megazyme
amylose/amylopectin assay kit (K-AMYL 07/11) and resistant starch assay kit (KRSTAR 08/11) respectively according to manufacturer instructions using three biological
and two technical repeats.
2.2.4 Amylose content determination by gel permeation chromatography
To measure the amylose content using Gel Permeation Chromatography (GPC),
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10 mg of acetone-washed dried granular starch was used with three biological and two
technical repeats. In 4 ml glass sampling bottles, starch was dissolved in 200 µL of 90%
DMSO with continuous stirring (50 rpm) on a magnetic stirrer overnight. 1600 µL of
boiling HPLC grade water was added to the dissolved starch. The dissolved starch was
cooled down from 90oC to room temperature and 200 µL of 0.01N sodium acetate buffer
(pH 5.5) was added. To debranch the starch, 3 µL of isoamylase enzyme (59000U/mg of
protein) (Hayashibara, Japan) was added and the mixture was stirred over night at 50 rpm
at room temperature. The enzyme was inactivated by placing the sampling bottles in a
hot water bath (90-95 oC) for 5 min. The mixture was cooled down at room temperature
and 200 µL of 5M NaOH was added and mixed well. The completely debranched starch
mixture was filtered through a 0.45 µm nylon filter (Sarstedt). 1 ml of the filtered sample
was injected into the CL 6B column (concentration of the starch solution was ~5 mg
starch/ml) and 1 ml fraction was collected in glass test tubes. 1 ml of 5% phenol and 5 ml
of 37 % sulphuric acid were added to each test tube, mixed, and the samples cooled.
Absorbance was measured at 490 nm on a Beckman Coulter spectrophotometer.
2.2.5 Measurement of amylose by iodine binding
Apparent amylose content of starches was determined with three biological and
two technical repeats by the method proposed by Chrastil (1987), with certain
modifications. In 20 mg acetone dried starch, 1M NaOH (2 ml) and distilled water (4 ml)
were added in a screw cap tube. The tube was capped and heated in a water bath at 95 ºC
for 30 min with occasional mixing on a vortex mixer (Eppendorf®). The solution was
cooled to room temperature and an aliquot of 0.2 ml in duplicates was added to 5 ml of
0.5 % trichloroacetic acid in a separate glass test tube. The solutions were mixed
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thoroughly and 0.1 ml of 0.1N I2-KI solution (1.27 g of I2 and 3 g of KI/L) was added to
each tube and mixed immediately. The resulting blue color was measured at 620 nm on a
Beckman Coulter spectrophotometer, after 30 min against a reference prepared without
starch. The amylose content was calculated using the standard curve prepared using
mixtures of pure potato amylose and amylopectin (over the range 0–100 % amylose).
2.2.6 Granule size distribution of different genotypes
Granule size, number and surface area of starch from different genotypes were
measured by means of laser scattering using the Master Sizer (Malvern Mastersizer 2000,
Malvern Instruments Ltd., UK). For each measurement, starch (± 100 mg) was weighed
into glass tubes and suspended with 10 ml of distilled water. Sample concentrations were
within equipment recommendations and the refractive indices of 1.31 for water and 1.52
for starch were used as the standard and distribution was measured as both the percentage
volume and percentage number.
2.2.7 Granule morphology
To study starch granule (A-, B- and C-)
morphology in detail, SEM was
performed on a field emission scanning electron microscope (S-4500, Hitachi, Tokyo,
Japan) as described by (Liu et al., 2007), equipped with Quartz PCI digital image
acquisition software (Quartz Imaging, Vancouver, BC, Canada). The acetone-dried
barley starch samples were sprayed on a metal plate covered with double-sided adhesive
tape. The samples were coated with gold using a Polaron SC500 sputter coater (Quorum
Technologies, East Sussex, UK). The samples were examined at 10 kV accelerating
voltage and representative micrographs were taken for each sample at different
magnifications according to requirement.
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2.2.8 Seed characteristics
2.2.8.1 Thousand grain weight (TGW)
1000 grains of each genotype were counted and weighed to 0.1 mg using a
tabletop electrical balance (Denver Instrument, Bohemia, NY, USA). The mean of three
replicates was used in statistical analysis (de Rocquigny, 2011).
2.2.8.2 Starch content
The amount of starch in barley seeds from the different genotypes using three
biological repeats was determined as described for wheat endosperm (Tetlow et al.,
1994).
2.2.9 High-performance anion exchange chromatography
To measure the chain length distribution of barley starch, High-Performance
Anion Exchange Chromatography (HPAEC) was used as described by (Liu et al., 2007).
2.2.10 Statistical analysis
For one way analysis of variance (ANOVA) and least significant difference
(LSD), Statsoft’s Software, Statistica (http://www.statsoft.com) was used. For principal
component analysis (PCA), XLSTAT (http://www.xlstat.com/en/) was used and biplots
were generated.
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2.3 Results
2.3.1 Amylose content
Barley starch can be classified into normal (25-27 % amylose), waxy (< 5 %
amylose), and high (> 35 %) amylose (Bhatty and Rossnagel, 1992; Bhatty and
Rossnagel, 1998; Izdorczyk et al., 2000). However in this study, depending upon amylose
content, the genotypes were divided into four groups (I), waxy (< 5 %), (II), low amylose
(5-20 %), (III), normal (20-27 %) and (IV), high amylose (> 30 %), as shown in the
Figure (2.1). Significant variation in amylose content was observed among and within
different groups of genotypes as shown in Figure (2.1). Group (I) contains all the waxy
genotypes which either lack or have inactive (GBSS), the enzyme which is solely
involved in amylose biosynthesis. Groups II & III (with low and normal amylose,
respectively) include normal established varieties, advanced lines and breeding material.
Group (IV) consists of mutants with mutations in starch synthase (SS) III (ssiii), starch
branching enzyme (SBE) IIa and IIb (sbeiia- and sbeiib-) or a mutation at the sex1 locus
(Appendix 1). Figure (2.1) shows all the genotypes in group IV have increased amylose
content compared to reference genotype. The sbeiia- and sbeiib- have similar amounts (30
%) of amylose. The genotypes with mutation at the sex1 locus (083611-118 sex1,
083611-124 sex1, 020113-385 sex1) also possessed high (35%) amylose content. The
amo1 mutants (HAG amo1 and SB94983 amo1) exhibited (40 and 30 %) amylose
content respectively. The genotypes of series 081011-928 - 081011-932 originated from
the same amo1 parent but these genotypes showed variation in their amylose content. For
example, in genotypes 081011-928, 081011-929 and 081011-932, amylose content was
similar (± 45 %), however genotypes 081011-930 and 081011-931 possessed
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60
Waxy
Low amylose
Normal
High amylose
40
30
20
10
081011-932
081011-931
081011-930
081011-929
081011-928
SB94983 amo1
HAG amo1
sbeiia-
sbeiib-
020113-385 sex1
083611-124 sex1
AC Bacon
GB992033
083311-104
083611-118 sex1
Genotypes
OAC Kawartha
OAC Baxter
083211-122
Hard Barley
Emperor
AC Metcalfe
083511-118
083411-113
083511-109
083211-120
AC Alberte
Sunderland
Sloop
McGwuire
Soft Barley
Neopolis
CDC Rattan
0
CDC Fibar
% amylose
50
Figure 2.1: % amylose of barley genotypes.
The amylose content was measured by Megazyme amylose/amylopectin assay kit using
acetone washed starch. Different groups of genotypes based on % amylose content are
presented with different bars representing standard error mean. Waxy, low amylose
and normal genotypes have amylose content varying between 1-27 %. While all other
genotypes with amylose content >27% are included in high amylose group.
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significantly less (39 and 28 %) amylose at p < 0.05, respectively.
2.3.2 Resistant Starch (RS) Content
RS was measured for genotypes and divided into three different groups: (I),
Normal, genotypes having RS content < 5 % and < 27 % amylose. (II), High amyloseLow RS, genotypes having higher amylose > 30 % but with RS content < 5 %. (III), High
amylose- High RS, genotypes having high amylose > 27 % with RS content > 5 %, as
shown in Figure (2.2). The rationale for dividing the genotypes into different groups is
primarily based on the amount of RS, the lowest amount of RS in a high amylose
genotype was 5.6 %, all genotypes in this group have an amylose content > 27 % (Figure
2.1).
Group (I) includes waxy, normal barley and experimental material, all these
genotypes have RS content < 5 %, but apparent amylose content higher than expected.
The genotypes Neopolis, CDC Fibar and CDC Rattan are waxy genotypes (Figure 2.1)
but possess higher RS than indicated by amylose content alone (Figure 2.2)
demonstrating that amylopectin may also contribute to the total RS contents. The amount
of amylose is positively related to RS but the above mentioned waxy genotypes show that
in addition to the amylose, some features of amylopectin may also be important in
determining the proportion of RS. Genotypes in group (II) are apparently high in amylose
(35%, Figure 2.1) but low (1.4 %) in measureable RS (Figure 2.2). In group III, all the
high amylose genotypes (Figure 2.1) are also high in RS (Figure 2.2). The genotypes in
group III exhibit varying content of high amylose which correlate positively (r = 0.80, p <
0.05) with increased RS, though the relationship is not strictly proportionate. Two
genotypes 081011-930 and 081011-931 were of particular interest as they represent
- 82 -
16
14
12
Normal
High amylose- Low RS
High amylose- High RS
8
6
4
2
0
CDC-Fibar
Neopolis
CDC-Rattan
Soft Barley
Sloop
McGwuire
Sunderland
AC Alberte
083211-120
083511-109
083411-113
083511-118
Emperor
AC Metcalfe
083211-122
Hard Barley
OAC Baxter
OAC Kawartha
GB992033
083311-104
AC Bacon
083611-118 sex1
083611-124 sex1
020113-385 sex1
sbeiiasbeiibHAG amo1
SB94983 amo1
081011-928
081011-929
081011-930
081011-931
081011-932
% RS
10
Genotypes
Figure 2.2: % RS of barley genotypes.
The RS content measured by Megazyme resistant starch assay kit using acetone washed
starch. Different groups of genotypes based on % RS content are presented. The normal
group includes genotypes which have amylose content < 27 % and RS content < 5 %.
High amylose- Low RS group include those genotypes which have amylose content > 27
% but RS content < 5 %. High amylose- High RS group includes all those genotypes which
have amylose content > 27 % and are also high in RS > 5 %.
- 83 -
different selections from the same cross involving an amo1 parent. The genotype 081011930 had an intermediate level of RS (6.5 %) whilst 081011-931 was a “normal” type with
(3 % RS).
2.3.3 Comparison of different methods for determination of amylose content
Three different methods were used to measure the amylose content i.e.
biochemical assay, iodine binding and GPC. PCA showed that the three methods
correlated well (Megazyme and iodine binding, r = 0.94, p < 0.05; Megazyme and GPC, r
= 0.67, p < 0.05; iodine binding and GPC, r = 0.60, p < 0.05), (Appendices 2 and 3).
2.3.4 Physical characteristics of seed
2.3.4.1 Seed characteristics
Seed characteristics including seed length, width and thickness were measured.
Different genotypes showed significant differences in different seed parameters. Seed
length varied from 7.1 to 9 mm among different genotypes. All high amylose genotypes
showed a higher seed length (≥ 7.5 mm) than normal genotype (7.4 mm), except sbeiiawhich possessed 7.1 mm seed length. In the case of seed width, the down regulated lines
of branching enzymes IIa and IIb, one amo1 mutant (HAG amo1) and all the sex1
mutants showed lower seed width (≤ 3.6 mm) compared with wild-type (3.7 mm) while
all other high amylose/ RS genotypes had greater seed width (≥ 3.8 mm) than the wildtype (Table 2.1). Normal genotype had the highest seed thickness (2.9 mm). All the sex1
mutants with shrunken endosperm had lowest (2 mm) seed thickness (Table 2.1).
2.3.4.2 Thousand grain weight (TGW)
The genotypes showed significant variation in grain weight. The genotype OAC
Baxter had a TGW of 63.1 g. Other genotypes exhibited lower seed weight than the
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Table 2.6: Physical characteristics of seed
Genotype
Type
Rows
Seed Characteristics (mm)
SL
SW
ST
Length:
Width
TGW
% Starch
Content
7.4f
3.7c
2.9a
2.0f
63.1a 100.0
C
6
g
d
cd
f
7.1
3.6
2.6
2.0
46.2c 95.8
C
2
7.5ef
3.6d
2.6de
2.1e
48.5c 84.4
C
2
d
e
f
c
8.2
3.4
2.0
2.4
43.9c 48.0
H
6
9.0a
3.4e
2.0f
2.6a
37.1d 55.9
H
2
cd
f
f
b
8.3
3.3
2.0
2.5
33.5d 61.1
C
6
8.6ab
3.4e
2.5e
2.5b
55.9b 93.9
C
6
8.6ab
3.9a
2.7bc
2.2d
59.2ab 104.0
H
2
ab
a
e
d
8.6
3.9
2.5
2.2
56.9ab 117.0
H
2
081011-928
8.6ab
3.9a
2.7bc
2.2d
60.5ab 116.4
H
2
081011-929
bcd
a
b
d
8.5
3.9
2.7
2.2
59.0ab 116.5
H
2
081011-930
7.8e
3.9a
3.0a
2.0f
60.0ab 110.1
C
2
081011-931
bcd
b
b
d
8.4
3.8
2.8
2.2
57.2ab 117.9
H
2
081011-932
SL= Seed length, SW= Seed width, ST= Seed thickness, TGW= Thousand grain weight
OAC Baxter
sbeiiasbeiib083611-118 sex1
083611-124 sex1
020113-385 sex1
HAG amo1
SB94983 amo1
Table 2.1, shows physical characteristics of seed, type indicates whether seed is covered
(C) or hulless (H). Rows represent rows of fertile spikelets on the barley spike. % starch
content of wild-type (OAC Baxter) was used as a reference and taken as 100 % to which
the genotypes were compared. Data present means of three replicates. Different letters
in columns following each mean value indicate whether genotypes are significantly
different at (p<0.05), genotypes sharing same alphabetical letter for a given character
are not significantly different. For seed length, width and thickness three biological
repeats with hundred independent observations for each character were taken.
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normal genotype and all sex1 lines (both covered and hulless) had the lowest TGW at <
45 g (Table 2.1).
2.3.4.3 Starch content
Starch is an important constituent of cereal seed and final yield. Different
genotypes showed significant variation in their starch contents. The starch content of the
reference genotype OAC Baxter was used as a reference and taken as 100 % to which the
other genotypes were compared. The shrunken mutants (083611-118 sex1, 083611-124
sex1, 020113-385 sex1) possessed lowest starch content < 48 % compared to reference
genotype. The sbeiia-, sbeiib- and HAG amo1 mutants were lower (95.8, 84.4 and 93.9 %,
p < 0.05) in starch content compared to wild-type. However, SB94983 amo1 and amo1
derived lines: 081011-928, 081011-929, 081011-930, 081011-931 and 081011-932
exhibited between 104.0 - 118.0 % starch content compared to the reference genotype
(Table 2.1). It should also be noted that these lines were hulless and so the starch was not
diluted by hull except 081011-931genotype which had covered seed.
2.3.5 Granule size, number and surface area distributions of different genotypes
The reference genotype, OAC Baxter, possesses a normal bimodal granule size
distribution with median granule size of 3.6 and 20.5 µm for B- and A-granules,
respectively (Figure 2.3A), whereas all high amylose genotypes with variable
amylose/RS contents have a unimodal granule size distribution (Figure 2.3A & B). The
sbeiia-, sbeiib- and sex1 mutants (083611-118 sex1, 083611-124 sex1, 020113-385 sex1)
exhibited average (6.6 -7.5 µm) size for B granules (Figure 2.3A). The amo1 mutants
(HAG amo1 and SB94983 amo1) possessed average B-granule size varying between 5.7
- 6.9 and 6.8 - 7.7 µm, respectively. The amo1 derived lines 081011-928, 081011-929
- 86 -
and 081011-932 exhibited average (5.2 - 6.7 µm) size for B granules, while two other
lines, 081011-930, 081011-931 have (5.9 - 7.2 and 7.5 - 8.7 µm) average B granule size,
respectively (Figure 2.3B). For large A-granules, the sbeiia-, SB94983 amo1, 083611-118
sex1 and 081011-930 exhibited the same average (17-19 µm) size (Figure 2.3A & B).
The sbeiib- and 081011-931 have average size (17.5-19.5 µm) for A-granules. While,
HAG amo1, 083611-124 sex1 and 020113-385 sex1, 081011-928, 081011-929 and
081011-932 exhibited the same average (16-18 µm) size A-granules.
The reference genotype OAC Baxter showed bimodal granule number distribution
in which B- and C-granules contributed almost 98 % to total granule number and Agranules constituted only 2 % of total granule number. The reference genotype OAC
Baxter also showed bimodal starch granule surface area distribution. However, all high
amylose/RS genotypes exhibited unimodal granule number and surface area distributions
(data not shown).
2.3.6 Contribution of A-, B- and C-granules in total mass of starch
Amount of different types of granules (A-, B- and C-) is important in
characterizing starch from different mutants. In this study different genotypes showed
noticeable differences in the amount of different types of granules (Figure 2.4). The
reference genotype OAC Baxter has a higher amount (11 %, p < 0.05) of C-granules
while all high amylose/RS mutants have very small or no C-granules. In the case of Bgranules, reference genotype and genotype 081011-931 have lower amount (24 and 37 %,
p < 0.05) of B-granules and higher amount (66 and 63 %, p < 0.05) of A-granules,
respectively, in contrast high amylose/RS genotypes have higher amount (> 50 %) of Band lower amount (< 50 %) of A-granules (Figure 2.4). In some genotypes (083611-124
- 87 -
Figure (2.3A & B): Granule size distribution.
Granule size distribution was measured by means of laser scattering using the
Mastersizer (Malvern Mastersizer 2000, Malvern Instruments Ltd., UK). Acetone washed
starch (± 100 mg) was suspended in 10 ml of distilled water and used according to
manufacturer instructions.
- 88 -
% Amount of starch
100
80
60
40
20
OAC Baxter
sbeiiasbeiib03611-118 sex1
083611-124 sex1
020113-385 sex1
HAG amo1
SB94983 amo1
081011-928
081011-929
081011-930
081011-931
081011-932
0
C- granules (1-5 mm)
B- granules (5-15 mm)
A- granules (15-45 mm)
Type of starch granules
Figure 2.4: Amount of A-, B- and C-granules in total mass of starch.
Determination of A-, B- and C-granules using a Mastersizer. Acetone washed starch (±
100 mg) was suspended in 10 ml of distilled water and used according to manufacturer
instructions (Malvern Instruments Ltd., UK).
- 89 -
sex1, HAG amo1, 081011-928, 081011-929 and 081011-932) amount of B-granules
exceeded 70 % as shown in Figure (2.4).
2.3.7 Granule morphology
The starch granules from different barley mutants not only vary in size but also in
shape, which was revealed by scanning electron microscopy. Generally, A-granules are
disc or lenticular, B-granules spherical and C-granules irregular shaped (Wei et al.,
2010). In this study starch granules of reference genotype showed the expected (reported)
morphology. All high amylose/RS genotypes exhibited altered granule morphology
(Figure 2.5). For example, in the sbeiia- and sbeiib- down regulated lines, round Agranules with tunnels were observed. In amo1 mutants and lines derived from amo1
parent, irregular A-granules with bumps were found in abundance. The sex1 mutants
exhibited round and flat A-granules with small ditches at the surface. In all the above
mentioned mutants morphology of B-granules did not change, however average size of
B-granules increased compared to reference genotype. The genotype (081011-931) which
has comparatively less amylose, exhibited bigger B-granules which resemble A-granules,
while its A-granules were not severely deformed compared to its counterparts. In most of
the high RS/amylose genotypes C-granules were either absent or present in very small
amounts. The accumulation of very high amount of B-granules in all high amylose/RS
genotypes shows that amount of amylose/RS is positively correlated (r = 0.89, p < 0.01)
to B-granules while negatively correlated (r = -0.80, p < 0.01) to A-granules as shown in
the Figure 2.9 A & B.
- 90 -
Figure 2.5: Morphology of starch granules from normal and high RS/amylose
genotypes observed by SEM.
For SEM analysis acetone dried barley starch samples were sprayed on a metal plate
covered with double-sided adhesive tape. The samples were gold coated with Polaron
SC500 sputter coater (Quorum Technologies, East Sussex, UK). The samples were
examined at 10 kV accelerating voltage and representative micrographs were taken for
each sample at same magnification. Scale bar for electron micrograph is given under
each panel. Each type of granule is presented by a colored arrow.
- 91 -
2.3.8 Amylopectin chain length distribution
The measured degree of polymerization (DP) of glucan chains in amylopectin
among different genotypes ranged from 6-50 DP with varying proportions of different DP
classes. The observed chain length distribution was divided into four groups as shown in
Table (2.2) (Liu et al., 2007). In group I, only two genotypes showed significant
differences (p < 0.05) for glucans in the range DP 6-12; genotype, 081011-931 has
highest proportion (46.3 %) and the genotype sbeiib- has lowest proportion (42.3 %),
(Table 2.2). There were no significant differences among genotypes for intermediate
chains with DP 13-24. A small proportion of longer chain glucans with DP 25-36 was
found in all genotypes with highest amount (8.8 %) in OAC Baxter and lowest (7.1 %) in
081011-931 (Table 2.2). The proportion of glucans chains with length of DP 37-50 was
lowest in all genotypes. However, different genotypes exhibited significant differences (p
< 0.05) relative to the reference genotype with some exceptions. For example, sbeiia- and
sbeiib- were not significantly different (p < 0.05) from waxy and the reference genotype,
respectively, but were significantly different from one another (Table 2.2).
To understand differences in individual classes of glucans DP among different
genotypes, difference plots of normal barley and mutants were produced (Figure 2.6). A
difference plot of (mutants - wild-type) showed that all genotypes exhibited differences in
glucan chains with DP 6-20. However, the extent of difference in particular glucan chain
groups in all genotypes compared to reference genotype was not very high. In the case of
longer chains with DP > 20, proportion of difference among different genotypes
compared to reference genotype was not statistically significant (Figure 2 6).
- 92 -
Table 2.2: Degree of polymerization (%) of amylopectin from barley genotypes
Genotype/chain length
OAC Baxter
CDC Fibar (waxy)
sbeiiasbeiibHAG amo1
SB94983 amo1
081011-928
081011-929
081011-931
081011-932
DP 6-12
43.3ab
45.2ab
42.3b
44.1ab
42.6ab
43.5ab
43.1ab
43.3ab
46.3a
44.3ab
DP-13-24
43.8a
43.6a
46.2a
43.7a
45.9a
46.8a
46.2a
46.3a
43.4a
45.7a
DP 25-36
8.8a
7.5cd
7.8bc
8.0bc
8.2ab
7.6bcd
8.1bc
7.7bcd
7.1c
7.4cd
DP 37-50
4.1a
3.6b
3.7b
4.2a
3.3c
2.1e
2.7d
2.7d
3.2c
2.6d
Avg. DP
20.36
19.62
19.87
20.18
19.59
18.57
19.09
19.04
19.25
18.79
Table 2.2, different groups of chain length with varying DP (s) have been presented.
Different letters in columns following each mean value indicate significance at (p<0.05),
genotypes sharing same alphabetical letter for a given group of chain length are not
significantly different.
- 93 -
The difference plot between different genotypes and waxy showed differences in
amylopectin chain length distribution among different high amylose genotypes, sbeiia-,
HAG amo1, SB94983 amo1 and 081011-929 relative to the amylose free genotype, CDC
Fibar (Figure 2.7). All genotypes exhibited a lower proportion of chain length with DP ≤
10 and a higher proportion of chain length with DP 10-20 compared to waxy with some
exceptions. In the case of chain length with DP > 20, no significant variations between
the proportions of different chain lengths were observed in all genotypes compared to the
waxy genotype (Figure 2.7).
- 94 -
Molar % difference
Figure 2.6: Difference plot of (different genotypes versus wild-type) of amylopectin
chain length distribution.
Amylopectin chain length distribution of barley genotypes was measured by HPAEC.
The chain length distribution of wild-type OAC Baxter was deducted from the chain
length distribution of different genotypes. The positive value shows that different
genotypes exhibited higher proportion of that specific glucan chain. While negative
value shows that OAC Baxter exhibited a higher proportion of that specific glucan chain.
X-axis is representing degree of polymerization (DP) between 6-50 and Y-axis represents
% moles of carbohydrates.
- 95 -
Molar % difference
Figure 2.7: Difference plot of (Different genotypes versus waxy) of amylopectin chain
length distribution.
Amylopectin chain length distribution of barley genotypes was measured by HPAEC.
The chain length distribution of waxy CDC Fibar was deducted from the chain length
distribution of different genotypes. The positive value shows that different genotypes
exhibited higher proportion of that specific glucan chain. While negative value shows
that CDC Fibar exhibited a higher proportion of that specific glucan chain. X-axis is
representing degree of polymerization (DP) between 6 - 50 and Y-axis represents %
moles of carbohydrates.
- 96 -
2.3.9 Principal component analysis (PCA)
PCA, along with simple correlation analysis (Figures 2.8 & 2.9) was performed to
determine relationships among different physiochemical parameters of starch and seed.
PCA illustrates more specific relationships among different parameters as compared to
linear correlation. Simple linear correlation shows that the amount of amylose/RS is
negatively correlated (r = -0.80, p < 0.01) with the proportion of A-granules of size 15-45
µm and B-granules (1.2-15 µm) are positively correlated (r = 0.89, p < 0.01) with
amylose/RS (Figure 2.9 A & B). Large granules of size (39-45 µm) and chain length of
DP 6-12 appear to be closely associated with seed thickness. Other seed parameters such
as, seed width are closely associated with % starch content and seed weight while seed
length and L/W ratio are not associated with any of the measured characters. The type of
the seed (covered or hulless) and rows of fertile spikelets on the spike (2 or 6 rows) do
not show an association with measured starch characters (data not shown).
Three different methods were used to measure the amylose content. PCA showed
that all the three methods are closely associated (Appendices 2 and 3). PCA was also
used to examine relationships among molecular structure and other physiochemical
properties of starch and it was revealed that overall the three methods used for amylose
determination, amylose content, RS content, amount of B-granule, B-granules of size (515 µm), and chain length of DP 13-24 are closely associated. Amylopectin content,
amount of A-granules and granules of size (15-30 µm) are also closely associated. PCA
also demonstrates that the amount of A- and B-granules is closely related to 15-30 µm
and 5-15 µm granules, respectively which means that these two granule sizes are in
higher proportion and contribute more to the total mass of starch.
- 97 -
Figure 2.8: PCA analysis shows association of different characters.
The name of each character is given in the PCA while different genotypes are presented
by (+). Different characters present in close vicinity are associated while different
characters opposing each other are negatively related. Genotypes have been clustered
into different groups depending upon their association with different characters and
each cluster is presented by a different circle: black, normal barley; Light green,
experimental lines; red, waxy; blue; Dark green, high amylose.
- 98 -
Figure 2.9(A & B): Linear correlation between A- and B-granules with % amylose.
% amylose was measured by using Megazyme amylose/ amylopectin assay kit and GPC.
Amount of A- and B-granules was determined by the Mastersizer (Malvern Mastersizer
2000, Malvern Instruments Ltd., UK). Correlation between amylose and large (15-45 µm)
and small (1-15 µm) granules was determined by Microsoft Excel.
- 99 -
2.4 Discussion
The foods we eat significantly affect our health. Type-2 diabetes, obesity, heart
disease and stroke were once thought to be caused by single gene mutations, but there is
now growing evidence attributing these conditions to a network of biological
dysfunctions and the food we eat is an important factor in that dysfunction (Vorster,
2009). Foods rich in resistant starch (RS) provide health benefits by preventing/reducing
certain diseases like diabetes, obesity, colon cancer, and cardiovascular diseases (Sharma
et al., 2008). RS-rich foods show low glycaemic index, and also reduce colorectal cancer
risk by promoting bowel health (Asare et al., 2011). Cereal grains are the best source of
different nutrients in balanced ratios and are also the best source of RS given the volume
of consumption in most diets. In the present study, a range of barley genotypes from
different sources were selected to screen for RS content and analyze different seed and
starch physicochemical characteristics to determine their contribution to RS. A group of
genotypes was identified which showed increased RS and that certain physical
characteristics of the seed and starch were important determinants of RS. All the high RS
genotypes (Figure 2.2) were also high in amylose (Figure 2.1), meaning that the increase
in amylose is directly related to increase in RS. Previously it was also reported that starch
with increased amylose is more resistant to α-amylase digestion (Crowe et al., 2000;
Emami et al., 2010). This correlation was true for all genotypes except two sex1 mutants
(083611-118 sex1 and 083611-124 sex1) which were high in amylose but low in RS
(Figures 2.1 & 2.2). The reason for apparent discrepancy is not known. However, it could
be argued that the possible reason of discrepancy of these two genotypes may arise from
the molecular structure of the starch because with respect to other physicochemical
- 100 -
characteristics these two genotypes were similar to other sex1 mutant and high amylose
genotypes. This finding also shows that in some cases, increase in amylose is not the only
factor in increased RS but the molecular structure of the starch and interplay between
amylose and amylopectin also plays an important role in determining RS. In addition to
amylose: amylopectin ratio, structure, the packing of amylopectin within the granule
(Benmoussa et al., 2007) amount of lipids and proteins (Copeland et al., 2009), and
complexes with long chain fatty acids (Putseys et al., 2010) are also important
contributors in RS. Similarly, structural aspect such as starch crystallinity (Jane et al.,
1997), and occurrence and perfection of crystalline region in both amorphous and
crystalline lamellae of the granule (Zhang et al., 2006) are also important characteristics
in determining the starch digestibility and significantly contribute to the RS. It should
also be noted that in barley these starches, high amylose, do gelatinize at near to normal
temperatures and that in the gelatinization process the RS is lost (but potentially gained
back with retrogradation). Different waxy genotypes showed higher RS than expected
from their amylose content, which also indicates that, apart from amylose, characteristics
of amylopectin are important in determining RS content. Other possible explanations of
high RS content in waxy genotypes may be due to presence of β-glucans. Barley is rich in
β-glucans and waxy genotypes accumulate even more β-glucans (Ajithkumar et al., 2005)
which may be adding to the RS content of the genotype.
To examine the contribution of different physical characteristics of seed in the
increased RS, PCA was used and showed that hulless or covered seed and barley types
such as two rows or six rows do not associate with either amylose/RS or any other seed
starch characters. Both of these characters were independent of different physiochemical
- 101 -
properties of starch. A negative relationship between amylose and starch content has been
reported previously (Morell et al., 2003; Boren et al., 2008; Regina et al., 2010; Asare et
al., 2011) which was also true for genotypes in the present study except 081011-928,
081011-929, 081011-930 and 081011-932 which were derived from amo1 mutant and
have more starch and amylose/RS than the reference genotype. These genotypes are of
significant importance in overcoming the problem of high amylose content associated
with low starch/ and grain yield. Other physical characteristics of seed such as seed
length (SL) was independent of all other characteristics measured, but seed thickness
(ST) and seed width (SW) were positively related to starch content and seed weight.
These characters contribute significantly to yield and some lines with increased
amylose/RS have increased seed width and seed thickness and may have higher yield
than the normal type as mentioned earlier. The sex1 mutants behaved differently in terms
of different seed and starch characters because all these mutants have shrunken
endosperm (starch mutants) and store more protein and have a different protein profile
(Bosnes et al., 1992).
In the present study it was found that granule size is negatively related to %
amylose/RS while granule number is positively related to % amylose/RS. All the high
amylose genotypes accumulated more small granules of size (5-15 µm). Reduction of
granule size in high amylose genotypes was related to unimodal granule size distribution
which shows that amylose also affects composition of starch granules. Two amo1 lines
081011-930 and 081011-931 derived from the same amo1 parent have lower amylose
than their counterparts, 081011-928, 081011-929 and 081011-932 (Figure 2.1). The
genotype 081011-930 has lower amylose, larger granule size (34 µm) and less altered
- 102 -
granule morphology than its counterparts (081011-928, 081011-929 and 081011-932).
The genotype 081011-931 has even lesser amylose than 081011-930, and in this
genotype granule size increased up to 39 µm and the proportion of granules with regular
morphology also increased (Figure 2.5). These observations suggest that amylose:
amylopectin are significantly correlated with granule size and granule size can therefore
be used as a diagnostic tool for the screening of high amylose genotypes.
To examine differences in the molecular structure of amylopectin from different
genotypes, chain length distribution of normal, waxy and increased amylose/RS
genotypes was determined. In maize and rice, high amylose/ RS contents are related to
longer modified amylopectin chains (Yoshimoto et al., 2002; Kubo et al., 2010) for
example in the ae- mutant of maize (Hilbert and MacMasters, 1946; Banks et al., 1974;
Klucinec and Thompson, 2002). However, in this study a higher proportion of long
chains in amylopectins from high amylose genotypes was not observed compared to waxy
and normal genotypes (Table 2.2). It shows that higher amylose content in barley does
not necessarily affect the structure of amylopectin at level of chain length distribution.
Therefore, amylopectin from different genotypes did not have much variation which has
also been reported by various authors (Macgregor and Morgan, 1984; Tester and
Morrison, 1990; Czuchajowscka et al., 1998; Schulman et al., 1995; Takeda et al., 1999;
Yoshimoto et al., 2000; Yoshimoto et al., 2002). Three different methods have been used
to measure amylose content and it was revealed that three methods were in agreement
(Appendices 2 and 3). This correlation may suggest that amylopectin from different
barley genotypes with varying amylose may be similar because differences in these
methods depend upon variations within amylopectin structure.
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By observing different physical properties of seed and starch it is suggested that
variation in granule size was more consistent in all increased amylose genotypes and this
character could be used as to identify high amylose genotypes.
2.5 Conclusion
The present study was focused on identifying genotypes high in RS/amylose and
their potential use in human diet. This study shows that in high amylose/RS genotypes,
starch exhibits a unimodal granule size distribution. Distinct division of granules into A-,
B- and C-, based on size is not possible because of decreased granule size, increased
amount of intermediate granules with altered granule morphology. Percentage RS is
positively related to the proportion of small granules. Amylose content and starch granule
size related strongly with some seed dimensions and physiochemical properties. In
general, production of starch with desired characteristics such as RS leads to reduction in
starch content and yield (Asare et al., 2011). The available high RS barley mutants have
reduced starch contents with increased amounts of fibre, lipids and phosphate (Morell et
al., 2003; Boren et al., 2008; Regina et al., 2010). Genotypes 081011-928, 081011-929
and 081011-932 are potentially useful for further breeding programs to develop
genotypes high not only in RS but also having increased seed size, starch content and
yield equal to or in some cases higher than normal or wild-type.
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Chapter 3 is prepared as a research article to be submitted for publication
- 105 -
Chapter 3: Post-translational modification, protein-protein
interactions among enzymes of starch biosynthesis and their
affect on physical properties of starch in high-amylose barley
genotypes
- 106 -
3.1 Introduction
Starch accumulates in cereal endosperm as an energy reserve for the next
generation and is used globally as a human food, livestock feed as well as numerous
important industrial applications including biofuels. Starch is found in higher plants,
mosses, ferns and some microorganisms (Keeling and Myers, 2010) and is an insoluble
polyglucan composed of two polymers of glucose, amylose and amylopectin. Amylose is,
essentially, a linear molecule with a molecular weight varying between (105-106 Da), in
which glucose residues are joined via α-1, 4 linkages with very few α-1, 6 linkages, and
typically constitutes upto 20-30 % of total starch. Amylopectin has a molecular weight of
(107-109 Da) constitutes 70-80 % of total starch, containing linear chains of various
degree of polymerization. Almost 5 % of the glucose units in amylopectin are joined by α
-1, 6 linkages, which introduce branches in the amylopectin in a non-random fashion.
Starch exists in the form of structurally well organized granules in which amylopectin
exhibits non-random distribution of linear chains and a clustered arrangement of branch
linkages which gives rise to a high degree of structural organization. This conserved
architecture of amylopectin is responsible for the semi-crystalline water insoluble starch
granule (James et al., 2003). Amylopectin is required for normal size and shape of the
granules, whereas granules with varying low, or no, amylose retain the same shape and
size as granules with normal amylose content (Keeling and Myers, 2010). In Festucoides,
such as wheat, oats and barley, granules can be divided into B (1-15 µm, round) and Agranules (> 15 µm, lenticular) (Evers, 1973; Bechtel et al., 1990; Bechtel and Wilson,
2003; Wilson et al., 2006), synthesis of which is developmentally regulated (Langeveld et
al., 2000; Bechtel and Wilson, 2003). The amylose: amylopectin ratio, and the size and
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shape of granules, are important parameters which impact the end use of starch (Wei et
al., 2010).
The organization of the starch granule is a complex process involving several
classes of enzyme, each with isoforms, which include adenosine diphosphate glucose
pyrophosphorylase (AGPase), granule bound starch synthase (GBSSI and GBSSII),
soluble starch synthases (SSI, SSIIa, SSIII, SSIV), starch branching enzymes (SBEI,
SEBEIIa and SBEIIb), starch debranching enzymes (DBEs) such as isoamylases (ISAI,
ISAII and ISA III), pullulanase (PU), and starch phoporylase (SP) (Tsai and Nelson,
1969; James et al. 1995; Ball and Morell, 2003; Patron and Keeling, 2005; Leterrier et al.,
2008). These different enzymes and their isoforms are differentially distributed in the
soluble and starch granule fractions of plastids (Tetlow et al., 2011).
Recent evidence has demonstrated that many enzymes involved in starch
biosynthesis are subjected to post translational modification by protein phosphorylation,
and also interact to form heteromeric protein complexes (Hennen-Bierwagan et al., 2008;
Tetlow et al., 2008; Liu et al., 2009, 2012b). In wheat GBSS, SSI and SSIIa are
phosphorylated in their starch granule bound state (Grimaud et al., 2008; Tetlow et al.,
2008a; Bancel et al., 2010). In wheat amyloplasts phosphorylation of SBEIIb, SSIIa and
SP has also been reported by radioactive labelling of amyloplasts with γ 32P-ATP (Tetlow
et al., 2004).
Co-immunoprecipitation of SBEIIb, SBEI, and SP provided direct evidence of
multi-enzyme complex formation in soluble extracts of wheat endosperm (Tetlow et al.,
2004), dependent upon the phosphorylation of target proteins. Other enzyme complexes,
containing SSI, SSIIa, SSIII, SBEIIa, and/or SBEIIb, in various combinations have also
- 108 -
been reported (Hennen-Bierwagen et al. 2008, Tetlow et al., 2008b). Hennen-Bierwagen
et al. (2009) also reported a novel complex in maize endosperm containing SSIII, SSIIa,
SBEIIa, SBEIIb, large and small subunits of AGPase, and pyruvate phosphate dikinase
(PPDK). Liu et al. (2009) reported that, in maize, a null mutant of SBEIIb (ae-) contained
a novel protein complex comprising SSI, SSIIa, SBEI, SBEIIa and SP which became
entrapped in the starch granule. Another ae- allele was described which expressed a
catalytically inactive SBEIIb protein. Although this protein was not able to bind glucan
substrate, it was still found as a granule associated protein, as a result of being able to
form a heteromeric protein complex leading to the entrapment of inactive protein within
the starch granule (Liu et al., 2012a). In related work (Liu et al., 2012b) reported that a
point mutation in SSIIa in a su2- mutant of maize, led to loss SSI and SBEIIb, as well as
SSIIa from starch granule. These observations suggest that single gene mutations affect
partitioning of several proteins between the soluble and granule bound fractions of
amyloplasts and impact amylopectin fine structure. They further imply that alteration in
the protein fingerprint of the granule may reflect variations in protein-protein interactions
in the stroma. Such changes in the granule proteome arising from allelic variations also
give rise to variation in granule structure and composition (Liu et al., 2012a). The present
study examines the relationship between the granule proteome and starch properties in
several varieties of barley with defined mutations, and relates this to variation in proteinprotein interactions in the stroma. Variation in the granule proteome of A- and B-granules
was also investigated with a view to understanding the relationship between granule type,
its proteome and mechanisms underpinning granule size and morphology.
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3.2 Materials and Methods
3.2.1 Plant material
The genotypes used in this study included, wild-type, starch branching enzyme
(sbeiia- and sbeiib-) down regulated lines, ssiia- (sex6) mutant, amo1 (ssiii-) mutant and a
waxy (amylose free) mutant lacking GBSS (Table 3.1). The sources of mutant seed were,
sbeiia- and sbeiib- (Regina et al., 2010), sex6 (Morrell et al., 2003), HAG amo1 (Banks et
al., 1971). The seeds for OAC Baxter (reference genotype) and Neopolis (waxy) were
obtained from Plant Agriculture, University of Guelph. The plant material was grown in
the glasshouse at the University of Guelph under conditions previously described for
growing wheat (Tetlow et al., 2008).
3.2.2 Isolation of starch granules
Mature dry barley seeds weighing ≈ (55) g were completely ground to flour with a
Retsch® MM301 homogenizer in liquid nitrogen. 50 g of flour was suspended in 150 ml
buffer containing 100 mM Tricine-KOH, pH 7.8, 1 mM Na2-EDTA, 1mM DTT and 5
mM MgCl2 at 4 oC. The suspension was vortexed (Eppendorf) for 5-10 minutes to make a
uniform suspension and left for 5-10 minutes on ice. The well-mixed suspension was
sieved through six-layers of cheese cloth to remove debris and bran. Buffer was added to
wash traces of starch from the cheese cloth. The sieved milky suspension, 450-500 ml,
containing starch, fine pieces of debris and bran was centrifuged at 16000 g for 15 min.
The supernant was discarded and the pellet resuspended in 100 ml buffer containing 50
mM TRIS-acetate, pH 7.5, 1 mM Na2-EDTA, and 1 mM DTT (wash buffer). The
resuspended pellet was centrifuged at 6000 g for 5 min. This washing step was repeated
5-7 times until a thick yellow layer of debris was left on the top of starch. The yellow
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layer, containing very fine pieces of debris and bran, was completely removed with a
spatula whilst minimizing loss of starch. After removing debris the starch was again
washed and centrifuged twice (see above) in wash buffer. The purified starch was washed
three times, each with 45 ml of 99 % acetone (termed acetone-washed starch) followed
by three washes, each with 45 ml of 2 % (w/v) sodium dodecyl sulphate (SDS) in water,
to remove proteins bound to the surface of starch granule. This was followed by 3
washings with 45 ml distilled water and the starch finally dried under vacuum
(Eppendorf, vacufugeTM) at 25 oC for 3 h.
3.2.3 Separation of A- and B-type starch granules
A- and B- starch granules were separated based on a method previously described
by Peng et al. (1999). Approximately 0.5 g of the acetone-washed starch was suspended
in 5 ml of dH2O. This starch suspension was then carefully laid on top of 10 ml of 70 %
(v/v) Percoll in dH2O in a 15 ml tube, followed by centrifugation at 10 g for 10 min at
room temperature. The larger A-granules were centrifuged through the Percoll pad and
precipitated at the bottom of the tubes, whereas the smaller B- granules remain in
suspension. After centrifugation, the supernatant (containing B- granules) was carefully
removed. The pellet containing A-granules was washed twice with dH20, by resuspension
and centrifugation at 4000 g for 5 min. The resulting pellet was resuspended in 5 ml dH20
and laid on top of 10 ml of 70 % (v/v) Percoll. Centrifugation through Percoll with
subsequent washing in dH2O was repeated 3 times. A-granules were then centrifuged 3
times through 100 % Percoll for 10 min at 10 g. All supernants from each Percoll
centrifugation step were pooled and centrifuged at 4000 g for 5 min. The resulting pellet
- 111 -
was washed twice with dH20, resuspended and centrifuged at 4000 g for 5min, and
comprised B- granules.
3.2.4 Extraction of amyloplasts and preparation of endosperm whole cell extracts
Barley endosperm amyloplasts were isolated as described by Tetlow et al.
(2008b). Endosperm whole cell extracts were prepared as described previously (Tetlow et
al., 2003).
3.2.5 Isolation of starch granule-bound proteins
The isolation of protein bound within the internal matrix of the starch granules (as
opposed to proteins present on the surface of starch granules, which can be removed by
extensive washing with SDS) was carried out as previously described by (Tetlow et al.,
2004 and Liu et al., 2009). The acetone and SDS-washed starch, either from mature seed,
fresh amyloplast lysate, or whole cell extract, was washed twice with 1 % (w/v) SDS, to
remove any traces of proteins attached to the surface of starch granules. To extract the
starch granule-bound proteins, equivalent amounts of starch (50mg) were boiled in 1 ml
SDS loading buffer containing 62.5 mM TRIS-HCl, pH 6.8, 2 % (w/v) SDS, 10 % (w/v)
glycerol, 5 % (v/v) β-mercaptoethanol and 0.001 % (w/v) bromophenol blue. Boiled
samples were centrifuged at 13,000 g for 10 min and the supernatant used to determine
total granule bound protein content (see below), and for SDS–PAGE. To determine the
equivalence of starch, 20 mg of starch was washed twice with 1% SDS, and 5 times with
50 mM CH3COONa, pH 4.8. Following washing, the starch was boiled in 250 µl sodium
acetate (pH 4.8) buffer at 95 oC for 6-7 min, and gelatinized starch was left at room
temperature to cool. 25 µl (containing 15 U) of amyloglucosidase (AMG) and α-amylases
(Sigma-Aldrich) prepared in acetate buffer was added to the gelatinized starch and left at
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37 oC overnight. Following digestion the sample was centrifuged at 13,000 g for 15 min.
Glucose was assayed using hexokinase and glucose-6-phosphate dehydrogenase coupled
to NADPH production measured at 340 nm (Deeg et al., 1980).
3.2.6 SDS-PAGE and immunoblotting of total starch, A and B granules
Equivalent amounts of starch, A- and B- granules (see above) were used to extract
protein as described earlier. For SDS–PAGE and immunodetection, either SDS gels with
10 % (w/v) acrylamide or pre-cast NUPAGE Novex 4–12% BISTRIS acrylamide
gradient gels (Invitrogen Canada, catalogue No. NP0335BOX) were used following the
manufacturer’s instructions. Gradient gels were run at room temperature in a MOPSbased running buffer prepared according to the manufacturer’s instructions (Invitrogen).
For
immunodetection,
following
electrophoresis,
gels
were
transblotted onto
nitrocellulose membranes (Pall Life Science), and blocked at room temperature in 1.5 %
(w/v) BSA for 15 min with gentle shaking. Different antibodies to wheat proteins were
used according to specifications described earlier (Li et al., 1999; Rahman et al., 2001;
Morell et al., 2003; Regina et al., 2005; Bresolin et al., 2006; Tetlow et al., 2008). In this
study antibodies to wheat proteins were used because these antibodies cross reacted with
barley target proteins, having a higher degree of amino acid sequence homology.
3.2.7 Co-immunoprecipitation
Co-immunoprecipitation experiments were performed as described by Liu et al.
(2009), with some modification. SSI, SSIIa, SBEIIa, and SBEIIb antibodies were used
for the co-immunoprecipitation experiments with amyloplast lysates (1 ml, 1-1.25 mg.
ml-1 protein) and endosperm whole extract (1 ml, 1.5-2 mg. ml-1 protein) from different
genotypes. The antibody and amyloplast lysate mixture was incubated at room
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temperature on a rotator for ~1 h. Immunoprecipitation was performed by adding 50 µl of
protein A-Sepharose beads (Sigma-Aldrich), pre-made as a 50 % (w/v) slurry with
phosphate-buffered saline (PBS) containing 137 mM NaCl, 10 mM Na2HPO4, 2.7 mM
KCl, 1.8 mM KH2PO4, pH 7.4 at room temperature for 50 min. The protein A-Sepharoseantibody-protein complex was centrifuged at 1000 g for 5 min at 4 oC in a refrigerated
micro-centrifuge. Each supernatant was stored to check the presence of unbound proteins.
Pellets were washed five times (1.4 ml each) with PBS, followed by four times washing
with (HEPES) buffer containing 10 mM HEPES-NaOH, pH 7.5, and 150 mM NaCl.
Washed pellets from all genotypes were boiled in SDS loading buffer and separated by
SDS-PAGE by loading equal amounts on a 10 % gel, followed by immunoblot analysis
with different antibodies. In order to exclude the possibility that different proteins (SSs,
SBEs and SP) co-immunoprecipitated together as a result of binding to the same glucan
chain, plastid lysates and endosperm whole cell extracts used for co-immunoprecipitation
were pre-incubated with glucan-degrading enzymes such as amyloglucosidase (EC
3.2.1.3, Sigma product number A7255, from Rhizopus) and α-amylase (EC 3.2.1.1,
Sigma product number A2643, from porcine pancreas), 5 U each for 20 min at 25 oC.
Following amyloglucosidase/a-amylase digestion of glucans, released glucose was
measured as described previously (Tetlow et al., 1994). To check the phosphorylation
dependent complex formation, amyloplast lysates from different genotypes were prepared
according to protocol described by Liu et al. (2009)
3.2.8 Detection of SS and SBE activity following non-denaturing gel electrophoresis
To locate SS and SBE activities following non-denaturing gel electrophoresis,
zymograms were run as described previously (Liu et al., 2012; Tetlow et al., 2004, 2008)
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with some modifications For each zymogram, protein samples were mixed with a buffer
containing 62.5 mM Tris-HCl, pH 6.8, 10 % (w/v) glycerol, and 0.001 % (w/v)
bromophenol blue. 300 µg protein was loaded into each well of 5 % (w/v)
polyacrylamide gels in (375 mM Tris-HCl, pH 8.8, containing 10 mg of Acarbose
(Prandase, Bayer) the α-amylase inhibitor). Gels for determination of SS activity
contained 0.3 % (w/v) amylopectin (Sigma-Aldrich) as primer in the gel. Following
electrophoresis gels were incubated for 48–72 h in a buffer containing (50 mM
glycylglycine, pH 9.0, 20 mM DTT, 100 mM (NH4)2SO4, 0.5 mg ml-1 BSA, and 4 mM
ADP-Glc (Sigma-Aldrich). To detect SBE activity, the gel contained 0.2 % (w/v)
maltoheptaose (Sigma-M7755) and 1.4 U of rabbit muscle extracted phosphorylase a
(Sigma-Aldrich, catalogue No. P-1261). Following electrophoresis, gels were washed
three times each for 15 min with buffer containing (20 mM MES-NaOH, pH 6.6 and 100
mM Na-citrate) followed by incubation in a buffer containing (20 mM MES-NaOH, pH
6.6, 100 mM Na-citrate, 45 mM Glc-1-P, 2.5 mM AMP, 1 mM Na2-EDTA and 1 mM
DTT) for 2–3 h at 28 oC in a shaking incubator. After incubation gels were washed with
water and were developed with Lugol’s solution and visualized immediately (Liu et al.,
2009).
3.2.9 Detection of SP activity
For detection of SP activity 3.75 % (w/v) polyacrylamide (stacking gel) and 12.5
% (w/v) polyacrylamide (resolving gel) were used. The non-denaturing gels were
prepared according to the protocol given above, except the resolving gel contained 0.05
% glycogen. Following electrophoresis, gels were incubated in buffer containing 100 mM
Na-citrate, pH 6.5, 0.1 % glycogen, 20 mM glucose-1-phosphate (sigma, G-7000), 2.5
- 115 -
mM AMP and 1 mM DTT. After incubation the activity bands were visualized by
Lugol’s solution.
3.2.10 Sliver staining
Silver staining was performed as described (Mortz et al., 2001) with some
modification. After electrophoresis, the SDS gel was kept in well-washed glass plates in
50 ml fixing solution (50 % Methanol [v/v], 5 % Acetic acid [v/v]) for 20 min on a
shaker, followed by washing buffer containing 50 % Methanol [v/v] for 10 min on a
shaker. The gel was then kept in distilled water at least for 1 h or overnight with
occasional changes of water. Sensitizing buffer (0.02 % Na2S2O3 [w/v]) was added for
1min, followed by two washes in distilled water for 1 min each. The gel was incubated in
ice-cold silver nitrate buffer (0.1 % AgNO3 [w/v]) for 20 min at 4 oC followed by two
washings with distilled water, each 1 min. The gel was immersed in developing solution
(2 % Na2CO3 [w/v], 0.04 % formalin [v/v]) for 3-5 min and then fresh developing buffer
(given above) was added, and the gel stained until the proteins bands were visualized.
Staining was stopped by adding 5 % acetic acid [v/v] for 5 min, and the gel transferred to
distilled water.
3.2.11 Pro-Q Diamond phospho-protein staining
Equal amounts of starch were loaded onto 4–12 % acrylamide gradient gels and
run according to the manufacturer’s instructions. Following electrophoresis, gels were
fixed in 50 % methanol, 10 % acetic acid overnight in dark. The gels were then washed 3
times with ddH20 and then incubated in Pro-Q Diamond stain according to the
manufacturer’s instructions (Molecular Probes) in the dark for 90 min. Gels were then
incubated 3 times each, for 30 min, in destaining solution containing 20 % acetonitrile,
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50 mM sodium acetate, pH 4.0 and washed with water 2 times each 5-7 min. Proteins
were visualised using a Typhoon scanner (Amersham Biosciences) with an excitation
wavelength of 530 nm and emission filter at 580 nm. A phospho-protein marker was used
as standard.
3.2.12 Starch gelatinization
Thermal analyses were performed as described by (Liu et al., 2007) using a
differential scanning calorimeter (2920 Modulated DSC, TA Instruments, New Castle,
DE). For gelatinization and retrogradation of starches, this system was equipped with a
refrigerated cooling system (RCS). Samples of starch granules were weighed into high
volume pans. A micropipette was used to add distilled water to make suspensions with 70
% moisture content. Approximately 20 mg of starch sample was used. Sealed pans were
equilibrated overnight at room temperature before heating in the DSC. Measurements
were taken at a heating rate of 10 °C/min from 5 to 180 °C. Calibration of the instrument
was done using indium and an empty pan as reference. To measure the enthalpy (ΔH) of
phase transitions from the endotherm of DSC, thermograms based on the mass of dry
solid, software (Universal Analysis, v.2.6D, TA Instruments) were used. Peak
temperature (Tp) of endotherms was also measured from DSC thermograms. For
retrogradation, the heated starch from the above procedure was cooled to 5 °C. Once the
temperature has reached 5°C, the sample was immediately stored at 5 °C. After two
weeks, stored samples were heated from 5 to 180 °C at 10 °C/min and, based on dry solid
mass, the enthalpy (ΔH) and peak temperature (Tp) of the endotherm were measured
from DSC thermograms.
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3.2.13 Estimation of amylose and RS content
Amylose and RS content of different genotypes have been measured with gel
permeation chromatography (GPC) as described (Chen and Christine, 2007) and
Megazyme RS assay kit according to manufacturer instructions with the procedure earlier
described (Goni et al., 1996). For each measurement three biological repeats were used.
3.2.14 Protein content
Protein content of amyloplasts lysate or whole cell extracts was determined using
the Bio-Rad protein assay (Bio-Rad Laboratories, Canada) according to the
manufacturer’s instructions and using BSA as a standard.
3.2.15 Mass spectrometry
In-gel digestion with trypsin and peptides for MS was performed according to the
protocol described by Tetlow et al. (2008). Using a hybrid Q-TOF spectrometer
(Micromass), interfaced to a Micromass CapLC capillary chromatograph, tandem
electrospray mass spectra were recorded previously described by (Tetlow et al., 2004).
3.2.16 Statistical analysis
For one way analysis of variance (ANOVA) and least significant difference
(LSD), Statsoft’s Software, Statistica (http://www.statsoft.com) was used. For principal
component analysis (PCA), XLSTAT (http://www.xlstat.com/en/) was used and biplots
were generated.
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3.3 Results
3.3.1 Physiochemical properties of mutant starches in barley
3.3.1.1 Starch, amylose and RS content
All the mutants used in this study had lower starch content than the wild-type
genotype (Table 3.1). Neopolis is a waxy (amylose free) genotype. All the other mutants
in this study possess increased amylose and RS content compared to wild-type (Table
3.1).
3.3.1.2 Starch gelatinization
Gelatinization of starch is an important parameter which contributes significantly
in determining different uses of starch and is an important diagnostic of alterations in
starch structure/architecture (Bhattacharyya et al., 2004). Gelatinization of starch from
different high RS genotypes was measured by DSC, demonstrated by two endothermic
peaks. Table 3.2 shows differences among genotypes with respect to onset (To)
gelatinization temperature. The highest onset gelatinization temperature (59.7 oC) is
shown by the waxy genotype, while the reference barley genotype (OAC Baxter)
demonstrated the lowest (55.8 oC) onset temperature. For (To) all mutants with altered
amylose content, exhibited values between waxy and normal barley. In the case of peak
temperature (Tp), genotypes also exhibited significant differences. The waxy genotype
exhibited the highest value (67.8 oC) of (Tp) while the reference genotype the lowest
(62.4 oC) Tp. For completion temperature (Tc), all genotypes presented significant
variation compared to reference genotype. In contrast to (To and Tp) the highest Tc (78.9
o
C) was shown by the high amylose barley genotype, HAG amo1, whilst the reference
genotype exhibited the lowest value (73.4 oC, Tc). Variations were also observed
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Table 3.7: Physiochemical properties of starch
Genotype
Predicted %
%
mutation amylose RS
OAC Baxter
Normal 24.3
1.9
Neopolis
Waxy
2.0
1.3
sbeiiasbeiia30.7
7.3
sbeiib
sbeiib
31
12.6
sex6
ssiia
HAG amo1
ssiii
35.6
5.9
Starch
content (%)
100a ±1.20
92b±1.20
94ab±1.95
83c±2.00
95ab ±1.10
Mutations in genes involved in starch/amylopectin biosynthesis (SBEIIa, SBEIIb, SSIIa and
SSIII) are as indicated. OAC Baxter was used as a reference genotype. Neopolis is a waxy
genotype lacking GBSS. % amylose and % RS contents were measured by GPC and
Megazyme RS kit respectively. Starch content (means of three replications, ± indicates
standard deviation). Starch content of reference genotype OAC Baxter was taken as 100
% and other mutants were compared to reference genotype. Alphabetical letters in
each starch content column represent existence of significant differences at (p < 0.05).
- 120 -
regarding transition enthalpy (∆H) for different genotypes. The waxy barley showed
maximum ∆H while high amylose genotypes exhibited significantly lower ∆H compared
to normal barley except sbeiia- which did not show a significant difference compared to
the reference genotype. In the second phase of gelatinization where disruption of
amylose–lipid complex occurs, the sbeiia- genotype exhibited the highest onset
temperature (96.3 oC) To. However, different genotypes with variation in amylose
content did not show significant differences. A similar trend was observed for Tp among
the different genotypes investigated. For Tc and ∆H, HAG amo1 exhibited the highest
values, while sbeiib- exhibited lowest (Tc) values, and normal barley exhibited the lowest
∆H respectively. The waxy genotype did not show any value for different phases of
disruption of the amylose–lipid complex, indicating that waxy starch has a very low
amount of amylose-lipid complex (Yoshimoto et al., 2002). In the second phase of
gelatinization of retrograded starch no significant variations were observed for various
transition temperatures between different genotypes.
3.3.2 Biochemical characterization of different mutants
3.3.2.1 Amyloplast stromal proteins
Amyloplasts from developing barley endosperm from the various genotypes were
isolated (18-25 DAP). Amyloplast lysates were used to detect the presence of different
proteins involved in starch biosynthesis in the amyloplast stroma using various peptidespecific antibodies. Immunological analysis of amyloplast lysates from different mutants
showed that some did not express the protein responsible for the mutation. The ∆sbeiiaexhibited reduced expression of SBEIIa protein (Figure 3.1). In the ∆sbeiib- a small
amount of expressed SBEIIb protein was also detectable. Similarly,
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Table 3.8: DSC data of different genotypes
Initial Heating Summary
Genotype
OAC-Baxter
Neopolis
sbeiiasbeiibHAG amo1
Starch Gelatinization
To
Tp
Tc
∆H
(Co)
(Co)
(Co)
(J/g)
55.8e 62.4d 73.4e 10.8b
59.7a 67.8a 77.3b 13.5a
57.9b 65.3c 76.9c 11.3b
56.7d 65.3a 75.9d 8.7c
57.4c 66.4b 78.9a 8.9c
Melting of amylose-lipid complex
To
Tp
Tc
(Co)
(Co)
(Co)
∆H(J/g)
95.9ab 103.7ab 110.3c
1.1c
96.3a 104.0a
110.5b
1.5b
ab
b
d
95.8
103.3
110.2
1.6b
b
b
a
103.2
95.4
110.6
1.9a
Re-Heating Summary
Genotype
OAC-Baxter
Neopolis
sbeiiasbeiibHAG amo1
Melting of retrograded starch
To
Tp
Tc
∆H
(Co)
(Co)
(Co)
(J/g)
39.0b 52.5a 66.7a 3.1a
40.3ab 53.9a 66.8a 3.5a
41.2a 53.8a 65.5ab 3.4a
41.4a 53.4a 63.6b 3.9a
melting of amylose-lipid complex
To
Tp
Tc
(Co)
(Co)
(Co)
∆H(J/g)
94.6b 104.0b
110.1a
1.1c
97.5a 104.9a
109.9a
1.2c
c
b
a
92.4
103.9
110.0
1.3b
c
c
a
91.5
103.6
110.0
1.5a
Thermal analyses of starch from different genotypes were performed as described by
(Liu et al., 2007) using a differential scanning calorimeter (2920 Modulated DSC, TA
Instruments, New Castle, DE). In this analysis Neopolis was used as waxy genotype.
Alphabetical letters in each column represent significant differences among genotypes
for a given character at (p<0.05).
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immunological analysis revealed that the sex6 mutant lacks SSIIa protein in the soluble
fraction (Figure 3.1). The amo1 (ssiii) mutant (HAG amo1) exhibited similar amount of
SSIII protein to that of the reference genotype. The amo1 mutant (HAG amo1) does not
lack SSIII protein. However, molecular analysis of this mutant indicates the mutant ssiii
has a leucine to arginine residue substitution in a conserved domain compared to the
wild-type protein (Li et al., 2011) resulting in a reduction of SSIII activity compared to
wild-type. Although immunoblotting is not quantitative, it can be used to provide a
qualitative impression of the amount of different proteins among different genotypes.
Immunological characterization of amyloplast lysates revealed that all other proteins
(SSI, SSIIa, SSIII, SSIV, SBEI, SBEIIa, SBEIIb, ISO1, AGPase large subunit, AGPase
small subunit) other than known mutations were present in comparable amounts to the
reference genotype (Figure 3.1). Based on immunological detection of SDS-denatured
proteins variation was found in the content of different proteins in the soluble fraction of
certain genotypes. For example, the sex6 mutant had comparatively more SSI protein
than the reference genotype (Figure 3.1). The branching enzyme mutants, sbeiia- and
sbeiib- appeared to possess less soluble SP compared to the reference genotype.
3.3.2.2 Detection and estimation of SS activity
SS activity was visualized on zymograms and compared with the reference
genotype. Among all soluble SSs, SSI appears to exhibit highest detectable activity on
zymograms (Figure 3.2A). The identity of different SS’s was determined by
immunoblotting similar zymogram gels with SS isoform specific antibodies (Figure
3.2B-E). Significant variations were not observed in other SS activities which were far
less pronounced than SSI. It is noticeable that in branching enzyme mutants (sbeiia- and
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Figure 3.1: Immunological characterization of endosperm amyloplast lysates from
different barley mutants.
Amyloplast lysates (~1.3 mg protein/ml) were prepared from developing barley
endosperms at 18–25 DAP. Aliquots of soluble (stromal) proteins were separated on 10
% polyacrylamide gels and electroblotted onto nitrocellulose membranes. Immunoblots
were developed with peptide-specific anti-wheat antibodies as described in Methods.
Left hand side indicate cross-reactions of each of the antibodies with its corresponding
target protein, and the name of each genotype is given. The approximate molecular
mass for each protein, based on its SDS–PAGE migration and predicted mass, is given on
right hand side.
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Figure 3.2. Detection of SS activity.
Non-denaturing electrophoresis was performed in 5% (w/v) polyacrylamide gels
containing 0.3% (w/v) amylopectin. Approximately 300 µg protein from whole cell
extracts of developing endosperm was loaded onto each lane. Following
electrophoresis, gels were incubated for 48–72 h at 30 oC as described (Methods).
Activity of SS was visualized by staining gels with I2–KI (A). The identity of SSI was
determined by coupling non-denaturing PAGE with immunoblotting, and protein
detected by using specific antibodies (B). Arrows indicate the activity band
corresponding to SSI protein in zymogram (2A) and presence of SSI in immunoblot
incubated with SSI antibody (2B). C, D and E represent immunoblots incubated with
SSIIa, SSIII and SSIV antibodies respectively.
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sbeiib-), the activity of the major SS band (SSI) is significantly reduced (absent)
compared to the reference genotype (Figure 3.2A) even though the proteins is readily
detected (Figure 3.2B).
The sex6 mutant exhibited higher SSI activity compared to the reference genotype
(Figure 3.2A), and is associated with an increase in the amount of detectable soluble SSI
protein (Figure 3.2B).
3.3.2.3 Detection and estimation of SBE activity
SBE activity was detected by zymogram analysis (Figure 3.3A) and isoforms
identified by using peptide-specific antibodies (Figure 3.3B-D). SBE mutants did not
show clear changes in SBE activity compared to reference genotype. However, an
activity band of SBE corresponding to the loss of SBEIIb protein is clearly lost in the
sbeiib- mutant (Figure 3.3A). In the sex6 mutant an activity band corresponding to SBEIc
was significantly increased compared to wild-type (Figure 3.3A). The putative SBEI
activity was only visible when higher concentration of (phosphorylase a) was used and
gels were incubated in Lugol’s solution > 15 min (data not shown). On the zymogram,
activity of SP (Pho1) can also be seen as a blue band, and the results suggest that activity
of soluble SP has been significantly reduced (absent) in the sbeiib- mutant (Figure 3.3A).
3.3.2.4 Detection and estimation of starch phosphorylase (SP) activity
Given the previous observation with the sbeiib- mutant (Figure 3.3A), the effects
on SP activity were studied directly. In sbeiib, negligible Pho1 activity was detected on
zymograms (Figure 3.4A). However, Pho1 protein is clearly present in the soluble
fraction of (sbeiib-) amyloplasts (Figure 3.4B). By contrast, activity and protein content
of Pho1 remained unaffected in the sbeiia- mutant (Figure 3.4A). Thus mutation in either
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Figure 3.3: Detection of SBE activity and protein.
Non-denaturing electrophoresis was performed in 5% (w/v) polyacrylamide gels
containing 0.2% (w/v) maltoheptaose and 1.4 U of rabbit muscle extracted
phosphorylase a. Approximately 300 µg protein from whole cell extracts was loaded
onto each lane. Following electrophoresis, gels were incubated for 3 - 3.5 h at 28oC.
Activity of SBE was visualized by staining gels with I2-IK. Activity of SP is detected as dark
blue bands. Migration of SBE isoforms was determined by immunoblotting with specific
antibodies (3B, SBEIIa; 3C, SBEIIb; 3D, SBEI and SBEIc). Name of each genotype shown.
Arrows indicate specific activity band in zymogram and presence of respective proteins
in immunoblots incubated with different antibodies.
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Figure 3.4: Detection of SP protein and activity in different genotypes.
(A) Activity of SP (Pho1) is visualized as a dark blue band. (B) Immunodetection of SP
(Pho1 and Pho2). (C) Identification of PHO1 in amyloplasts extract. Non-denaturing
electrophoresis was performed in 3.75 % (w/v) polyacrylamide (stacking gel) and 12.5 %
(w/v) polyacrylamide (resolving gel containing 0.05 % glycogen). Approximately 300 µg
protein from whole cell or amyloplasts extracts of developing endosperm was loaded
onto each lane. Following electrophoresis, gels were incubated according to methods
and the activity bands were visualized by Lugol’s solution.
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SBEIIa or SBEIIb gives rise to different pleiotropic effects on SP activity. In HAG amo1
and sex6, activity of Pho1 was readily detected and comparable to the reference genotype
OAC-Baxter (Figure 3.4A). Interestingly, in HAG amo1 and sex6 mutants Pho1 protein
could not be detected in immunoblots of non-denaturing gels (Figure 3.4B), even though
it was readily detected following denaturation and SDS-PAGE (Figure 3.1). Cytosolic SP
(Pho2) is found in small amounts in barley endosperm (Figure 3.4B), though activity is
barely detected in any genotype under the conditions employed (Figure 3.4A).
Confirmation of the identity of plastidial SP (Pho1) was determined in amyloplast lysates
in which activity of Pho1only was observed (Figure 3.4C).
3.3.3 Protein-protein interactions among different proteins of starch biosynthesis
Co-immunoprecipitation was used to determine physical interactions among
enzymes involved in starch biosynthesis in the different barley mutants studied here,
using antibodies to SSI, SSIIa, SBEIIa and SBEIIb.
Immunoprecipitation of SSI in OAC Baxter (reference genotype) was
accompanied by co-precipitation of SSIIa, SBEIIb and small amount of SBEIIa (Figure
3.5A). A similar pattern of co-immunoprecipitation was observed in the branching
enzyme mutant sbeiia-. However, in the sbeiib- mutant, SBEIIa more clearly coimmunoprecipitated with SSI and SSIIa. In the amo1 mutant (HAG amo1) the SBEIIa
and SBEIIb could both be co-precipitated with SSI and SSIIa. However, in the sex6
mutant, the SSI antibody did not result in co-immunoprecipitation of the other proteins
(Figure 3.5A). Other proteins SSIII, SSIV, SBEI, SBEIc, Isoamylases and SP were not
immunoprecipitated by anti-SSI in any genotypes studied.
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Protein complexes from different genotypes were also co-immunoprecipitated
with SBEIIa antibodies. In the reference genotype, OAC Baxter, SSI, SSIIa and SBEIIb
co-precipitated with SBEIIa antibody (Figure 3.5B). In the sbeiia- mutant, not
surprisingly, the SBEIIa antibody did not co-precipitate other proteins. In the sbeiibmutant, SBEIIa antibody precipitated SSI, SSIIa, SBEI, SP and SBEIIa (Figure 3.5B). In
the amo1 mutant (HAG amo1) the co-immunoprecipitation pattern of SBEIIa was similar
to that seen with anti-SSI in this mutant. In the sex6 mutant, the SBEIIa antibody did not
result in co-immunoprecipitation of other proteins, unlike the situation observed in other
genotypes (Figure 3.5B).
The co-immunoprecipitation study was further extended by using an antibody to
SBEIIb. In the reference genotype SSI, SSIIa and SBEIIa were co-precipitated by antiSBEIIb (Figure 3.5C). In the sbeiia- mutant, SBEIIb antibody precipitated SSI, SSIIa,
SBEI, SP and SBEIIb. Consistent with the reduced expression of SBEIIb protein, in
sbeiib- no other proteins were co-precipitated by SBEIIb antibody. In HAG amo1 a
similar pattern of co-immunoprecipitation was observed to that seen with SSI and SBEIIa
antibodies. In the sex6- mutant, the SBEIIb antibody did not co-precipitate other proteins
(Figure 3.5C). Other proteins SSIII, SSIV and Isoamylases were not found as a part of
immunoprecipitated protein complexes.
3.3.3.1 Phosphorylation dependent protein-protein interactions
Previously it was reported that the formation of protein complexes is dependent
upon phosphorylation of different components of a protein complex (Tetlow et al., 2004;
Tetlow et al., 2008; Hennen-Bierwagen et al., 2009; Liu et al., 2009). To study similar
phenomenon in barley, ATP and alkaline phosphatase (APase) treatments were employed
- 130 -
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Figure 3.5: Co-immunoprecipitation of stromal proteins from amyloplasts of different
barley genotypes with SSI, SBEIIa and SBEIIb antibodies.
Aliquots (0.75-1 ml) of amyloplast lysates (0.8–1.3 mg protein ml/ml) prepared from
endosperms of different genotypes at 18–25 DAP were incubated with specific anti-SSI
(5A), anti-SBEIIa (5B) and anti-SBEIIb (5C) antibodies at 25 oC for 1h, and then
immunoprecipitated with protein A–Sepharose beads. The protein A–Sepharose–
antibody–antigen complexes were washed several times to remove non-specifically
bound proteins, boiled in 200 µl of SDS loading buffer, and 25 µl was loaded in each lane
of 10 % polyacrylamide gels. Following electrophoresis, gels were electroblotted onto
nitrocellulose, and developed with various anti-barley antibodies as indicated. D),
Phosphorylation dependent protein complex formation in the reference genotype of
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barley. Amyloplast lysates were pre-treated with 1 mM ATP (for phosphorylation) or
APase (for dephosphorylation) and co-immunoprecipitation performed as indicated. E),
Amyloplast lysates from sbeiia- and sbeiib- were treated with 1 mM ATP and APase and
co-immunoprecipitation was performed with anti-SSI antibody.
in co-immunoprecipitation experiments similar to those described in the previous section.
In all treatments with ATP or APase the ability of the respective antibody to precipitate
its target protein was not affected (Figure 3.5D). In amyloplast lysates of the reference
genotype, interaction between SSI, SSIIa, SBEIIa and SBEIIb as described earlier with
SSI, SBEIIa and SBEIIb antibodies were enhanced by pre-treatment of lysate with 1 mM
ATP (Figure 3.5D). Similarly these interactions were undetectable following
dephosphorylation with APase (Figure 3.5D). In the reference genotype, SP and SBEI
were not co-precipitated by any of the antibodies used (Figure 3.5) whether treated with
ATP and APase or not. Identical ATP and APase treatments were also used in coimmunoprecipitation experiments with endosperm amyloplast lysates from sbeiia-, sbeiib, sex6 and amo1 mutants. In these mutants, protein-protein interactions were enhanced by
the presence of ATP, but no differences were observed in composition of different
protein complexes based on their ability to be co-immunoprecipitated. Anti-SSI coimmunoprecipitation data for sbeiia- and sbeiib- mutants illustrates no difference in the
pattern of other co-precipitated proteins with ATP or APase treatments compared to the
reference genotype (OAC Baxter) (Figure 3.5E).
3.3.4 Analysis of starch granule-associated proteins
Following extensive washings with buffer, SDS and acetone, granule associated
proteins were extracted (see methods). In barley, starch is composed of two types of
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starch granules large A-type and small B-type granules, which were separated using the
method described of Peng et al. (1999), and proteomic analysis was performed by
immunodetection (Figures 3.6A, B & C). The purity of A- and B- granules was
confirmed by light and electron microscopy. As an illustration the micrograph of the
reference genotype OAC Baxter are presented in the Appendices (15). Granule proteome
analysis revealed that in the reference genotype and in HAG amo1: SSI, SSIIa, SSIII,
SBEIIa and SBEIIb were all present in total starch, and independently in A and B
granules (Figures 3.6A & B). Other than these proteins, an additional polypeptide of
approximately 110 kDa was also observed in all genotypes except sex6 (Figure 3.7A).
This 110 kDa protein was identified as SP in the sbeiia- and sbeiib- mutants. This SP was
also only present in A granules (and therefore total starch) (Figures 3.6A & B). In the
sbeiia- and sbeiib- mutants, along with SP, SBEI was also detected in total starch.
Fractionation of granules shows that SSI, SSIIa, SSIII, and whichever SBEII protein is
expressed, are present in A- and B-granules, whereas SBEI and SP are only present in Agranules (Figures 3.6A, B & C). In all genotypes, SBEIc was only detected in A-granules
(and therefore, also, total starch). In the sex6 mutant, little or no protein was detected in
granules apart from GBSS the latter being present in all genotypes in both types of
granules.
3.3.4.1 Phosphorylation state of the starch granule proteome
Previously it has been shown that protein complex formation is dependent upon
protein-phosphorylation (Tetlow et al., 2004; Liu et al., 2009, 2012b) and some proteins
entrapped with in starch granules are also phophorylated (Grimaud et al., 2008; Liu et al.,
2009, Bancel et al., 2010). To investigate the phosphorylation state of different granule-
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Figure 3.6: Starch, A- and B-granules bound proteins.
Starch granules were isolated from mature barley grains by grinding seeds and
preparing starch granules as described Materials and Methods. The purified, acetone
washed starch was used to separate large A and small B granules with Percoll gradient
centrifugation method from all genotypes. The separated A, B granules and starch were
washed extensively to remove proteins loosely bound to the granule surface. A 50 mg
aliquot of purified starch, A and B granules were boiled in 1 ml of SDS loading buffer,
and 40 µl of the supernatant from the boiled sample loaded onto 4–12 % acrylamide
gradient gels and 10 % acrylamide SDS gels. Following electrophoresis, gels were
electroblotted to nitrocellulose membrane and developed with various specific antiwheat antibodies as indicated (A): SSI, SSIIa, SSIII, SSIV, (B): SBEI, SBEIIa, SBEIIb, (C): SP,
ISO1 and GBSS. Each Figure is labelled with respective antibody and type of granules
used either starch, A- or B-.
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bound proteins, Pro-Q Diamond staining was used. GBSS is the most abundant protein in
the starch granule and this protein showed very strong staining for protein
phosphorylation in all genotypes including, surprisingly, the waxy genotype (Figure
3.7B) which has significantly reduced amount of GBSS (Figure 3.7A). In barley, granule
bound SSIIa, SBEIIa and SBEIIb migrate similarly on SDS gels (Figure 3.7A) and all
appear to be phosphorylated (Figure 3.7B) which makes it difficult to identify individual
phosphorylated proteins. However, study of branching enzyme mutants (sbeiia- and
sbeiib) showed that SSIIa and SBEIIb are phosphorylated in their granule bound states.
Similarly SSI also appeared to be phosphorylated (Figure 3.7B). In the branching enzyme
mutants sbeiia- and sbeiib-, SP is also granule localized and phosphorylated (Figure
3.7B). Other proteins SSIII and SBEIc were entrapped in the granule (Figure 3.7A) but
did not appear to be phosphorylated. Physiochemical and biochemical data of remaining
amo1 and sex1 mutants are presented in Appendices.
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Figure 3.7: Starch granule bound proteome phospho-proteome.
To determine granule bound phospho-proteome equal amounts of starch from all
genotypes were run onto 4–12% acrylamide gradient gels, and stained with Pro-Q
Diamond. Following Pro-Q Diamond staining the same gel (B) was subject to silver
staining (A) to visualize all bands. The bands visualized with silver stain are numbered as
(1, SSIII; 2, SBEIc; 3, SP/unknown; 4, 5, 6, SBEIIb; SSIIa; SBEIIa; SBEI 7, SSI and 8, GBSS).
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3.4 Discussion
This study presents a detailed biochemical characterization of different barley
genotypes possessing mutations in isoforms of starch synthesizing enzymes resulting in
high-amylose starches and compared to a reference and a waxy genotype. Barley starch is
composed of two types of granules (A- and B-) and the results suggest that association of
different proteins within the growing starch granules is a reflection of the formation of
stromal functional heteromeric protein complexes, involved in amylopectin biosynthesis,
which become granule-localized. Furthermore, as will be discussed, there is evidence that
different protein complexes are partitioned into A and B granules differentially.
All the mutants have increased “apparent amylose” compared to reference
genotype and waxy (Table 3.1). Interestingly genotypes with similar high amylose
content showed differences in RS content, with the sbeiib- mutant exhibiting significantly
higher RS than sbeiia-. In maize lack of SBEIIb leads to longer internal chains length and
less frequently branched outer chains in amylopectin compared to wild-type starches
(Hilbert and MacMasters, 1946; Banks et al., 1974; Klucinec and Thompson, 2002),
giving rise to starches which are characterized as “high amylose” even though the
modification is in amylopectin. In barley, elimination of either SBEIIa or SBEIIb does
not result in a significant alteration in the number of branches in the amylopectin
molecules nor in higher proportions of longer glucan chains (Ahmed et al., 2013,
manuscript in preparation). Interestingly, however, the branch frequency of amylose was
found to be increased in sbeiib- (Regina et al., 2010). The apparent reason for these
differences between cereals may be the expression pattern of SBE isoforms which are
different in barley endosperm than other cereals like maize and wheat. In maize,
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expression of SBEIIb has been estimated as 50 times higher than SBEIIa (Gao et al.,
1997). In contrast SBEIIb is present at much lower levels than SBEIIa in wheat
endosperm (Gao et al., 1997; Morell et al., 1997; Regina et al., 2005). However, in barley
endosperm both proteins are expressed at approximately equal levels (Sun et al., 1998).
Therefore it is suggested that in barley loss of either of SBEII isoform is, to some degree,
compensated by the expressed isoform. Yao et al. (2004) and Regina et al. (2010)
suggested an inhibitory role of SBEIIb upon SBEI, and in the absence of SBEIIb, SBEI
potentially adds more branches onto amylose. It is possible that this branched amylose
with longer glucan chains is more resistant to α-amylase digestion and consequently
sbeiib- exhibits increased RS content compared to sbeiia-, as observed in the present
study. In the sbeiia- mutant, amylopastidic SP (Pho1) has normal activity while in sbeiibits activity is undetectable. These variations did not affect starch granule phenotype
drastically, although the starch content of sbeiib- was significantly reduced compared
with the sbeiia- mutant (Table 3.1).
Our results indicate that the reference genotype has lower (To) and higher (∆H)
than high amylose genotypes, with the exception of sbeiia- which has higher (∆H) than
reference genotypes. Previously You and Izydorczyk, (2007) reported that (∆H) values
were associated with the amount of double helical domains of amylopectin and single
helical structure (amylose–lipid complexes). Similarly others reported that (∆H) values
reflect the loss of double helical order rather than loss of X-ray crystallinity (Zheng et al.,
1998; Morrison et al., 1993; Cooke and Gidley, 1992; Li et al., 2001; Yoshimoto et al.,
2000; You and Izydorczyk, 2007). Tester and Morrison, (1990) proposed that (∆H)
provides an estimate of crystallinity, that (Tp) can be used to measure the crystallite
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quality, and that (To) has a significant correlation with average branch chain length (Jane
et al., 1999; Yuan et al., 1993; Wang et al., 1993 and Shi and Seib, 1992). The apparent
loss of soluble SSI activity in SBEII mutants, even though the protein is present, may
have contributed to a reduction in the proportion of short chains in amylopectin, leading
to a reduction in crystallinity and consequently smaller (∆H) values compared to
reference genotype. In this regard the effect of loss of SBEIIb was more severe than
SBEIIa with respect to (∆H) (Table 3.2). These observations suggest that SBE mutations
in barley are having different effects on starch structure and organization compared to the
reference genotype.
Western blot analysis of whole cell extracts, amyloplast stroma and starch
granules showed that the sex6 mutant lacks SSIIa protein consistent with previous results
(Morrell et al., 2003). Similarly sbeiia- demonstrates reduced expression of SBEIIa
protein while sbeiib- has small amount of expressed SBEIIb protein. It has previously
been reported that HAG amo1 does not lack SSIII protein but has a leucine to arginine
residue substitution in a conserved domain at amino acid 1480 compared to the wild-type
protein (Li et al., 2011). This substitution results in reduction in the activity of SSIII
protein compared to wild-type (Li et al., 2011). The waxy genotype is not a null mutant
and has significantly reduced expression of GBSSI. Mutations in SBEIIa and SBEIIb
appear to result in intriguing pleiotropic effects with respect to the measureable activities
of SP (Pho1) and SSI.
Both sbeiia- and sbeiib- mutants appear to possess less soluble plastidial SP
protein compared to reference genotype (Figure 3.1). Previously (Liu et al., 2009)
observed a decrease in SP activity in soluble extracts of a maize ae- mutant lacking
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SBEIIb and wild-type. In SBEII mutants, activity of SSI was not detected (Figure 3.2A)
even though the SSI protein was present in similar amounts to the reference genotype
(Figure 3.2B). This suggests that SSI activity is in some way dependent on the presence
of both SBEII isozymes, since each genotype is mutated in only one or other isoform. In
the case of branching enzyme activities, the activity of SBEIIb appeared reduced in the
sbeiib- mutant on zymograms, but the effect on SBEIIa activity in the sbeiia- mutant was
less obvious (Figure 3.3). In sbeiib- plants, negligible activity of plastidial SP (Pho1) was
found (Figure 3.4A) even though plastidial SP protein was present (Figure 3.4B).
Whereas, in the sbeiia- mutant activity of SP (Pho1) remained unaltered (Figure 3.4A).
This suggests that the presence of SBEIIb is required for SP (Pho1) activity. In previous
reports of ae- mutants of maize and rice, a decrease in soluble SS activity, particularly
SSI, from whole cell extracts compared to wild-type has been observed (Nishi et al.,
2001; Nakamura et al., 2012). However, in contrast to these observations Liu et al. (2009)
reported increased SS activities from amyloplast stromal proteins of the ae- mutant of
maize compared to wild-type, and a decrease in plastidial SP (Pho1) activity from aemutant. Nishi et al. (2001) observed no change in SP activity in the endosperm of rice aemutants compared to wild-type. In amo1 and sex6 plants, pleiotropic affects on SP are
intriguing since in these mutants SP activity was readily detected on non-denaturing gels
(Figure3. 4A), where as the protein itself was not detectable (Figure 3.4B) even though
the presence of the SP protein was confirmed under denaturing conditions (Figures 3.1 &
3.6). This could arise from conformational changes or perhaps by association with other
proteins leading to the antigenic epitopes being masked under non-denaturing conditions.
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Given the pleiotropic effects observed on enzymes activities, we investigated
whether any differences in protein-protein interactions occurred in the genotypes studied.
Co-immunoprecipitation experiments with different antibodies resulted in purification of
different types of protein complexes. For example, in the reference genotype, a protein
complex containing SSI, SSIIa, and either of SBEIIa or SBEIIb could be precipitated
with SSI, SBEIIa and SBEIIb antibodies (Figure 3.5). It is argued that either SBEIIa or
SBEIIb can interact with SSI and SSIIa, resulting in the formation of two distinct protein
complexes in the reference genotype. In the branching enzyme mutants sbeiia- and sbeiib, co-precipitation of SSI, SSIIa, SBEI and SP was observed when using anti-SBEIIb or
anti-SBEIIa antibodies (Figure 3.5). Importantly it was found that SBEI and SP were also
granule -localized in these two mutants and were present only in A-granules and not in B
granules (Figure 3.6). In these two mutants, a protein complex comprising of SSI, SSIIa
and whichever SBEII is expressed, was purified with anti-SSI antibody. Anti-SSI
antibody did not co-precipitate SBEI and SP (as mentioned earlier) in these mutants,
suggesting that two types of enzyme complexes are formed. One complex comprising
SSI, SSII, and whichever of the SBEII proteins is available is found in A- and Bgranules. A second multi-enzyme complex comprising SBEI and SBEIIb (or IIa) and SP,
similar to that previously reported in wheat (Tetlow et al., 2004), is only found in Agranules. This is distinct from the SSI/SSII/SBEII complex, consistent with the
observation that SSI antibodies do not precipitate SP or SBEI. SP/SBEI containing
complexes became entrapped in the A-granules but not B-granules (Figure 3.6). The
observation that the two types of stromal multi-enzyme complexes described are
differentially partitioned between the two types of starch granules implies that they have
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distinctive roles in the formation of A- and B- granules at least in the branching enzyme
mutants.
In the amo1 mutant, a protein complex containing SSI, SSIIa, SBEIIa and SBEIIb
was co-precipitated with anti-SSI, anti-SBEIIa and anti-SBEIIb antibodies. As antiSBEIIa precipitates SBEIIb protein and vice versa, so it could be argued that may be
SBEIIa and SBEIIb form a dimer which is precipitated with either of the SBEII
antibodies in addition to distinct SSI/SSIIa/SBEIIa or SSI/SSIIa/SBEIIb protein
complexes. There is also a possibility that SBEIIa and SBEIIb form a larger protein
complex with SSI and SSIIa, which was co-precipitated with anti-SSI, anti-SBEIIa and
anti-SBEIIb antibodies. In the sex6 mutant, none of the proteins appear to interact and
none are detected (apart from GBSS) in the granule, or are present only at much lower
amounts (Figures 3.5 & 3.6).
Analysis of granule bound proteins revealed that SSI, SSIIa, SBEIIa, SBEIIb and
GBSSI are granule localized in different barley mutants. In SBEII mutants, it appears the
remaining SBEII isoform which is expressed is found in higher amounts in the granule in
comparison to the reference line possibly indicating its role as a “substitute” for the
missing protein or as a part of complex with SBEI and SP (Figure 3.6). In sbeiib- plants,
more SP appears to be partitioned to the granule than for sbeiia- barley (Figure 3.6C).
This may contribute to loss of detectable soluble SP (Pho1) activity in zymograms of
sbeiib- mutants (Figure 3.4). SBEIc was found only in the A-granules in all genotypes (as
shown previously Peng et al., 2000). SSI, SSIIa, SBEIIa, SBEIIb and GBSSI are also
routinely found within the starch granules of other cereals like maize, wheat, barley and
rice (Rahman et al., 1995; Morell et al., 2003; Boren et al., 2004; Regina et al., 2005;
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Umemoto and Aoki, 2005; Liu et al., 2009, 2012a, 2012b). A small amount of SSIII and
an unknown protein of 110 kDa were also found in the starch granules of wild-type and
the mutants studied here. All of these starch granule associated proteins, which are
involved in starch biosynthesis, are also components of identified soluble protein
complexes in wheat and maize amyloplasts (Tetlow et al., 2008; Hennen-Bierwagen et
al., 2008; Liu et al., 2009, 2012a, 2012b). In the sex6 mutants, due to lack of SSIIa, no
soluble protein complex was detected (Figure 3.5A) and therefore none of the above
mentioned proteins was found in the granule except GBSSI. Thus it is suggested that
SSIIa plays an important role in trafficking other proteins to the granule by forming
protein complexes. Similar results have been reported recently by Liu et al., (2012b) in
the sugary2 (su2) mutant of maize. A summary of different protein complexes and their
association with granules is presented in Figure (3.8).
3.5 Conclusion
This study has provided a detailed biochemical analysis of barley mutants with
mutations in key amylopectin synthesizing enzymes and their relationship to altered
physiochemical properties of starch. In different mutants, amylopectin synthesizing
enzymes form phosphorylation-dependent functional protein complexes in various
combinations in the amyloplast stroma compared to the reference genotype. These
protein complexes become entrapped in the starch granules and are reflected as
alterations in the starch granule proteome. Due to loss of either SBEII isoform SP and
SBEI become part of a heteromeric protein complex. This protein complex containing SP
and SBEI is arguably involved primarily in the synthesis of A-granules, reflected in the
A-granule proteome, and not B-granules. Such alterations may also affect the
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physiochemical properties of starch, since sbeiib- mutants have significantly higher RS
content than sbeiia- even though the apparent amylose content is similar. This could be
due to an inhibitory role of SBEIIb on SBEI. In the amo1 mutant HAG amo1 protein
complex comprising SSI, SSIIa, SBEIIa and SBEIIb was co-precipitated. In this mutant
starch with altered physiochemical properties was observed compared to reference
genotype. Similarly mutation in SSIIa prevented formation of heteromeric protein
complexes which is reflected in the lack of other amylopectin synthesizing enzymes in
the granule proteome of this mutant.
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Figure 3.8: Protein–protein interactions formed between amylopectin-synthesizing
enzymes in barley endosperm.
In the reference genotype (A), the major forms of SBEII (SBEIIa and SBEIIb) form
distinct, phosphorylation-dependent protein complexes with SSI and SSIIa. In the
branching enzyme mutants (B & C), two distinct protein complexes consisting of
SSI/SSIIa and expressed SBEII, and SP/SBEI and expressed SBEII are formed and became
entrapped in granules. SP/SBEI/SBEII is partitioned to A-granules only. Loss of SSIIa leads
to the absence of other amylopectin synthesizing enzymes from the granules (D). In
amo1 mutant (E) protein complex consisted of SSI, SSIIa, SBEIIa and SBEIIb was coprecipitated. It is possible that SBEIIa and SBEIIb form distinct complexes with SSI and
SSIIa as described for reference genotype. It is possible that SBEIIa and SBEIIb form a
dimer which is co-precipitated along with the above mentioned distinct complexes, or
that SBEIIa and SBEIIb form a larger complex with SSI and SSIIa. A- and B- granules are
presented as grey circles. In the sex6 mutant severely deformed A-granules are
presented. Coloured dots present different protein complexes and their distribution in
A- and B- granules. GBSS is present in both A- and B- granules and not represented here.
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Chapter 4: Physiochemical and biochemical properties of
starch and its relationship to granule size distribution in barley
genotypes from diverse genetic back grounds
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4.1 Introduction
The semicrystalline, water-insoluble starch granule is of significant agricultural
and commercial importance. This water-insoluble, osomotically inactive granule confers
advantages to plants for short- and long-term carbon reserves. This stored starch not only
provides energy reserves for the next generation but is also used as food, feed, biofuels
and raw material for many industries. Starch is made up of two glucan polymers: amylose
and amylopectin. Amylose is sparsely branched and contributes only 20-30 % of the total
starch and makes the amorphous portion of the granule while amylopectin is highly
branched and the major constituent (70-80 %) of starch granules forming partially
semicrystalline structures (Hizukuri, 1996; Lemke et al., 2004). In amylopectin, the
distribution of glucan chains and branch point clustering allow short linear chains to pack
together efficiently as parallel, left-handed, double helices. The organized array of
clusters is the basis of the semicrystalline nature of much of the starch granule, resulting
in a water-insoluble granule. The size, shape and number of starch granules vary within
and among species (Shapter et al., 2008).
In cereals, such as wheat and barley, starch granules can be distinguished based
on size and shape as, A-type (15-45 µm, lenticular), B-type (5-15 µm, round) and C-type
(1-5 µm, polygonal). Along with other features, granule morphology has an important
impact on starch physiochemical properties (Da Silva et al., 1997; Lindeboom et al.,
2004) and granule size determines most of the potential food and industrial applications
of starch (Ji et al., 2004). Starch with a greater proportion of small granules is suitable for
use as a fat substitute, paper coating and as a carrier in cosmetics (Lindeboom et al.,
2004). However, starch with predominantly large granules can be used in the
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manufacture of biodegradable plastic films, carbonless copy paper and in other industries
(Lindeboom et al., 2004). Starches extracted from different barley genotypes vary widely
in structure, composition and properties (Kang et al., 1985; Morrison et al., 1993; Lorenz
& Collins, 1995; Song & Jane, 2000; Yoshimoto et al., 2000). Besides variations in
morphology, size, and origin, large and small starch granules in wheat and barley also
show differences in characteristics and properties with regard to chemical composition.
These include amylose content, amylose-lipid complex and phosphorus contents (Raeker
et al., 1998; Shinde et al., 2003; Geera et al., 2006; Ao and Jane, 2007), molecular
structure (Sahlstrom et al., 2003), gelatinization temperature, and retrogradation (Peng et
al., 1999; Singh and Kaur, 2004). Large A-granules of wheat show an increased enthalpy
of gelatinization, lower gelatinization temperatures, increased retrogradation (Peng et al.,
1999; Singh and Kaur, 2004) and softer textured flours compared to smaller B-granules
(Gaines et al., 2000). Wheat starches possessing different granule sizes exhibited
different degrees of susceptibility to enzymatic hydrolysis, as well as thermal and pasting
properties (Morrison & Gadan, 1987; Peng et al., 1999; Liu et al., 2007; Raeker et al.,
2007). Along with granule size, differences in amylose content, protein content, and
branch chain length of amylopectin in A- and B-type starch granules are also major
factors responsible for differences in digestibility and other functional properties of starch
(Liu et al., 2007).
The biosynthesis of starch is controlled by different enzyme classes: adenosine
diphosphate glucose pyrophosphorylase (AGPase), five classes of starch synthases and
two classes of starch branching enzymes. Starch synthases can be distinguished as
granule-bound starch synthase (GBSS) and soluble starch synthases (SSI, SSII, SSIII and
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SSIV). Different SS classes have different isoforms, two GBSS isoforms (GBSSI and
GBSSII), three SSII isoforms (SSIIa, SSIIb and SSIIc) two SSIII isoforms (SSIIIa and
SSIIIb) and two SSIV isoforms (SSIVa and SSIVb) (Hirose and Terao, 2004; Fujita et
al., 2007). Starch branching enzymes (SBEs) in cereals include SBEI, SBEIIa and
SBEIIb. In addition, starch debranching enzymes (DBEs) such as isoamylases (ISOI,
ISOII and ISOIII) and pullulanase (PU), and starch phosphorylase (SP [Pho1 and Pho2])
are also involved in starch metabolism (Ball et al., 1996; Zeeman et al., 1998).
GBSS is primarily involved in amylose biosynthesis, while other classes of
enzymes such as SSs, SBEs, and DBEs are primarily responsible for amylopectin
biosynthesis. The relative amounts of SS and SBE isoforms differ among different organs
of the same plant or among species (Ball and Morell, 2003; Li et al., 2003; Patron and
Keeling, 2005; Leterrier et al., 2008). The integrated activities of these different enzymes
result in semicrystalline water insoluble starch granule. The interaction between starch
synthesizing enzymes is a complex process, partly due to cyclic substrate-product
relationships. For example, SSs produce linear glucan chains which become substrates
for SBEs to create branches in amylopectin, while shorter glucan chains of DP 6-7,
produced during branch formation, become substrates for SSs (Zeeman et al., 2007; Liu
et al., 2009). Similarly the product of SBE activity is also a substrate for DBEs
(Szydlowski et al., 2011). Apart from their interdependence, these proteins are also
subjected to post-translational modifications such as, protein phosphorylation and protein
complex formation (Tetlow et al., 2004, 2008; Liu et al., 2009, 2012a). Previously, it was
suggested that during granule formation these complexes become entrapped as functional
protein complexes (Liu et al., 2009). The above observations illustrate that starch
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biosynthesis is a complex process which is controlled at multiple levels of organization.
Thus, it is important to understand the biochemical basis of starch physiochemical
properties, and the regulation of its biosynthesis in the endosperm, as key steps to
improve and modify starch properties for a wider variety of applications (Stamova et al.,
2009). An understanding of the genetic and biochemical bases of multimodal starch
granule size distribution is of great interest, in crop species like wheat and barley because
each type of granule has varied physiochemical properties and consequently different end
uses (Sahlstrom et al., 1998; Lindeboom et al., 2004; Yonemoto et al., 2007). There are a
number of factors involved in the differentiation of starch granules, including multiple
and complex genetic controls and biochemistry, size and numbers of plastids,
environmental
conditions
during
seed
development,
availability
of
malto-
oligosaccharides and the integrated effect of all these factors (Shapter et al., 2008).
Recently, understanding the synthesis of barley starch has gained more attention
due its ability to replace other starches and because of its physiochemical properties and
potential to be used in different industries (Asare et al., 2011). Although much
information is available regarding differences in physiochemical properties of starches
with varied granule sizes, little is known about the underlying biochemical processes. In
this study, barley genotypes with different proportions of large and small granules,
possessing different physiochemical properties, have been used to examine the
relationship among the starch granule proteome and the physiochemical properties of
starch. As described earlier, from a wide range of genotypes from different genetic back
grounds, amylose/RS contents were measured (Chapter 2). From these genotypes, a
group of high amylose mutants with known mutations in amylopectin synthesizing
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enzymes was selected for detailed biochemical analyses (Chapter 3). The remaining
genotypes with no apparent mutations in amylopectin synthesizing enzymes, but with
variations in starch physiochemical properties were selected in this study for detailed
physiochemical analyses of starch and analysis of granule proteome.
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4.2 Material and Methods
4.2.1 Plant material
Nineteen different barley genotypes from different genetic backgrounds were
employed. Table (4.1) outlines the different characteristics/description of the barley
genotypes. Different barley genotypes were grown at the University of Guelph under
glasshouse conditions in a soil medium containing, Turface soil (Profile Products),
Turface MVP (Profile Products), lime, peat moss, and Nutricote (14-14-14; Morton’s
Horticultural Products) in a ration of 3:1:0.01:1:0.01 (w/w). Plant growth conditions were
maintained at a temperature of 15-25 oC. Barley was also grown at the University of
Guelph fields, Elora, under natural conditions on a clay loam soil. Each year, planting of
barley started in the first week of May.
4.2.2 Isolation of starch granules
Mature dry barley seeds weighing ≈ (55) g were completely ground to flour with a
homogenizer (Retsch® MM301) in liquid nitrogen. Equal amounts of flour (≈ 50 g) were
suspended in 150 ml of rupturing buffer containing 100mM Tricine-KOH, pH 7.8, 1mM
Na2-EDTA, 1mM DTT and 5mM MgCl2 at 4oC. The suspension was vortexed
(Eppendorf, vortex) for 5-10 minutes to make a uniform suspension and left for 5-10
minutes on ice. The well mixed suspension was sieved through six-layers of cheese cloth
to remove debris and bran. Excess buffer was added to wash traces of starch from the
cheese cloth. The sieved milky suspension ≈ 450-500 ml containing starch, fine debris
and bran was centrifuged at 16000 g for 15 min. The supernant was discarded and pellet
was washed in ≈ 100 ml wash buffer containing 50 mM TRIS-acetate, pH 7.5, 1 mM
Na2-EDTA, and 1 mM DTT. The resuspended pellet was centrifuged at 6000 g for 5 min.
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This washing step was repeated 5-7 times until a thick yellow layer of debris was left on
the top of starch. The yellow layer, containing very fine pieces of debris and bran, was
completely removed with a spatula minimizing starch losses. After removing debris, the
starch was again washed and centrifuged twice with washing buffer. Purified starch was
washed three times with acetone, followed by three washes with 2 % (w/v) sodium
dodecyl sulphate (SDS), 3 times with distilled water, and then dried using a speed
vacuum (Eppendorf, vacufugeTM) at 25 oC for 3 h.
4.2.2.1 Separation of A- and B-type starch granules
A- and B-/C-type starch granules were separated using a method previously
described by Peng et al. (1999). Approximately 0.5 g of the dried, acetone washed starch
was suspended in 5 ml of dH20. This starch suspension was then carefully laid on top of
10 ml, 70 % (v/v) Percoll in a 15 ml tube, avoiding precipitation through the underlying
Percoll, followed by centrifugation at 10 g for 10 min at room temperature. The larger Agranules passed through the Percoll gradient and precipitated at the bottom of the tubes
(along with some B-granules). The majority of the smaller B-/C-granules remained in
suspension. After centrifugation, supernatant (containing B-granules) was removed to
fresh tubes. The pellet, containing A- and some of B-granules, was washed (twice) with
dH20 by resuspension and centrifugation at 4000 g for 5 min. After centrifugation the
supernant was discarded and the pellet was resuspended in 5 ml of dH20 and laid on top
of 10 ml of 70 % (v/v) Percoll. This process of A- and B-/C-granule separation (70 %
(v/v) Percoll gradient centrifugation at 10 g for 10 min, and washing of the pellets in
dH20) was repeated for 3 cycles, and supernants from each cycle were pooled. The pellet
containing predominantly A-granules was then further purified by centrifugation through
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100 % (v/v) Percoll as described for 70 % Percoll, to produce a homogeneous A-granule
population in the resultant pellet. Similarly, the supernant from this step was pooled with
the supernant from 70 % Percoll gradient separation and centrifuged at 4000 g for 5 min,
and the supernatant discarded. The pellet was washed (twice) with dH20 by resuspension
and centrifugation at 4000 g for 5 min. This pellet comprised B-/C-granules. To separate
C-granules of size < 5 µm, sufficient amount (≈ 1 g) of partially purified B-granules
were suspended in 50 ml of water and left for ≈ 40-50 min to precipitate A-granules and
most of the B-granules, so that very small C-granules (<5 µm) remained in the supernant.
The supernant from this suspension was collected in a separate tube, centrifuged and
washed in the same way as described earlier. These three types of granules were used for
further experiments.
4.2.3 Isolation of starch granule-bound proteins
The isolation of protein bound within the internal matrix of the starch granules (as
opposed to proteins present on the surface of starch granules, which can be removed by
extensive washing with SDS) was carried out by the method previously described by
Tetlow et al. (2004) and Liu et al. (2009). The acetone and SDS washed starch, either
from mature seed, amyloplast lysates, or whole cell extracts was washed with 1 % (w/v)
SDS. This washing step was repeated to remove any traces of proteins attached to the
surface of starch granules. To extract the starch granule-bound proteins, an equivalent
amount of starch (≈ 50 mg) was boiled in 1000 µl SDS loading buffer containing, 62.5
mM TRIS-HCl, pH 6.8, 2 % (w/v) SDS, 10 % (w/v) glycerol, 5 % (v/v) βmercaptoethanol and 0.001 % (w/v) bromophenol blue. Boiled samples were centrifuged
at 13 000 g for 10 min and the supernatant used to determine total granule-bound protein
- 156 -
content, as well as for SDS–PAGE and immunodetection of granule-bound proteins.
4.2.4 SDS-PAGE and immunodetection of granule-bound proteins
For proteomic analysis of starch A- and B-granules, equivalent amounts of starch,
A- and B-granules were used to extract protein in the same way as described in section
(4.2.3). For SDS–PAGE and immunoblotting, either SDS gels with 10 % (w/v)
acrylamide or pre-cast NUPAGE Novex 4–12 % BISTRIS acrylamide gradient gels
(Invitrogen Canada,
catalogue
No.
NP0335BOX)
were
used,
following
the
manufacturer’s instructions. At room temperature, gradient gels were run in a MOPSbased running buffer prepared according to the manufacturer’s (Invitrogen) instructions.
For immunoblotting, following electrophoresis, gels were transblotted onto nitrocellulose
membranes (Pall Life Science), and blocked at room temperature in 1.5 % (w/v) BSA for
15 min with gentle shaking. The SSI, SSIIa, SSIII, SSIV, SBEIc, SBEIIa, SBEIIb, SP,
ISO1 and GBSS wheat-antibodies were used at dilutions as follows: anti-SSI 1:2000,
anti-SSII 1:2000, anti-SSIII 1:2000, anti-SSIV 1:2000, anti-SBEI 1:5000, anti-SBEIIa
1:5000, anti-SBEIIb 1:5000, anti-SP 1:1000 and anti-ISO 1:1000 in 1.5 % (w/v) BSA.
Different antibodies to wheat proteins were used according to specifications described
earlier (Li et al., 1999; Rahman et al., 2001; Morell et al., 2003; Regina et al., 2005;
Bresolin et al., 2006; Tetlow et al., 2008). Alkaline phosphatase (APase)-conjugated goat
anti-rabbit IgG (Sigma) was used as a secondary antibody.
4.2.5 Silver staining
Silver staining was performed according to a procedure described earlier Mortz et
al. (2001). Following electrophoresis, the gel was kept in a well-cleaned glass plate in 50
ml fixing solution buffer containing, 50 % methanol (v/v), 5 % acetic acid (v/v) for 20
- 157 -
min on a shaker, followed by washing in washing buffer (50 % methanol [v/v]) for 10
min on a shaker. Following washing, the gel was kept in distilled water for at least 1 h, or
overnight, with occasionally changing water. The gel was then immersed in sensitizing
buffer containing 0.02 % Na2S2O3 (w/v) for 1 min and washed twice in distilled water for
1 min each. The gel was incubated in ice-cold silver nitrate buffer (0.1 % AgNO3 [w/v])
for 20 min at 4oC followed by two washings with distilled water 1 min each at room
temperature. Finally, the gel was developed by adding developing solution containing 2
% Na2CO3 (w/v), 0.04 % formalin (v/v) for 3-5 min and then fresh developing buffer was
added and gel was stained until the proteins bands were visualized. Staining was stopped
by adding a solution containing 5 % acetic acid [v/v] for 5 min and the gel was
transferred to distilled water.
4.2.6 Estimation of protein phosphorylation by Pro-Q diamond staining
Equal amounts (1.5 mg) of starch from all genotypes were loaded in each lane onto 4–12
% acrylamide gradient gels (Invitrogen) and were separated by SDS-PAGE according to
manufacturer’s instructions. Following electrophoresis, gels were fixed in 50 %
methanol, 10 % acetic acid overnight in dark. The gels were then washed three times with
ddH20 and then incubated in Pro-Q Diamond stain according to the manufacturer’s
instructions (Molecular Probes, Life Technologies) in the dark for 90 min. Gels were then
destained three times each for 30 min in distaining solution containing 20 % acetonitrile,
50mM sodium acetate, pH 4.0 and then washed with water two times each 5-7 min. Gels
were visualised using a Typhoon scanner (Amersham Biosciences, Artisan Technology
Group, IL, USA) with an excitation wavelength of 530 nm and emission filter at 580 nm.
- 158 -
A phospho-protein marker was used as standard and phophorylated bands appeared
black.
4.2.7 Measurement of total granule-bound protein content
To measure total granule-bound protein equivalent amounts (50 mg) of acetonewashed starch were used. Protein was extracted by boiling starch in 2 % (w/v) SDS for 5
min followed by centrifugation at 13500 g for 10 min (see above). Total granule-bound
protein in the supernatant from three biological repeats was quantified with Pierce® BCA
protein assay kit (Thermo Scientific, catalogue No. 23227) according to manufacturer
instructions, using BSA as a standard.
4.2.8 In-gel protein quantification
The amount of individual protein from starch, A- and B-granules was measured
following silver staining by using Molecular Imager® Gel Doc™ XR+ and Chemi Doc™
XRS+ Systems with Image Lab™ Software, version 3 (BioRad Laboratories Inc.)
according to manufacturer’s instructions, using protein markers (Bio Rad). In-gel protein
content was determined using 100-1500 ng BSA to generate a linear standard ranges for
calibration. Following electrophoresis of triplicate samples, gels were silver stained and
the intensity of each band determined by imaging and protein content calculated.
4.2.9 Starch gelatinization
Thermal analyses of starches were performed as described by Liu et al. (2007)
using a differential scanning calorimeter (DSC) (2920 Modulated DSC, TA Instruments,
New Castle, DE, USA). For gelatinization and retrogradation of starches, this system was
equipped with a refrigerated cooling system (RCS). Samples of starch granules were
weighed into high volume pans. A micropipette was used to add distilled water to
- 159 -
produce suspensions with 70 % moisture content. Approximately 20 mg of starch was
used per sample. The sealed pans were equilibrated overnight at room temperature before
heating in the DSC. Measurements were taken at a heating rate of 10°C/min from 5°C to
180°C. Calibration of the instrument was performed using indium and an empty pan as
reference. To measure the enthalpy (ΔH) of phase transitions from the endotherm of
DSC, thermograms based on the mass of dry solid, software (Universal Analysis, v.2.6D,
TA Instruments) were used. Peak temperature (Tp) of endotherms was also measured
from DSC thermograms. For retrogradation, the heated starch from the above procedure
was cooled to 5°C. Once the temperature reached 5°C, sample was immediately removed
from the DSC and stored at 5°C. After two weeks, stored samples were heated from 5 to
180°C at 10°C/min and, based on dry, solid mass, the enthalpy (ΔH) and peak
temperature (Tp) of the endotherm were measured from DSC thermograms.
4.2.10 High-Performance Anion Exchange Chromatography (HPAEC)
To measure the chain length distribution of barley starch, HPAEC was used as
described by (Liu et al., 2007). Isoamylase-debranched starch granules were dispersed in
2 ml of 90 % (v/v) DMSO (5 mg/ml) by stirring in a boiling water bath for 20 min. The
sample was left to cool to room temperature, followed by addition of 6 ml methanol,
mixing and incubation in an ice bath for 30 min. The suspension was centrifuged (1,000
× g for 12 min), and the pellet dissolved in 2 ml, 50 mM sodium acetate buffer (pH 3.5)
by continuous stirring in a boiling water bath for 20 min. The sample was removed and
equilibrated at 37 °C, after which 5 μL isoamylase (EN102, 68,000 U/mg of protein,
Hayashibara Biochemical Laboratories, Okayama, Japan) was added and digested for 22
h. To inactivate the enzyme, the sample was boiled for 10 min. The sample was cooled
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and 200 μL of debranched sample was diluted with 2 ml of 150 mM NaOH. The diluted
sample was filtered (0.45 μm nylon syringe filter) and injected into the HPAEC-PAD
system (50 μL sample loop). The HPAEC-PAD system consisted of a Dionex DX 600
equipped with an ED50 electrochemical detector with a gold working electrode, GP50
gradient pump, LC30 chromatography oven, and AS40 automated sampler (Dionex
Corporation, Sunnyvale, CA, USA).
4.2.11 Granule size distribution of different genotypes
Granule size, number and surface area of starch from different genotypes were
measured by means of laser scattering using the Mastersizer (Malvern Mastersizer 2000,
Malvern Instruments Ltd., Worcestershire, UK). For each measurement, acetone washed
starch (≈ 100mg) was weighed into glass tubes and suspended with 10 ml of distilled
water. Sample concentrations were within equipment recommendations and the refractive
indices of 1.31 for water and 1.52 for starch were used as standards and distribution was
measured as both the percentage volume and percentage number.
4.2.12 Granule morphology
To study starch granule morphology in detail, SEM was performed on a field
emission scanning electron microscope (S-4500, Hitachi, Tokyo, Japan) equipped with
Quartz PCI digital image acquisition software (Quartz Imaging, Vancouver, BC, Canada)
as described by Liu et al. (2007). The acetone dried barley starch samples were sprayed
on a metal plate covered with double-sided adhesive. The samples were coated with gold
using a Polaron SC500 sputter coater (Quorum Technologies, East Sussex, UK). The
samples were examined at 10 kV accelerating voltage and representative micrographs
were taken for each sample at different magnifications, according to requirement.
- 161 -
4.2.13 Thousand grain weight (TGW)
1000 grains of each genotype were counted and weighed to 0.1 mg using a
tabletop electrical balance (Denver Instrument, Bohemia, NY, USA). The mean of three
biological replicates was used in statistical analysis (de Rocquigny, 2011).
4.2.14 Starch content
The amount of starch in barley seeds from the different genotypes using three
biological replicates was determined as described by Tetlow et al., (1994) for wheat
endosperm.
4.2.15 RS Content
RS was measured by using, 100 mg acetone washed starch from three biological
replicates with Megazyme resistant starch assay kit (K-RSTAR 08/11, Megazyme
International, Ireland) following manufacturer’s instructions.
4.2.16 Amylose content
The apparent amylose content was measured by using, 25 mg acetone washed
starch from three biological replicates with Megazyme amylose /amylopectin assay kit
(K-AMYL
07/11,
Megazyme
International,
Ireland)
following
manufacturer’s
instructions.
4.2.17 Mass spectrometric analysis
In-gel digestion of protein bands was carried out with trypsin and peptides for MS
were prepared according to the protocol described by Tetlow et al., (2008). Using a
hybrid Q-TOF spectrometer (Micromass) interfaced to a Micromass CapLC capillary
chromatograph, Tandem electrospray mass spectra were recorded according to the
method previously described (Tetlow et al., 2004).
- 162 -
4.2.18 Statistical analysis
For one way analysis of variance (ANOVA) and least significant difference
(LSD), Statsoft’s Software, Statistica (http://www.statsoft.com) was used. For principal
component analysis (PCA), XLSTAT (http://www.xlstat.com/en/) was used and biplots
were generated.
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4.3 Results
4.3.1 Physical characteristics of seed
4.3.1.1 Seed morphology characteristics
Seed characteristics, including seed length, width, thickness and length to width
ratio were measured. Different genotypes showed significant differences in different
parameters of seed. Seed length varied from 7.3 to 9.4 mm among different genotypes
with lowest seed length exhibited by McGwuire and highest by genotype 083211-122,
respectively. Seed width of different genotypes varied between 3.4 to 4.4 mm. Similarly,
differences in seed thickness among different genotypes were observed. Details of
different seed parameters are shown in Table (4.1).
4.3.1.2 Thousand grain weight (TGW)
Significant differences were observed among genotypes for thousand grain weight
Table (4.1). The genotype 083211-120 possessed highest 72.8 g TGW while genotype
Emperor had the lowest, 40.9 g TGW. Both waxy genotypes (CDC Fibar and CDC
Rattan) had lower TGW than the reference genotype (OAC-Kawartha).
4.3.1.3 Starch content
Starch content of wild-type (OAC Kawartha) has been taken as a reference and all
other genotypes were compared to it. All genotypes possessed higher starch content than
wild-type except 083311-104, which contained less starch. Genotype 083511-118
possessed highest (139.8 %) starch (Table, 4.1).
4.3.1.4 Internal seed structures and starch packing within endosperm
To observe internal seed structures and starch packing within endosperm, cross
sections along seed length from all genotypes were made and stained with iodine (Figure
- 164 -
Table 4.1: Physiochemical properties of seed and starch of barley genotypes.
Genotype
T
R
OAC Kawartha
McGuwire
GB992033
Sloop
CDC Fibar
CDC Rattan
Soft Barley
Hard Barley
AC Metcalfe
083211-120
083211-122
083311-104
083411-113
AC Alberte
AC Bacon
Emperor
Sunderland
083511-109
083511-118
C
H
C
C
H
H
H
H
C
C
C
C
H
H
H
H
C
H
H
6
2
2
2
2
2
2
2
2
2
2
6
6
2
6
2
2
2
2
Amylose
(%)
25.0 ±1.1
18.1 ±1.0
25.3 ±0.6
16.0 ±1.0
1.0 ± 0.3
2.3 ± 0.6
12.0 ±1.1
24.2 ±1.3
23.3 ±1.5
21.3 ±1.5
23.3 ±0.6
26.3 ±0.6
22.0 ±1.3
20.0 ±2.4
27.2 ±1.8
22.7 ±1.5
18.1 ±1.8
21.3 ±1.6
22.0 ±1.4
RS (%)
1.8 ±0.5
1.8 ±0.4
1.6 ±0.4
1.9 ±0.4
1.9 ±0.5
1.3 ±0.4
1.0 ±0.1
2.2 ±0.6
1.6 ±0.5
2.0 ±0.7
1.8 ±0.6
2.1 ±0.8
2.3 ±0.6
2.0 ±0.7
2.5 ±0.7
2.0 ±0.7
2.2 ±0.6
2.0 ±0.6
2.6 ±0.9
Seed Characteristics (mm)
SL
SW
ST
7.9 ±0.7 3.8 ±0.1 2.9 ±0.2
7.3 ±0.4 3.7 ±0.2 2.9 ±0.1
7.5 ±0.3 3.7 ±0.1 2.9 ±0.1
7.6 ±0.4 3.9 ±0.1 2.6 ±0.2
7.6 ±0.7 3.6 ±0.3 2.8 ±0.2
7.8 ±0.6 3.7 ±0.2 2.7 ±0.1
7.5 ±0.5 3.6 ±0.1 2.7 ±0.1
8.7 ±0.6 3.6 ±0.2 2.6 ±0.1
8.2 ±0.4 3.9 ±0.2 2.8 ±0.2
8.9 ±0.4 4.1 ±0.2 2.9 ±0.2
9.4 ±0.3 4.3 ±0.1 2.8 ±0.1
7.7 ±0.6 3.8 ±0.2 2.8 ±0.1
8.2 ±0.6 3.8 ±0.2 2.7 ±0.2
8.5 ±0.3 3.4 ±0.2 2.5 ±0.2
8.6 ±0.5 3.4 ±0.2 2.6 ±0.2
8.9 ±0.7 3.4 ±0.1 2.4 ±0.2
8.1 ±0.4 3.6 ±0.1 2.8 ±0.1
8.5 ±0.6 4.1 ±0.2 2.8 ±0.2
8.2 ±0.4 4.4 ±0.2 3.2 ±0.1
SL:
SW
2.1
2.0
2.0
1.9
2.1
2.1
2.1
2.4
2.1
2.2
2.2
2.0
2.1
2.5
2.5
2.6
2.2
2.1
1.9
1000 grain
weight (g)
57.7 ±1.1
47.9 ±0.6
56.1 ±0.9
59.7 ±1.1
46.1 ±0.1
46.0 ±0.9
48.9 ±0.7
48.6 ±0.6
56.8 ±0.5
72.8 ±1.9
73.3 ±1.8
55.9 ±0.9
54.7 ±0.6
49.8 ±0.4
54.8 ±0.7
40.9 ±0.5
54.7 ±0.8
51.9 ±0.7
61.7 ±0.8
% Starch
content
100.0
119.7
106.8
105.5
121.1
123.1
119.2
118.4
101.6
111.6
112.1
97.3
103.3
107.9
131.1
106.3
119.7
103.3
139.8
T= Type, C= Covered, H= Hulless, R= Rows, SL:SW= Seed length to seed width ratio
SL= Seed length, SW= Seed width, ST= Seed thickness, TGW= Thousand grain weight
Table 4.1: Physical characteristics of seed and physiochemical characteristics of starch. Type (T) indicates whether seed is covered (C)
or hulless (H). Rows (R) represent number of rows (2 or 6) of fertile spikelets on the barley spike. Amylose and resistant starch (RS)
contents were measured using Megazyme kits. % starch content of the reference genotype, OAC-Kawartha wild-type was taken as
100 %. Data represent means of three biological replicates for amylose (%), RS (%) and % starch content. For SL, SW, ST and SL: SW
data from hundred independent observations with three biological replicates are presented. For 1000 grain weight, data from
thousand independent observations with three biological replicates are presented. ± standard deviation.
- 165 -
4.1). In all genotypes, seeds were fully stained internally showing uniform distribution of
starch within the seed except waxy genotypes CDC Fibar and CDC Rattan and lowamylose genotype Soft Barley (12 % amylose) which did not stain due to reduced
iodine-binding (Figure 4.1). Most of the genotypes possessed thin pericarp except
genotypes Sloop, AC Alberte (hulless) and AC Bacon (hulless) which possessed
comparatively thicker pericarp.
4.3.2 Physiochemical properties of starch
4.3.2.1 Amylose and resistant starch (RS) contents
Differences with respect to amylose content were observed among different
genotypes. OAC Kawartha (which represents normal, wild-type barley) has 25 %
amylose. Most of the genotypes possessed normal amylose content with certain variations
compared to wild-type as shown in the Table (4.1). The Soft Barley genotype has 12 %
amylose and is characterized as a low amylose genotype. Two genotypes (CDC Fibar and
CDC Rattan) possessed < 5 % amylose and are characterized as waxy genotypes. All
genotypes showed < 5 % RS content.
4.3.2.2 Granule Size, number and surface area distributions of different genotypes
Differences among the barley genotypes were observed for granule size,
number and surface area distributions. The reference genotypes OAC Kawartha and
McGwuire exhibited bimodal granule size, number and surface area distributions
(Figures 4.2, 4.3 & 4.4A & B). However in all other genotypes, unimodal distribution
was observed. In all genotypes maximum granule size was upto 45 µm, except in
genotypes, Sloop and Emperor in which maximum granule sizes upto 39 µm and 34 µm
were observed respectively. Figures (4.3A & 4.5) show that in normal genotypes granules
- 166 -
Figure 4.1: Iodine-staining of barley grains (cross section).
To observe internal structure and starch packing with in seed from different genotypes,
cross sections along the length of seed were made and incubated in Lugol’s solution.
- 167 -
of size < 5µm in diameter represents > 90 % of total granule number but contributes < 15
% to total starch mass.
4.3.2.3 Contribution of A-, B- and C-granules in total mass of starch
Barley starch granules can be divided into three major classes based on size and
shape which include C-type (< 5 µm, polygonal), B-type (5-15 µm, round) and A-type
(15-45 µm, lenticular). Figure (4.5) shows that genotypes OAC Kawartha and McGwuire
possess considerable amounts of C-granules unlike the other genotypes studied.
However, based on size distribution all genotypes contain considerable amount of Bgranules in which genotypes sloop and CDC Fibar possessed highest amount of Bgranules compared to wild-type. A-granules constituted the major portion of total starch
mass in all genotypes and among all genotypes 083411-113 possessed the highest amount
at 89.76 % of A-granules (Figure, 4.5).
- 168 -
20
O A C K a w a r th a
M c G w u ir e
G B 992033
S lo o p
C D C F ib a r
C D C R a tta n
S o ft B a r le y
H a r d B a r le y
A C M e tc a lfe
0 8 3 2 1 1 -1 2 0
A
18
0 8 3 2 1 1 -1 2 2
0 8 3 3 1 1 -1 0 4
B
16
0 8 3 4 1 1 -1 1 3
% Volume of granules
18
16
14
12
10
8
6
4
2
A C A lb e r te
A C B acon
Em peror
S u n d e r la n d
0 8 3 5 1 1 -1 0 9
0 8 3 5 1 1 -1 1 8
14
12
10
8
6
4
2
B -g r a n u le s
45.1
39-45
34-39
30-34
26-30
22-26
19-22
17-19
15-17
13-15
11.0-13.0
8.7-10
7.5-8.7
6.6-7.5
5.7-6.6
5.0-5.7
4.3-5.0
3.8-4.3
10.0-11.0
C -g r a n u le s
3.3-3.8
2.8-3.3
2.5-2.8
2.1-2.5
1.9-2.1
1.6-1.9
1.4-1.6
0
1.2-1.4
% Volume of granules
0
20
A -g r a n u le s
G r a n u l e siz e in m m
Figure 4.2 (A & B): Starch granule size distribution of different genotypes.
Granule size distribution in normal, low amylose and waxy starches from various
genotypes. Granule size distribution was measured by means of laser scattering using
the Mastersizer (Malvern Mastersizer 2000, Malvern Instruments Ltd., UK). Starch (≈100
mg) was suspended in 10 ml of distilled water and used according to manufacturer
instruction.
- 169 -
22
083211-122
083311-104
083411-113
AC Alberte
AC Bacon
Emperor
Sunderland
083511-109
083511-118
B
20
18
16
14
12
10
8
6
4
2
C-granules
B-granules
45.1
39-45
34-39
30-34
26-30
22-26
19-22
17-19
15-17
13-15
11.0-13.0
10.0-11.0
8.7-10
7.5-8.7
6.6-7.5
5.7-6.6
5.0-5.7
4.3-5.0
3.8-4.3
2.8-3.3
2.5-2.8
2.1-2.5
1.9-2.1
1.6-1.9
1.4-1.6
0
1.2-1.4
% Number of granules
OAC Kawartha
McGwuire
GB992033
Sloop
CDC Fibar
CDC Rattan
Soft Barley
Hard Barley
AC Metcalfe
083211-120
A
3.3-3.8
% Number of granules
24
22
20
18
16
14
12
10
8
6
4
2
0
24
A-granules
Granule size in mm
Figure 4.3 (A & B): Starch granule number distribution of different genotypes.
Granule number distribution in normal, low amylose and waxy starches from various
genotypes. Granule number distribution was measured by means of laser scattering
using the Mastersizer (Malvern Mastersizer 2000, Malvern Instruments Ltd., UK). Starch
(≈100 mg) was suspended in 10 ml of distilled water and used according to
manufacturer instruction.
- 170 -
22
O A C K aw a rtha
M cG w u ire
G B 9 920 33
S lo op
C D C Fiba r
C D C R a ttan
S oft Barley
H a rd B a rley
A C M etca lfe
0 832 11-12 0
% Surface area of granules
20
18
16
14
12
10
8
6
A
4
2
0
22
18
16
14
12
10
8
6
4
2
C -g ra nules
B -gra nules
45.1
39-45
34-39
30-34
26-30
22-26
19-22
17-19
15-17
13-15
11.0-13.0
10.0-11.0
8.7-10
7.5-8.7
6.6-7.5
5.7-6.6
5.0-5.7
4.3-5.0
3.8-4.3
3.3-3.8
2.8-3.3
2.5-2.8
2.1-2.5
1.9-2.1
1.6-1.9
1.4-1.6
0
1.2-1.4
% Surface area of granules
B
08 321 1-122
08 331 1-104
08 341 1-113
A C A lberte
A C B acon
E m pe ro r
Su nderland
08 351 1-109
08 351 1-118
20
A -gra nules
G ra nule size in m m
Figure 4.4 (A & B): Starch granule surface area distribution of different genotypes.
Granule surface area distribution in normal, low amylose and waxy starches from
various genotypes. Granule surface area distribution was measured by means of laser
scattering using the Mastersizer (Malvern Mastersizer 2000, Malvern Instruments Ltd.,
UK). starch (≈100 mg) was suspended in 10 ml of distilled water and used according to
manufacturer instruction.
- 171 -
% Amount of granules
100
80
60
40
OAC Kawartha
McGwuire
GB992033
Sloop
CDC Fibar
CDC Rattan
Soft Barley
Hard Barley
AC Metcalfe
083211-120
20
0
% Amount of granules
100
80
60
40
C-granules
B-granules
A-granules
B- granules
A- granules
083211-122
083311-104
083411-113
AC Alberte
AC Bacon
Emperor
Sunderland
083511-109
083511-118
20
0
C- granules
Type of starch granules
Figure 4.5: Starch granule distribution into (A-, B-, and C-) classes.
A-granules (15-45 µm); B-granules (5-15 µm) and C-granules (< 5 µm).
- 172 -
4.3.2.4 Granule surface morphology
Granule surface morphology of different genotypes was observed using
scanning electron microscopy (SEM). In all genotypes, no differences were observed in
the morphology of small granules up to 15 µm in size. However differences were
observed in the morphology of A-granules from different genotypes. In all genotypes Agranules were lenticular except; Hard Barley, 083411-113, AC Alberte, AC Bacon and
Sunderland. In these genotypes, A-granules with altered morphology were observed
(Figure, 4.6). The extent of alteration was different in these genotypes, for example, in
genotype 083411-113, A-granules were severely deformed and lost lenticular shape
(Figure, 4.6). However in all other genotypes, with altered granule morphology (Hard
Barley, AC Alberte, AC Bacon and Sunderland), the lens shape of A-granules was
retained although grooves or ditches were observed in the granules. In waxy genotypes
CDC Fibar and CDC Rattan, no difference was observed in the starch granule
morphology compared to the reference genotype.
4.3.3 Starch gelatinization (Thermal properties)
To measure the thermal properties of starch, six genotypes were selected from
above mentioned genotypes based on certain parameters. The selected genotypes
included OAC Kawartha (reference genotype); CDC Fibar (waxy); Soft Barley (low
amylose, with normal amount of B-granules of size, 5-15 µm); Hard Barley (normal
amylose, with normal amount of B-granules of size, 5-15 µm); AC Metcalfe and 083411113 (normal amylose, with significantly reduced amount of small granules of size, 1-10
µm). To measure the thermal properties of starch derived from selected genotypes,
differential scanning calorimetry (DSC) was employed. Table (4.2) shows that significant
- 173 -
Figure 4.6: Surface morphology of starch granules from different genotypes observed
by SEM.
For SEM analysis acetone dried barley starch samples were sprayed on a metal plate
covered with double-sided adhesive tape. The samples were gold coated with Polaron
SC500 sputter coater (Quorum Technologies, East Sussex, UK). The samples were
examined at 10 kV accelerating voltage and representative micrographs were taken for
each sample at same magnification. Scale bar for electron micrograph is given under
each panel. Each type of granule is presented by a colored arrow.
- 174 -
differences were present among different genotypes with respect to onset (To)
gelatinization temperature where highest gelatinization temperatures (59.3 and 59.1 oC)
were exhibited by waxy genotype CDC Fibar and low amylose genotype Soft Barley,
respectively. Genotype 083411-113 exhibited lowest (54.7 oC) onset gelatinization
temperature (To). Wild-type, Hard Barley and AC Metcalfe exhibited lower (To) than
waxy genotype. In the case of peak temperature (Tp), genotypes also exhibited significant
differences with highest (Tp) possessed by CDC Fibar and lowest by Hard Barley. The
Soft Barley genotype also possessed higher (Tp) than wild-type. Similarly genotypes
CDC Fibar and 083411-113 possessed highest completion temperature (Tc) of starch
gelatinization. Genotype 083411-113 exhibited significantly higher (Tc-To) of 21.6 oC
compared to wild-type, Soft Barley and Hard Barley. Significant variations were also
observed regarding transition enthalpy (∆H) for different genotypes in which highest
(∆H) 13.3 and 13.2 J/g was exhibited by CDC Fibar and 083411-113, respectively, and
lowest (∆H) 9.8 J/g was exhibited by Soft Barley (Table, 4.2). In the second phase of
gelatinization where disruption of amylose-lipid complex took place, different genotypes
showed interesting variations, for example, Hard Barley and AC Metcalfe possessed
highest and wild-type showed lowest onset temperature (To) of melting amylose-lipid
complex. Hard barley also exhibited highest peak temperature (Tp) and genotypes Soft
Barley and AC Metcalfe possessed lowest (Tp) values indicative of melting amylose–
lipid complex. Soft Barley has highest and OAC Kawartha, AC Metcalfe and 083411-113
exhibited lowest completion temperatures (Tc). Genotype Soft Barley exhibited highest
(Tc-To) values indicative of degree of branching of amylopectin compared to all other
genotypes. Different genotypes also exhibited significant variation in transition enthalpy
- 175 -
(∆H). CDC Fibar and 083411-113 possessed highest values for transition enthalpy (∆H)
(13.3 and 13.2 J/g), respectively. Low amylose genotype Soft Barley exhibited lowest
(∆H) 9.8 J/g. The waxy genotype (CDC Fibar) did not show any value for all transition
phases of melting amylose-lipid complex, indicating waxy starch has very low amount or
no amylose-lipid complex as previously shown by Yoshimoto et al. (2000).
In the 2nd phase of gelatinization of retrograded starch, no significant variations
have been observed for different phases of gelatinization. However, the genotype Soft
Barley, which is a low amylose genotype, did not show any value for melting of
retrograded starch as was also observed for the waxy genotype CDC Fibar. Interestingly,
Soft Barley showed comparable values to that of other genotypes for different phases of
melting of the amylose-lipid complex (To, Tp, Tc) of retrograded starch. However, Soft
Barley showed a very low value of ∆H compared to other genotypes (Table, 4.2).
- 176 -
Table 4.2: Thermal properties of starch.
Initial Heating Summary
Melting of amylose-lipid complex (oC)
Genotype
Starch Gelatinization (oC)
To
Tp
Tc (Tc-To) ∆H (J/g) To
Tp
Tc
(Tc-To) ∆H(J/g)
c
c
ab
b
b
c
ab
c
OAC Kawartha 56.4 62.7 73.2 16.9
10.4
93.3 102.6 108.1 14.8b
1.4a
a
a
a
ab
a
d
c
d
d
CDC Fibar
59.3 67.4 77.4 18.2
13.3
0.00 0.00
0.00
0.0
0.0c
Soft Barley
59.1a 65.4b 73.5ab 14.4b
9.8b
93.6bc 101.8b 111.6a 18.0a
0.5b
d
e
b
b
ab
a
a
b
bc
Hard Barley
55.8 60.9 70.7 14.9
11.3
95.9 103.1 109.9 13.9
1.5a
AC Metcalfe
57.5b 62.9c 75.2ab 17.6ab
11.8ab
95.3a 102.2b 108.2c 12.9c
1.1a
e
d
a
a
a
ab
ab
c
bc
083411-113
54.7 61.7 76.3 21.6
13.2
94.7 102.6 108.1 13.3
1.6a
Re-Heating Summary
Genotype
Melting of retrograded starch (oC)
melting of amylose-lipid complex (oC)
To
Tp
Tc (Tc-To) ∆H (J/g) To
Tp
Tc
(Tc-To) ∆H(J/g)
OAC Kawartha 39.5a 52.4a 66.5a 27.1a
2.9b
91.2a 101.4b 1110.9a 19.8a
1.3a
b
b
b
b
c
b
d
c
d
CDC Fibar
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0c
Soft Barley
0.0b 0.0b 0.0b 0.0b
0.0c
91.2a 99.8c 105.7b 14.6c
0.7b
a
a
a
a
ab
a
a
a
bc
Hard Barley
39.4 52.1 66.2 26.8
3.2
93.3 104.2 109.9 16.5
1.3a
AC Metcalfe
39.5a 52.3a 68.2a 28.7a
3.9ab
91.7a 103.1a 109.9a 18.3ab
1.1ab
083411-113
39.5a 52.1a 66.4a 26.9a
4.1a
92.6 103.1a 109.5a 16.9bc
1.1ab
Table 4.2, Thermal analyses of starch from different genotypes. DSC was performed according to the method described by
Liu et al. (2007) using a differential scanning calorimeter (2920 Modulated DSC, TA Instruments, New Castle, DE). In this
analysis CDC Fibar represents a genotype waxy and Soft Barley a low amylose genotype. The data are presenting mean of
two replicates. Values in the same column followed by the same letter are not significantly different at p < 0.05.
- 177 -
4.3.4 Amylopectin chain length distribution (CLD)
Amylopectin structure and the proportion of chain lengths with different degrees
of polymerization (DP) (chain length distribution) are important for determining the
functionality of starch. These features contribute to texture, stability, uniformity and
freeze-thaw properties of starch (Chibbar et al., 2005). Amylopectin chain length
distribution of selected genotypes, OAC Kawartha, Sloop, CDC-Fibar, 083211-122 and
083411-113 (Section, 4.3.3) was measured. The measureable degree of polymerization in
different genotypes ranged from 6 DP to 50 DP with varying proportions of different DP.
The measured DP has been divided into four groups as shown in Table 4.3. Interestingly,
all genotypes showed no significant differences in first three DP ranges (6-12, 13-24 and
25-36) as shown in Table (4.3). However, in case of group IV which is represented by
chains of DP > 37, genotypes showed significant differences. Interestingly, the waxy
genotype CDC Fibar possessed the highest amount of longer chains, while low amylose
(Soft Barley) and normal amylose genotypes (Hard Barley and 083411-113) exhibited the
lowest amount of longer chains of DP > 37. There was no significant difference in
average chain length distribution for any genotype.
To understand differences in individual DP among different genotypes, difference
plots of : different genotypes versus “reference genotype” OAC-Kawartha; different
genotypes versus “waxy genotype” CDC Fibar; and different genotypes versus “low
amylose genotype” Soft Barley, have been produced. Compared to reference genotype
(OAC-Kawartha) all other genotypes studied have a lower proportion of DP 6, except
Hard Barley, which has a higher proportion of short chains with DP 6. CDC Fibar has a
relatively higher proportion of chains with DP 7-9 and long chains with DP > 37 and a
- 178 -
lower proportion of chains with DP 10-17 and DP 29-33. Soft Barley has lower
proportion of chains with DP 6-8 and intermediate and long chains beyond DP 21 and has
higher proportion of chains with DP 9-16 compared to wild-type. Genotype AC Metcalfe
has greater proportion of chain lengths with DP 7-10 and lower proportion of some
chains with DP > 11 (Figure 4.7). Genotype 083411-113 also exhibited interesting results
in which proportion of chains with DP 7-11 has increased and the proportion of all chains
with DP > 12 decreased (Figure 4.7).
A second set of difference plots: different genotypes versus waxy was generated,
and it was found that all genotypes have a relatively higher proportion of chains with DP
6 compared to waxy except Soft Barley. OAC Kawartha (WT) has lower proportion of
chains with DP 7-9, all other chains with different DP were either higher in proportion or
exhibited minor differences compared to waxy. Soft Barley also has a smaller proportion
of chains with DP 7-9 and chains with DP > 33 and a higher proportion of chains with
DP 10-33, with some exceptions (Figure 4.8). Similarly, Hard Barley, 083411-113 and
AC Metcalfe have lower proportion of longer chains and higher proportion of small to
intermediate chains compared to waxy (Figure 4.8).
Difference plot of different genotypes versus low amylose genotype showed that,
compared to the low amylose genotype, all other lines exhibited variations in chains with
DP 6-18. All of the genotypes had a higher proportion of small chains with DP < 10 and a
lower proportion of chains with DP 10-18 compared to the low amylose variety. With
reference to chains with DP > 18 all genotypes exhibited minor differences compared to
the low amylose genotype (Figure 4.9).
- 179 -
Table 4.3: Amylopectin chain length in different barley genotypes.
Genotypes
OAC Kawartha
CDC Fibar
Soft Barley
Hard Barley
AC Metcalfe
083411-113
6-12
44.2a ±2.6
45.2a ±2.2
44.9a ±2.3
46.9a ±1.2
45.8a ±1.3
47.8a ±1.5
13-24
44.4a ±2.0
43.7a ±0.8
45.1a ±1.9
44.2a ±1.1
44.0a ±1.1
43.1a ±1.1
DP
25-36
8.1a ±0.7
7.5a ±1.2
7.5a ±0.3
6.7a ±0.5
7.5a ±0.6
6.6a ±0.3
37-50
3.2ab ±0.1
3.7a ±0.2
2.6c ±0.2
2.3c ±0.3
2.7bc ±0.4
2.5c ±0.1
Avg. CL
19.5a ±0.2
19.6a ±0.5
18.9a ±0.2
18.4a ±0.1
18.9a ±0.2
18.5a ±0.1
Table 4.3, different groups of chain length with varying DP (s) are presented in four
different classes. The data are presented as % moles of carbohydrate. Values in the
same column followed by the same letter are not significantly different at p < 0.05. ±
standard deviation.
- 180 -
Molar % difference
Figure 4.7: Difference plot of (other genotypes versus wild-type) for amylopectin
chains with different DP.
Amylopectin chain length distribution of barley genotypes was measured by HPAEC. The
chain length distribution of wild-type OAC Kawartha was deducted from the chain
length distribution of other genotypes. The negative value shows that reference
genotype exhibited a higher proportion of that specific glucan chain, while positive
value shows that other genotypes exhibited a higher proportion of that specific glucan
chain.
- 181 -
Molar % difference
Figure 4.8: Difference plot of (other genotypes versus waxy) for amylopectin chains
with different DP.
Amylopectin chain length distribution of barley genotypes was measured by HPAEC. The
chain length distribution of waxy genotype CDC Fibar was deducted from the chain
length distribution of other genotypes. The negative value shows that waxy genotype
exhibited a higher proportion of that specific glucan chain, while positive value shows
that other genotypes exhibited a higher proportion of that specific glucan chain.
- 182 -
Molar % difference
Figure 4.9: Difference plot of (other genotypes versus low amylose) for amylopectin
chains with different DP.
Amylopectin chain length distribution of barley genotypes was measured by HPAEC. The
chain length distribution of low amylose genotype Soft Barley was deducted from the
chain length distribution of different genotypes. The negative value shows that low
amylose genotype exhibited a higher proportion of that specific glucan chain, while
positive value shows that other genotypes exhibited a higher proportion of that specific
glucan chain.
- 183 -
4.3.5 Biochemical characterization of barley genotypes
4.3.5.1 Amyloplast stromal proteins (soluble proteins)
Amyloplasts from developing barley endosperm were isolated (18-25 DAP) from
selected genotypes, OAC Kawartha, Sloop, CDC-Fibar, 083211-122 and 083411-113
(Section 4.3.3). Amyloplast lysates were used to detect the presence of different proteins
involved in starch biosynthesis in the amyloplast stroma using a range of peptide specific
antibodies, which include SSI, SSIIa, SSIII, SSIV, SBEI, SBEIc, SBEIIa, SBEIIb, ISO1,
SP, AGPase large subunit and AGPase small subunit. These proteins were observed at
their expected sizes on SDS-gels based on known sequences. Immunological analysis of
amyloplast lysates from these genotypes showed that similar amounts of SSI, SSIIa,
SSIII, SSIV, SBEI, SBEIc, SBEIIa, SBEIIb, ISO1, SP, AGPase large subunit, AGPase
small subunit are present when compared to wild-type (data not shown).
4.3.5.2 Analysis of starch granule-associated proteins
Granule associated proteins are those which remain entrapped within the starch
granule after extensive washing with, buffer, SDS and acetone.
4.3.5.2.1 Total amount of starch granule-bound protein
The total amount of starch granule-associated protein (µg of protein/50 mg of
starch) was measured. Genotype 083511-118 exhibited highest amount (109 µg) while
waxy genotype CDC Rattan exhibited lowest amount (26 µg) of total granule-bound
protein compared to reference genotype OAC Kawartha (91 µg). All other genotypes
studied exhibited minor differences compared to reference genotype (Table 4.4).
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4.3.5.2.2 Individual isoforms of starch granule-associated proteins
The starch granule proteome of different genotypes was analysed. Barley starch
has trimodal granule size distribution comprising of A-, B- and C-type granules. These
A- B- and C-granules were separated (Methods 4.2.2.1) from all genotypes using a
method described by Peng et al. (2000) and differences in the proteome of these three
types of granules have been determined. To isolate starch granule-bound proteins, buffer,
SDS and acetone washed starch, A- B- and C-granules were used and starch granulebound proteins were extracted (see methods). These proteins were analysed by silver
staining and immunoblotting using antibodies against various enzymes of the
biosynthetic pathway. Along with other previously described granule-bound proteins,
such as GBSS, SSI, SSIIa and SBEIIb other proteins were also found to be associated
with starch granules, such as SSIII, SBEIc, SBEIIa. Immunoblot analysis revealed that all
genotypes under study have comparable amounts of granule-bound SSI, SSIIa, SSIII,
SBEIc, SBEIIa, SBEIIb and GBSS, except genotypes CDC Rattan and Soft Barley which
have reduced amount of GBSS (Figure 4.10). Although different proteins have been
found to be granule-associated, other isoforms of some proteins like SSIV, SBEI,
Isoamylases and SP, were not detected within the starch granule (Figure 4.10). No
qualitative differences have been found in the starch granule proteome of all genotypes
except CDC Rattan and Soft Barley. However, detailed analyses of the proteome of Aand B-granules revealed that B-granules lack SBEIc protein, and is only present in Agranules (Figure 4.10). In certain genotypes AC Metcalfe, 083411-113, AC Bacon,
083511-109 and 083511-118, due to presence of low amounts of B-granules, immunoblot
analysis was not performed. Similarly in all genotypes immunodetection of C-
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Table 4.4: Amount of total starch granule-bound protein (µg/ 50 mg of starch).
Genotype
OAC Kawartha
McGwuire
GB992033
Sloop
CDC Fibar
CDC Rattan
Soft Barley
Hard Barley
AC Metcalfe
083211-120
Protein
91b ±5.5
89b ±3.7
87c ±11.6
90b ±4.4
74c ±6.6
26d ±4.2
31d ±6.7
83bc ±8.7
82bc ±6.2
91b ±8.9
Genotype
083211-122
083311-104
083411-113
AC Alberte
AC Bacon
Emperor
Sunderland
083511-109
083511-118
Protein
89b ±9.6
90b ±5.0
88b ±6.5
86bc±7.0
88bc ±8.2
89b ±11.2
88bc ±10.6
88bc ±11.0
109a ±12.4
Table 4.4. Total granule-bound protein was determined from 50 mg of acetone-washed
starch using Pierce® BCA protein assay kit. The data for each genotype represent means
of three biological replications. Values in the same column followed by the same letter
are not significantly different at p < 0.05. ± standard deviation.
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Figure 4.10: Immunodetection of granule-bound proteins in A- and B-starch granules.
Starch granules were isolated from mature barley grains, the purified, acetone washed
starch was used to separate large A- and small B-granules. 50 mg aliquot of purified
starch, A- and B-granules were boiled in 1 ml of SDS loading buffer, and 40 µl of the
supernatant from the boiled sample loaded onto 4–12% acrylamide gradient gels and
10% acrylamide SDS gels. Following electrophoresis, gels were electroblotted to
nitrocellulose membrane and developed with various specific anti-wheat antibodies.
Each figure is labelled with respective antibody and predicted molecular weight of
protein it is cross reacting with (right) and type of granules (left).N/A B-granules were
not available.
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granules proteome was not performed due to lower amount of these granules.
4.3.5.2.3 Quantitation of individual granule-bound proteins
To measure the amount of individual protein entrapped within A- and B-granules of
starch, silver stained gels were used and the intensity of each band was measured (see
Methods). The identity of each band was confirmed by immunoblotting similar gels with
different antibodies (see above). Silver staining revealed that all genotypes have a distinct
protein band of approximately (110 kDa) size. However this protein was not identified by
immunoblotting with antibodies available. Interestingly this 110 kDa protein was only
present in A-granules (Figure, 4.12 a & b). Quantitative analysis revealed that GBSS was
the most abundant protein in A- and B-granules of all genotypes, except CDC Rattan and
Soft Barley (Figure, 4.11). In genotypes, CDC Rattan and Soft Barley small amounts of
GBSS were present compared to the reference genotype SSIIa was the next most
abundant protein in all genotypes. However certain proteins like SSIII and unknown
protein of 110 kDa were present in small amounts in all genotypes.
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Genotypes
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Amount of protein (ng)
Amount of protein (ng)
Genotypes
Figure 4.11: Quantitation of individual granules bound proteins.
Starch granules were isolated from mature barley according to themethod describe
earlier (Peng et al., 2000). Large A- and small B-granules were separated and granulebound proteins were extracted. Equal amounts of starch, A- and B-granules were loaded
onto 4–12% acrylamide gradient gels. Following electrophoresis gels were visualized by
silver staining. Different proteins were identified by immunoblotting using specific
antibodies and some of proteins were also identified by Q-TOF-MS analysis. Amount of
individual protein was determined by measuring the intensity of each band with
Molecular Imager® Gel Doc™ XR+ and Chemi Doc™ XRS+ Systems with Image Lab™
Software, version 3 (BioRad laboratories Inc.). The measured intensity was compared
with known amount of BSA protein (100-1500 ng) which was run as standard in each
gel. Y-axis is presenting the amount of protein in (ng) while X-axis represents genotypes
in number form which are as follow, (1, OAC Kawartha; 2, McGwuire; 3, GB992033; 4,
Sloop; 5, CDC Fibar; 6, CDC Rattan; 7, Soft Barley; 8, Hard Barley; 9, AC Metcalfe; 10,
083211-120; 11, 083211-122; 12, 083311-104; 13, 083411-113; 14, AC Alberte; 15, AC
Bacon; 16, Emperor; 17, Sunderland; 18, 083511-109; 19, 083511-118. Protein quantity
in C-granules was not determined because of very low amount of proteins in these
granules.
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4.3.5.2.4 Proteomic analysis of C-granules
In order to determine the proteome of C-granules, purified B-granules were used to
obtain C-granules (see Section 4.2.2.1). The size of these granules was determined by
light and electron microscopy. In Figure (4.13) OAC Kawartha is presented as an
illustration for size determination of different types of purified granules. Silver staining
revealed that C-granules exhibited significant qualitative and quantitative differences
compared to A- and B-granules. Particularly the amount of individual proteins was
decreased in C-granules of all genotypes compared to A- and B-granules (4.12c).
4.3.5.3 Phosphorylation of proteins in starch granule
Pro-Q diamond staining was employed to study phosphorylation of different granulebound proteins and it was found that some of the starch granule proteins were
phosphorylated. GBSS is the most abundant protein in the starch granule and this protein
showed very strong indication of protein phosphorylation in all genotypes (Figure 4.14).
After GBSS, the second most abundant protein in barley granule was SSIIa and the data
suggest that SSIIa in the starch granules is also phosphorylated in all genotypes (Figure
4.14). The other major granule-bound protein which showed evidence of phosphorylation
was SBEIIb (Figure 4.14). SSI, which is also found within the starch granules, showed
evidence of phosphorylation. In all genotypes a band of 110 kDa was observed but this
band was not found to be phosphorylated. In the barley starch granules along with the
above mentioned proteins, other proteins (SSIII and SBEIIa) were also present, but these
proteins were not phosphorylated. Overall, GBSS1, SSI, SSIIa, and SBEIIb were found
to be phosphorylated in their granule-bound state.
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Figure 4.12: Granule-bound proteins (starch, A- and C-granules) visualized by silver
staining.
Figure 12, Detection of individual starch synthesizing enzyme bound with in starch, Aand C-granules from different genotypes of barley. No differences were observed
between A- and B-granules, therefore, silver stained gels of B-granules are not
presented. Equal amounts (1.5 mg) of starch, A- and C-granules were loaded onto 4–12
% acrylamide gradient gels. Following electrophoresis, proteins were visualized by silver
staining. Protein markers, molecular mass (left hand side) kDa. Right hand side-different
bands are indicated by numbers, (1, SSIII; 2, SBEIc; 3, unknown; 4, 5, 6, SSIIa, SBEIIa,
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SBEIIb; 7, SSI; 8, GBSS; 9, degraded GBSS and SSs ). SSIIIa was present in small amounts
in starch and A-granules in all genotypes.
Figure 4.13: Analysis of starch composition and granule morphology by light
microscope (A, B & C) and electron microscope (D & E).
Separation of A- and B-/C-granules was performed according to method described
earlier (Peng et al., 2000). Total starch containing, A-, B- and C-granules (A). Purified Agranules (B). Purified B-/C-granules (C). Electron micrograph of total starch, with A-, Band C-granules (D). Purified B-/C-granules (E). The reference genotype, OAC Kawartha is
presented as an illustration for A- and B-/C-granules purity confirmation in all genotypes
studied here. The scale bar on panels A, B and C represents 20 µm. Scale bar for electron
micrograph is given under each panel. Each type of granule is presented by a colored
arrow.
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Figure 4.14: Phosphorylation of starch granule associated proteins.
To determine phosphorylation of granule-bound proteins equal amounts (1.5 mg) of
starch from all genotypes were run onto 4–12% acrylamide gradient gels. Following
electrophoresis the gels were stained with Pro-Q Diamond according to the
manufacturer’s instructions (Molecular Probes) in the dark for 90 min. Following
distaining and washing, the gels were visualised using a Typhoon scanner (Amersham
Biosciences) with an excitation wavelength of 530nm and emission filter at 580nm. After
Pro-Q Diamond staining the same gels were subject to silver staining to visualize all
bands. The bands visualized with silver stain are numbered as (1, SSIII; 2, SBEIc; 3,
unknown; 4, 5, 6, SSIIa, SBEIIa, SBEIIb; 7, SSI; 8, GBSS).
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4.4 Discussion
The present study investigated the physiochemical properties and the biochemical
markers of starch biosynthesis from various barley genotypes in an attempt to identify
biochemical characteristics responsible for useful starch phenotypes. To determine
possible interactions between different physiochemical properties of the seed and starch,
Principal Component Analysis (PCA) was performed (Table 4.5). It was found that seed
characteristics and type such as: hulless or covered seed; two rows or six rows; seed
length, width, thickness and seed length; and width ratio, did not correlate with different
physiochemical properties of starch, e.g. amylopectin chain length distribution and
gelatinization temperature. However, seed length, width and thickness showed a positive
correlation with thousand grain weight, in which seed width showed a higher correlation
(r = 0.78, p < 0.01) with TGW. PCA also demonstrates that RS (r = -0.79, p < 0.01) and
amylose (r = -0.52, p < 0.01) contents are negatively related to starch content, while RS is
positively (r = 0.59, p < 0.01) related to amylose content. Similar results have been
reported earlier in barley (Asare et al., 2011). Analysis of different sizes of granules
demonstrates that A-granules are a major contributor to the total mass of starch, by
weight, while B-granules contributed in total granule number. Previously, it was reported
by Evers et al. (1973) that small size starch granules account for > 90 % of total granule
number but contribute < 30 % in total starch weight of wheat endosperm. Raeker et al.
(1998) also reported that small granules of size 1–10 µm make up to 99 % of total
granule number in wheat. PCA also demonstrated that starch content was negatively (r =
-0.57, p < 0.01) correlated with total granule-bound protein. In waxy or low amylose
genotypes, lower iodine binding was observed due to lack of amylose resulting in the
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Table 4.5: Correlation between different physiochemical properties of starch determined by principal component analysis.
Variables
SL (mm)
SW (mm)
ST (mm)
L:W
TGW
Starch Content
% B Granules
% A granules
RS
Amylose
TGBP
SL
(mm)
1
0.22
-0.26
0.64
0.44
-0.46
-0.39
0.39
0.37
0.41
0.32
SW
(mm)
0.22
1
0.71
-0.60
0.78
-0.27
-0.19
0.19
0.19
0.15
0.32
ST
(mm)
-0.26
0.71
1
-0.72
0.51
-0.08
-0.14
0.14
0.14
0.05
0.27
L:W TGW
0.64
0.44
-0.60
0.78
-0.72
0.51
1 -0.25
-0.25
1
-0.19 -0.59
-0.17 -0.26
0.17
0.26
0.16
0.40
0.22
0.34
0.01
0.40
Starch
Content
-0.46
-0.27
-0.08
-0.19
-0.59
1
0.33
-0.33
-0.79
-0.52
-0.57
%B
%A
Granules granules
-0.39
0.39
-0.19
0.19
-0.14
0.14
-0.17
0.17
-0.26
0.26
0.33
-0.33
-1.00
1
-1.00
1
-0.39
0.39
-0.36
0.36
-0.14
0.14
RS
Amylose
0.37
0.41
0.19
0.15
0.14
0.05
0.16
0.22
0.40
0.34
-0.79
-0.52
-0.39
-0.35
0.39
0.36
0.59
1
0.59
1
0.84
0.69
TGBP
0.32
0.32
0.27
0.01
0.40
-0.57
-0.14
0.14
0.84
0.69
1
SL= Seed length, SW= Seed width, ST= Seed thickness, L:W= length to width ratio, TGW= Thousand grain weight, RS= Resistant
starch, TGBP= Total granule-bound protein
Principal Component Analysis (PCA) was used to determine relationship between different physiochemical properties of starch and
biplots and correlation table were generated. But only correlation table with selected variables are shown in the Table (4.5).
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dilution of dark blue color (Figure, 4.1). However, in the low amylose genotype, Soft
Barley relatively dark blue color compared to waxy genotypes was observed which
indicates the presence of a certain amount of amylose or extra-long unit chains (ELCs) in
this genotype, even though GBSS is low or absent (Figure 4.1).
PCA also shows that different gelatinization temperatures are positively
correlated with amylopectin chain length with different DP. Starch gelatinization is
demonstrated by two endothermic peaks. The first endothermic peak shows the transition
phase of starch upon heating from an ordered granule structure to a random coil state in
the presence of excess water. The second peak represents the disruption of the amyloselipid complex (Kugimiya, 1980). Similarly, for retrograded starch, the transition phase is
represented by two endothermic peaks. The first peak shows the retrogradation of
retrograded starch to a random coil state upon heating, and the second peak shows
disruption of amylose-lipid complex. The results indicate that the waxy genotype has a
higher ∆H and To than normal amylose genotypes. In contrast, amylose extender (ae)
and waxy amylose extender (wx ae) of rice and maize showed higher gelatinization peak
temperature (Tp) and transition enthalpy (∆H) than wild-type, and waxy because of their
many long branch-chain of ae amylopectin (Kubo et al., 2010; Shi et al., 1995; Cooke
and Gidley, 1992). Our results indicate that, in barley, amylopectin chain length
distribution from different genotypes possessing varying proportions of amylose does not
exhibit significant variations in chain length with DP 6-36, compared to the reference
genotype or waxy cultivars (Tables 4.1 & 4.3). This is consistent with previous reports
showing that the amylopectins of different barley genotypes, possessing varied amylose
content are similar in structure (Yoshimoto et al., 2002, Schulman et al., 1995; Takeda et
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al., 1999; Yoshimoto et al., 2000 Czuchajowscka et al., 1998; MacGregor & Morgan,
1984; Song and Jane, 2000; Tester and Morrison, 1990). Thus similar amylopectin
structure appeared to be characteristic for barley cultivars with altered amylose content.
A difference plot (waxy -WT) shows that long amylopectin glucan-chains are in higher
proportion in waxy barley compared to reference genotype and other genotypes (Figure
4.8 ). Previously, You and Izydorczyk, (2007) reported that ∆H values were associated
with the amount of double helical domains of amylopectin and single helical structure
(amylose–lipid complexes). Similarly, others (Zheng et al., 1998; Morrison et al., 1993;
Cooke and Gidley, 1992; Li et al., 2001; Yoshimoto et al., 2000; You and Izydorczyk,
2007) reported that (∆H) values reflect the loss of double helical order rather than loss of
X-ray crystallinity. Tester and Morrison (1990) and Li et al. (2001) reported that ∆H is an
estimation of crystallinity and Tp can be used to measure the crystallite quality, and To
has a significant correlation with average branch chain length (Jane et al., 1999; Yuan et
al., 1993; Wang et al., 1993 and Shi and Seib, 1992). Thus, the higher ∆H of the waxy
line is probably due to the higher proportion of amylopectin, because higher amylopectin
content will lead to greater crystallinity, and hence more energy will be required to break
these interactions. Similarly, waxy variety CDC Fibar exhibits the highest onset
temperature of gelatinization. This indicates that CDC Fibar has higher amounts of
crystallites and double helical domains than starches from other genotypes, since the
onset temperature (To) corresponds to the melting of crystallites of starch, mainly
derived from amylopectin. Based on the results it is argued that, in barley, increased
amylopectin provide more stability to the starch granule against gelatinization, as a result
of increased crystalline organization as oppose to other cereals. Different genotypes
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showed significant differences in Tc–To in line 083411-113, showed a wider Tc–To
value than other genotypes. Previously Biliaderis et al. (1980) proposed that the degree of
branching of amylopectin influences Tc–To i.e. the greater the degree of branching, the
wider the melting temperature range. It is also possible that Tc–To may also indicate the
degree of heterogeneity of the starch crystallites. Thus it can be speculated that 083411113 has more branched amylopectin, with a higher proportion of short glucan chains of
DP 6-12, as shown in Figures (4.7, 4.8 & 4.9). Li et al. (2001) reported that Tc–To is
influenced to a large extent by the proportion of small granules. For example, starches
with a high proportion of small granules exhibited the wider Tc–To (Li et al., 2001). In
contrast, our results show that 083411-113 has a significantly reduced proportion of small
granules (Figure 2B) and a wider “Tc–To” (Table 2) compared to wild-type and waxy,
(which have considerable amounts of B-granules but narrower Tc–To). Thus it can be
hypothesized that Tc-To is mainly influenced by chain length distribution.
Starch granule-associated proteins from all genotypes (starch, A- and B-granules)
were detected by immunoblotting with different antibodies (see Methods). Analysis of
granule-bound proteins did not reveal significant variation in the granule-associated
proteome of starch, A- or B-granules from different genotypes compared to normal
reference genotype, with the exception of waxy and low amylose genotypes (CDC Fibar
and Soft Barley) which have significantly reduced amount of GBSS. In all genotypes, a
core group of granule-associated proteins was detected including SSI, SSIIa, SBEIIa,
SBEIIb and GBSSI. These proteins (SSI, SSIIa, SBEIIb and GBSSI) are also routinely
found within the starch granules of other cereals like maize, wheat, barley and rice
(Rahman et al., 1995; Morell et al., 2003; Boren et al., 2004; Regina et al., 2005;
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Umemoto and Aoki, 2005; Liu et al., 2009, 2012a, 2012b). Along with these proteins,
other proteins (SSIII, SBEIc, unknown protein of 110 kDa) were also found in all
genotypes. SBEIc and an unknown protein of 110 kDa were absent from B-granules. The
amount of individual starch granule proteins involved in starch biosynthesis in the
granules was also measured from total starch, A- and B-granules from all genotypes (see
methods). Only minor differences were observed in the amount of different proteins.
Previously it was reported that alteration/mutation in specific starch biosynthetic enzymes
results in the alteration of starch granule proteome of mutants (Morell et al., 2003;
Grimaud et al., 2008; Li et al., 2011; Liu et al., 2012a, 2012b; Chapter, 3). However, in
this study such variations have not been found in the starch granule proteome, which
suggests that these genotypes do not have mutations in the major isoforms of different
proteins involved in starch biosynthesis (other than GBSS). Evidence suggests that most
of these starch granule-associated proteins are components of previously identified,
soluble protein complexes, and that the formation of these protein complexes is
phosphorylation dependent (Tetlow et al., 2008; Hennen-Bierwagen et al., 2008; Liu et
al., 2009, 2012a, 2012b; Chapter 3). These functional protein complexes become
entrapped within starch granules, resulting in soluble proteins becoming granule-bound
(Liu et al., 2009). Thus, association of different proteins within the growing starch
granules is, at least partially, a reflection of the formation of stromal functional
heteromeric protein complexes involved in amylopectin biosynthesis. Analyzing the
granule proteome of various genotypes, may provide an indication of the existence of
protein complexes in the stroma. Our results show that GBSS, SSI, SSIIa, and SBEIIb are
phosphorylated in their granule-bound state. It was also reported previously that in wheat,
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GBSS, SSI and SSII were phosphorylated in their starch granule-bound state (Bancel et
al., 2010). Similarly phosphorylation of GBSS, SBEIIb, and SP in maize and wheat was
reported by Grimuad et al. (2008) and Tetlow et al. (2008). It is also possible that some
granule-bound proteins are phosphorylated and granule-associated as free, noncomplexed forms.
Barley has a bimodal starch granule size distribution in which granules can be
divided into large A- and small B-granules or C-granules. The biochemical processes
underlying synthesis of A- and B-granules is not well understood. Peng et al. (2000)
reported that, in wheat, one peak of granule nuclei formation occurs before 15 DPA and
another after 15 DPA. In the second stage of development, A-granules grow larger than
10 µm, while B-granules lack this ability. The underlying reason for this is not known. In
barley, soluble SSs are thought to have a role in determination of granule size. For
example, a mutation at the barley shx locus results in reduced SSI activity and a reduction
in the size of A-granules, transforming the normal bimodal granule size distribution to
unimodal (Schulman and Ahokas, 1990; Tyynela and Schulman, 1993; Tyynela et al.,
1995). There is evidence that, along with starch biosynthetic enzymes, synthesis of Aand B-granules is controlled by other molecular factors. Stamova et al. (2009) reported
that the rate of the change in volume (%) of the A-granule was correlated with the
expression profiles of two unknown genes (BE422634, r = 0.832 and BE438268, r =
0.922), a putative DEAD box RNA helicase (r = 0.849), and chitinase2 (r = 0.811). The
number and volume distributions of B-granules, the ratio of B- to A-granules in terms of
their volume and number predominantly correlated with the changes in transcriptional
expression of storage protein genes (Stamova et al., 2009). The percent number of A-
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granules has been shown to be correlated with the expression profiles of genes encoding
several transcription factors and other proteins involved in carbohydrate metabolism such
as, Glucose-6-phosphate dehydrogenase and GBSSII (Stamova et al., 2009). In this study,
variation in granule size distribution and (%) amount of small granules in total mass of
starch in different barley genotypes have been found. However, no significant differences
have been observed in the granule proteome analysis of A- and B-granules from different
genotypes. Similarly, phospho-proteome analysis from different genotypes did not reveal
significant variations. Thus, it can be deduced that granule size distribution and %
amount of A-, B- and C-granules may be regulated by different factors other than
enzymes which become entrapped within the granule. Our results also show that very
small C-granules have significantly reduced amounts of SSI, SSIIa, SBEIIa and SBEIIb
proteins (Figure, 4.12c) per unit (1.5 mg) of starch. The possible reason for this reduction
in the amount of protein may be related to large surface area to volume ratio of very
small granules compared to large granules. However, more detailed molecular and
biochemical analyses are required to understand the regulation of synthesis of A- and Bgranules.
4.5 Conclusion
This study provides detailed physiochemical and biochemical analyses of
barley genotypes with varying proportions of A- and B-granules and amylose content.
Different physical and morphological properties of the seed did not correlate with
physiochemical and biochemical properties of starch. The detailed biochemical analyses
of granule-bound proteins did not show significant differences, except that B-granules
lacked SBEIc and an unknown granule protein of 110 kDa. The phospho-proteome
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analysis showed that GBSS, SSI, SSIIa, and SBEIIb are phosphorylated in their granulebound state in A- and B-granules. The ratios of different granule-bound proteins with
respect to SSI, SSIIa, SBEIIa and SBEIIb were not altered in different genotypes.
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Chapter 5: General discussion and future work
- 205 -
5.1 General Discussion
In this thesis, detailed analyses of the physiochemical properties of resistant
starch, and potentially important biochemical mechanisms, of RS biosynthesis, have been
investigated in barley. RS has significant beneficial effects on human health (Asare et al.,
2011). There are different types of RS (reference to Section, 1.3.2.1.1.1.1.1). RSII is most
important because it is naturally occurring starch in its granular form which is resistant to
digestion by α-amylase. The natural occurrence of RSII makes it possible to study
variations in the physiochemical properties of RS, as well as alterations in the
biochemical pathway of starch biosynthesis which give rise to the formation of RS.
Barley has proved a very useful model system of starch biosynthesis given its diploid
nature, ease of transformation, and large number of starch mutants and genotypes
available. Given the similarities in granule structure between wheat and barley, barley
serves as an ideal model system for studying cereal starch biosynthesis.
5.1.1 Analysis of the physiochemical properties of RS as determinant of increased
RS genotypes
To determine the relationship between seed and starch physicochemical
properties, and resistant starch, a wide range of barley genotypes from different sources
were selected to screen for their RS content. Analysis of these genotypes showed that
there are certain physico-chemical properties of the seed and starch which can be used as
an indication of RS content. A correlative analysis suggested that increased RS is
associated with higher amylose content. Previously it was also reported that amylose is
the principal component in the formation of RSII (Sajilata et al., 2006; Asare et al.,
2011). Previous reports suggested that during cooking when starch is gelatinized amylose
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leaches out of the swollen starch granules as coiled polymers which upon cooling
associate as double helices and form hexagonal networks (Jane and Robyt, 1984;
Haralampu et al., 2004). In waxy starches this network does not form where instead,
aggregate formation occurs which is more susceptible to α-amylase hydrolysis (Miao et
al., 2009). The results presented in Chapter 2 indicated that increased amylose is
associated with an alteration in proportion of small granules to large granules. All the
genotypes with increased amylose exhibited a higher proportion of small granules (Figure
2.4). Similarly in all the high amylose genotypes starch granules with altered morphology
were observed compared to waxy and reference genotypes (Figure 2.5). The data suggest
that these two characters can be used as physical markers for high amylose genotypes.
The major challenge in the development of high amylose genotypes is reduced yield. The
known high amylose mutants of barley, sex6 and sbeiia-/sbeiib- double mutant (> 60 %
amylose) exhibited reduced starch content (Morrell et al., 2003: Regina et al., 2010).
Similarly our data suggest that amylose content is negatively related to starch content and
seed size. None the less, during this study certain genotypes, 081011-928, 081011-929
and 081011-930, were identified which exhibited significantly higher amylose content
than the reference genotype while starch content and TGW were comparable to the
reference genotype (Table 2.1). It is proposed that these lines can be used for further
breeding programs to develop high amylose barley genotypes without loss of yield.
5.1.2 Regulation of starch biosynthesis in the barley endosperm is governed by the
formation of multi-enzyme protein complexes
In the recent past, several studies have shown the existence of physical
interactions between enzymes of starch biosynthesis. In a study by Tetlow et al. (2004a)
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with wheat endosperm, it was shown that SBEI, SBEIIb and SP form phosphorylationdependent multi-enzyme complexes. Similarly in the maize endosperm, the existence of
physical interactions among major isoforms of starch biosynthetic enzymes in the form of
protein complexes has also been reported (Hennen-Bierwagen et al., 2008; Liu et al.,
2009, 2012a, 2012b). These studies were further developed by determining the existence
of possible protein complexes in the barley endosperm (Chapter, 2, 3 & 4). Similar to
maize and wheat, protein-protein interactions between SSs and SBEs were also observed
in barley, suggesting that protein-protein interactions between starch biosynthetic
enzymes are a common feature in cereal endosperm.
Analysis of defined barley mutants revealed novel protein-protein interactions due
to single gene mutations. In the SBEII mutants, SBEI, SP and the expressed form of
SBEII, were clearly observed forming multi-enzyme complexes. However, since anti-SSI
antibody did not co-precipitate SBEI and SP, it can be argued that in the absence of one
or other isoform of SBEII, SBEI and SP interact with the remaining expressed form of
SBEII. Liu et al. (2009) also reported a similar protein complex in the ae mutant of maize
(lacking SBEIIb protein) in which SBEI, SBEIIa and SP were clearly observed, although
in that case they appeared to form multi-enzyme complexes with SSI and SSIIa which is
not the case here. In the amo1 mutant, both SBEIIa and SBEIIb were coimmunoprecipitated with SSI and SSIIa proteins. A number of possibilities might account
for this observation. It is possible that SBEIIa and SBEIIb were co-purified together as a
result of forming a dimer, or that SBEIIa and SBEIIb interact with SSI and SSIIa
independently. In the sex6 mutant (lacking SSIIa protein) interactions among different
isoforms of starch biosynthetic enzymes were not observed reinforcing the centrality of
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SSIIa in forming protein complexes (Liu et al., 2012b).
The importance of interactions among different enzymes of starch biosynthesis
pathway was revealed by the study of different mutants. For example, in either of the
branching enzyme II mutants studied, SSI activity was significantly decreased (Chapter
3). This may indicate that association, or presence, of both SBEII proteins is required for
SSI activity. Similarly, SP activity was not observed in the sbeiib- mutant (Chapter 3),
suggesting that in vivo association of SBEIIb with SP might confer a positive regulatory
effect on SP activity. Similarly, in the sex6 mutant (lacking SSIIa), SSI activity increased,
suggesting that SSIIa possibly has a negative regulatory effect on SSI activity. Detailed
biochemical analysis of granule proteins revealed that in the SBEII mutants, different
protein complexes are involved in the synthesis of A- and B-granules. It is argued that a
complex containing SSI/SSIIa and either form of expressed SBEII is involved in the
synthesis of A- and B-granules, whereas protein complexes containing SBEI and SP are
involved only in the synthesis of A-granules. This hypothesis was supported by detailed
biochemical analysis of starch granules in which SBEI and SP were only present in the
A-granules.
Functional association of key enzymes involved in starch biosynthesis within
multi-enzyme complexes may enhance the efficiency of amylopectin construction. For
example, products of SSs become the substrates of SBEs, and the products of SBEs in
turn become the substrates of SSs. Previously, abnormal amylopectin structures were
observed in the mutants lacking key starch biosynthetic enzymes (Yuan et al., 1993; Shi
and Seib, 1995; Klucinec and Thompson, 2002; Morell et al., 2003; Zhang et al., 2004;
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Delvalle et al., 2005; Regina et al., 2010; Liu et al., 2009, 2012a), which suggests a
higher level of organization indicative of multi-enzyme coordination.
5.1.3 Formation of multi-enzyme complexes containing starch biosynthetic enzymes
in barley endosperm is regulated by protein phosphorylation
The evidence suggests that the physical interactions between isoforms of starch
synthases (SSs) and branching enzymes (SBEs) in cereal endosperm amyloplasts are
driven by protein phosphorylation (Hennen-Bierwagen et al., 2008; Liu et al., 2009,
2012; Tetlow et al., 2004a, 2008). In barley, co-immunoprecipitation experiments
indicated that conditions that would be expected to favour protein phosphorylation (+
ATP) lead to an increase in protein complex formation. It also appears that SSI, SSIIa,
SBEIIb, SP and SBEI are phosphorylated in their granule-bound state arguing that it is
complexes carrying phosphorylated proteins which become entrapped in the starch
granule.
5.1.4 Physiochemical properties of seed and starch do not correlate with starch
granule proteome
As described earlier, using a wide range of genotypes from different genetic back
grounds, amylose/RS contents were measured (Chapter 2). From these genotypes,
detailed biochemical analyses of high amylose mutants with known mutations in
amylopectin synthesizing enzymes were performed (Chapter 3). Similarly, detailed
physiochemical analyses of starch and analysis of granule proteome of the remaining
genotypes where the source of variation was unknown was also performed (Chapter 4).
The results presented indicate that these genotypes exhibited variations in physiochemical
properties of seed and starch. However, analysis of the starch granule proteome did not
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reveal variation among different genotypes, except for waxy genotypes which could
account for this (Chapter 4). From these genotypes, OAC Kawartha, CDC- Fibar, Soft
Barley, Hard Barley, AC Metcalfe and 083411-113 were further selected on the basis of
parameters described in section (4.3.3). Analysis of soluble proteins (involved in starch
biosynthesis) from amyloplast lysates of these genotypes also did not reveal significant
variation. The starch granule proteome of genotypes with no characterized mutation in
amylopectin synthesizing enzymes did not correlate to variations in the physiochemical
properties of seeds and starch. This argues that the source of variation in physiochemical
properties of seed and starch for these cultivars lie elsewhere and are independent of
starch granule proteome. Results from chapter (2, 3 & 4) indicate that starch granule
proteome is not always indicative of variation in starch physiochemical properties.
However, the starch granule proteome can be used to identify mutations in starch
biosynthetic enzymes and variations in starch physiochemical properties, when mutations
are in the pathway of starch biosynthesis (Chapter 3).
This study also showed that in high amylose/RS genotypes, starch exhibits
unimodal granule size distribution due to an increased proportion of small granules
compared to large granules and % RS/amylose was positively related to the proportion of
small granules (Banks et al., 1971; You and Izydorczyk, 2002: Asare et al., 2011).
Amylose content and starch granule size related strongly with some seed dimensions and
physiochemical properties. Production of starch with desired characteristics such as high
RS is often associated with a reduction in starch content and yield. As stated earlier, this
study recommends genotypes, 081011-928, 081011-929 and 081011-932 for further
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breeding programs to develop genotypes high not only in RS, but also having starch
content comparable to normal or reference genotype.
5.2 Future Work
In this study an effort has been made to study the biochemical processes involved
in the synthesis of RS. Future work should be aimed at understanding the involvement of
post-translational modifications, particularly protein-protein interactions in the synthesis
of RS. For example, what protein kinases and phosphatases are involved and how would
their modification affect starch synthesis and the type of starch produced. Similarly,
experiments which analyse the physiological significance of the observed multi-enzyme
complexes in relation to the synthesis of A- and B-granules, are needed. Finally use of
transgenic plants with modifications which prevent specific protein-protein interactions,
and consequently the synthesis of particular classes of granule in vivo, would be a
powerful tool to study the physiological functions of protein complexes.
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REFERENCES
- 213 -
Abad MC, Binderup K, Rios-Steiner J, Arni RK, Preiss J, Geiger JH. 2002. The Xray crystallographic structure of Escherichia coli branching enzyme. The Journal of
Biological Chemistry, 277, 42164–42170.
Abel GJW, Springer F, Willmitzer L, Kossman J. 1996. Cloning and functional
analysis of a cDNA encoding a novel 139 kDa starch synthase from potato (Solanum
tuberosum L.). The Plant Journal, 10, 981–991.
Ajithkumar A, Andersson R, Christerson T, Åman P. 2005. Amylose and β glucan
content of new waxy barleys. Starch/Stärke, 57, 235–239.
Alexander RD, Morris PC. 2006. A proteomic analysis of 14-3-3 binding proteins from
developing barley grains. Proteomics, 6, 1886-1896.
Ao Z, Jane JL. 2007. Characterization and modeling of the A- and B-granule starches of
wheat, triticale, and barley. Carbohydrate Polymers, 67, 46–55.
Asare EK, Jaiswal S, Maley J, Baga M, Sammynaiken R, Rossnagel BG, Chibbar
RN. 2011. Barley grain constituents, starch composition, and structure affect starch in
vitro enzymatic hydrolysis. Journal of Agricultural and Food Chemistry, 59, 4743–4754.
Bae JM, Giroux M, Hannah L. 1990. Cloning and characterization of the brittle-2 gene
of maize. Maydica, 35, 317–322.
Baghurst PA, Baghurst KI, Record SJ. 1996. Dietary fibre, non-starch polysaccharides
and resistant starch–a review. Food Australia, 48, S3–S35.
Ball S, Guan HP, James M, Myers AM, Keeling P, Mouilee G, Buleon A, Colonna P,
Preiss J. 1996. From glycogen to amylopectin: a model for the biogenesis of the plant
starch granule. Cell, 86, 349–352.
Ball SG, Deschamps P. 2009. Starch metabolism. In The Chlamydomonas Sourcebook,
ed. DB Stern, EHH Harris, pp. 2–40. Amsterdam: Elsevier. 2nd ed.
Ball SG, Morell MK. 2003. From bacterial glycogen to starch: Understanding the
biogenesis of the plant starch granule. Annual Review of Plant Biology, 54, 207–233.
Ballicora MA, Iglesias A, Preiss J. 2004. ADP-glucose pyrophosphorylase: a regulatory
enzyme for plant starch synthesis. Photosynthesis Research, 79, 1–24.
Bancel E, Rogniaux H, Debiton C, Chambon C, Branlard G. 2010. Extraction and
proteome analysis of starch granule-associated proteins in mature wheat kernel (Triticum
aestivum L.). Journal of Proteome Research, 9, 3299–3310.
Banks W, Greenwood CT, Muir DD. 1974. Studies on starches of high amylose
content: part 17. A review of current concepts Starch/Stärke, 26, 289–300.
- 214 -
Baum B R, Baily LG. 1987. A survey of endosperm starch granules in the genus
Hordeum : a study using image analytic and numerical taxonomic techniques. Canadian
Journal of Botany, 65(8), 1563–1569.
Beatty MK, Rahman A, Cao H, Woodman W, Lee M, Myers AM, James MG. 1999.
Purification and molecular genetic characterization of ZPU1, a pullulanase- type starchdebranching enzyme from maize. Plant Physiology, 119, 255–266.
Bechtel DB, Wilson JD. 2003. Amyloplast formation and starch granule development in
hard red winter wheat. Cereal Chemistry, 80(2), 175–183.
Bechtel DB, Zayas I, Kaleikau L, Pomeranz Y. 1990. Size distribution of wheat starch
granules during endosperm development. Cereal Chemistry, 67, 59–63.
Beckles DM, Smith AM, Rees T. 2001. A cytosolic ADP-glucose pyrophosphorylase is
a feature of graminaceous endosperms, but not of other starch storing organs. Plant
Physiology, 125, 818–827.
Benmoussa M, Moldenhauer KAK, Hamaker BR. 2007. Rice amylopectin fine
structure variability affects starch digestion properties. Journal of Agricultural and Food
Chemistry, 55, 1475–1479.
Benmoussa M, Suhendra B, Aboubacar A, Hamaker BR. 2006. Distinctive sorghum
starch granule morphologies appear to improve raw starch digestibility. Starch-Starke,
58, 92–99.
Bennett MD, Smith JB, Barclay I. 1975. Early seed development in the Triticeae.
Philosophical Transactions of the Royal Society B: Biological Sciences. 272, 199–227.
Bertoft E, Kulp SE. 1986. A gel filtration study on the action of barley-amylase
isoenzyme on granular starch. Journal of the Institute of Brewing, 92, 69–72.
Sharma A, Yadav BS, Ritika BY. 2008. Resistant starch: physiological roles and food
applications. Food Reviews International, 24, 193–234.
Bhattacharyya P, Ghosh U, Chowdhuri UR, Chattopadhyay P, Gangopadhyay H.
2004. Effects of different treatments on physico-chemical properties of rice starch.
Journal of Scientific & Industrial Research, 63, 826-829.
Bhatty RS, Rossnagel BG. 1992. Zero amylose lines of hull-less barley. Cereal
Chemistry, 74, 190–191.
Bhatty RS, Rossnagel BG. 1998. Comparison of pearled and unpearled Canadian and
Japanese barleys. Cereal Chemistry, 75, 15–21.
- 215 -
Bhave MR, Lawrence S, Barton C, Hannah LC. 1990. Identification and molecular
characterization of Shrunken-2 cDNA clones of maize. The Plant Cell, 2, 581–588.
Biliaderis CG, Maurice TJ, Vose JR. 1980. Starch gelatinization phenomena studied by
differential scanning calorimetry. Journal of Food Science, 45, 1669–1674.
Birkett A, Muir J, Phillips J, Jones G, O’Dea K. 1996. Resistant starch lowers fecal
concentrations of ammonia and phenols in humans. The American Journal of Clinical
Nutrition, 63(5), 766-772.
Blauth SL, Kim KN, Klucinec JD, Shannon JC, Thompson DB, Guilitinan MJ.
2002. Identification of Mutator insertional mutants of starch-branching enzyme 1 (SBEI)
in Zea mays L. Plant Molecular Biology, 48, 287–297.
Blauth SL, Yao Y, Klucinec JD, Shannon JC, Thompson DB, Guilitinan MJ. 2001.
Identification of Mutator insertional mutants of starch-branching enzyme 2a in corn.
Plant Physiology, 125, 1396–1405.
Borén M, Mikkel A. Glaring HG, Helena O, Andreas B, Christer J. 2008. Molecular
and physicochemical characterization of the high-amylose barley mutant Amo1. Journal
of Cereal Science, 47, 79–89.
Borén, M, Larsson H, Falk A, Jansson C. 2004. The barley starch granule proteome:
internalized granule polypeptides of the mature endosperm. Plant Science 166, 617–626.
Bosnes M, Weideman F, Oslen OA. 1992. Endosperm differentiation in barely wildtype and sex mutants. The Plant Journal, 2, 661-674.
Bowsher CG, Scrase-Field EFAL, Esposito S, Emes MJ, Tetlow IJ. 2007.
Characterization of ADP-glucose transport across the cereal endosperm amyloplast
envelop. Journal of Experimental Botany, 58, 1321-1332.
Boyer CD, Preiss J. 1979. Properties of citrate-stimulated starch synthesis catalyzed by
starch synthase I of developing maize kernels. Plant Physiology, 64, 1039–1042.
Boyer CD, Preiss J. 1981. Evidence for independent genetic control of the multiple
forms of maize endosperm branching enzymes and starch synthases. Plant Physiology,
67, 1141–1145.
Brown PH, Ho THD. 1986. Barley aleurone layers secrete a nuclease in response to
gibberellic acid. Purification and partial characterization of the associated ribonuclease,
deoxyribonuclease, and 3- nucleotidase activities. Plant Physiology, 82, 801–806.
Brown RC, Lemmon BE, Olsen OA. 1994. Endosperm development in barley:
mircotubule involvement in the morphogenetic pathway. The Plant Cell, 6, 1241–1252.
- 216 -
Burton RA, Jenner H, Carrangis L, Fahy B, Fincher GB, Hylton C, Laurie DA,
Parker M, Waite D, van Wegen S, Verhoeven T, Denyer K. 2002. Starch granule
initiation and growth are altered in barley mutants that lack isoamylase activity. The
Plant Journal, 31, 97–112.
Buschiazzo A, Ugalde JE, Guerin ME, Shepard W, Ugalde RA, Alzari PM. 2004.
Crystal structure of glycogen synthase: homologous enzymes catalyze glycogen synthesis
and degradation. European Molecular Biology Organization Journal, 23, 3196–3205.
Busi M, Palapoli N, Valdez HA, Fornasari MS, Wayllace NZ, Gomez-Casati DF,
Parisi G, Ugalde RA. 2008. Functional and structural characterization of the catalytic
domain of the starch synthase III from Arabidopsis thaliana. Proteins, 70, 31-40.
Bustos R, Fahy B, Hylton CM, Seale R, Nebane NM, Edwards A, Martin C, Smith
AM. 2004. Starch granule initiation is controlled by a heteromultimeric isoamylase in
potato tubers. Proceedings of the National Academy of Sciences, 101, 2215–2220.
Campbell MR, Pollak LM, White PJ. 1994. Dosage effect at the sugary-2 locus on
maize starch structure and function. Cereal Chemistry, 71, 464–468.
Cao H, Imparl-Radosevich J, Guan H, Keeling PL, James MG, Myers AM. 1999.
Identification of the soluble starch synthase activities of maize endosperm. Plant
Physiology, 120, 205–216.
Carciofi M, Blennow A, Nielsen MM, Holm PB, Hebelstrup KH. 2012. Barley callus:
a model system for bioengineering of starch in cereals. Plant Methods, 8, 36.
Chadwick R. 2004. Nutrigenomics, individualism and public health. Proceedings of the
Nutrition Society, 63(1), 161–6.
Champ MJ. 2004. Physiological effects of resistant starch and in vivo measurements.
Journal of the Association of Official Analytical Chemists International, 87(3), 749-55.
Chen HM, Chang SC, Wu CC, Cuo TS, Wu JS, Juang RH. 2002. Regulation of the
catalytic behaviour of L-form starch phosphorylase from sweet potato roots by
proteolysis. Plant Physiology, 114, 506-515.
Chen MH, Christine JB. 2007. Method for determining the amylose content, molecular
weights, and weight- and molar-based distributions of degree of polymerization of amylose
and fine-structure of amylopectin. Carbohydrate Polymers, 69, 562-578.
Chibbar RN, Chakraborty M. 2005. Characteristics and uses of waxy wheat. Cereal
Foods World, 50, 121–126.
Chibbar RN, Ganeshan S, Baga M, Khandelwal RL. 2004. Carbohydrate Metabolism.
In Encyclopedia of Grain Science; Wrigley, C., Ed.; Elsevier: Oxford, U.K., 2004; pp
168–179.
- 217 -
Choi H, Kim W, Shin M, 2004. Properties of Korean amaranth starch compared to waxy
millet and waxy sorghum starches. Starch-Starke 56: 469–477.
Chrastil J. 1987. Improved colorimetric determination of amylose in starches or flours.
Carbohydrate Research 159, 154–158.
Coleman CE, Larkins BA. 1999. The prolamins of maize. In PR Shewry, R Case, eds,
Seed Proteins. Kluwer Academic Publishers, Dordrecht, The Netherlands, pp 109–139.
Colleoni C, Dauvillee D, Mouille G, Morell MK, Samuel M, Slomiany MC, Lienard
L, Wattebled F, d’Hulst C, Ball S. 1999. Biochemical characterization of the
Chlamydomonas reinhardtii α-1,4 glucanotransferase supports a direct function in
amylopectin biosynthesis. Plant Physiology, 120, 1005-1014.
Colleoni C, Myers AM, James MG. 2003. One- and two-dimensional native PAGE
activity gel analyses of maize endosperm proteins reveal functional interactions between
specific starch metabolizing enzymes. Journal of Applied Glycoscience, 50, 207–212.
Colonna P, Buleon A, Lemarie F. 1988. Action of Bacillus subtilis R-amylase on native
wheat starch. Biotechnology and Bioengineering, 31, 895–904.
Colquhoun IJ, Parker R, Ring SG, Sun L, Tang HR. 1995. An NMR spectroscopic
characterization of the enzyme--resistant residue from α-amylolysis of an amylose gel.
Carbohydrate Polymers, 27, 255–259.
Commuri PD, Jones RJ, 1999. Ultrastructural characterization of maize (Zea mays L.)
kernels exposed to high temperature during endosperm cell division. Plant Cell and
Environment, 22, 375–385.
Commuri PD, Keeling PL. 2001. Chain-length specificities of maize starch synthase I
enzyme: studies of glucan affinity and catalytic properties. The Plant Journal, 25, 475486.
Comparot-Moss S, Denyer K. 2009. The evolution of the starch biosynthetic pathway in
cereals and other grasses. Journal of Experimental Botany, 60, 2481–2492.
Comparot-Moss S, Kötting O, Stettler M, Edner C. Graf A. 2010. A putative
phosphatase, LSF1 is required for normal starch turnover in Arabidopsis leaves. Plant
Physiology, 152, 685–697.
Cooke D, Gidley MJ. 1992. Loss of crystalline and molecular order during starch
gelatinization: Origin of the enthalpic transition. Carbohydrate Research, 227, 103–112.
Copeland L, Blazek J, Salman H, Tang MC. 2009. Form and functionality of starch.
Food Hydrocolloids, 23, 1527–1534. Putseys
- 218 -
Cossegal M, Vernoud V, Depege N, Rogowsky PM. 2007. The embryo surrounding
region. In OA Olsen, ed, Endosperm, Vol 8. Springer-Verlag, Berlin/Heidelberg, pp 57–
71.
Craig J, Lloyd JR, Tomlinson K, Barber L, Edwards A, Wang TL, Martin C,
Hedley CL, Smith AM. 1998. Mutations in the gene encoding starch synthase II
profoundly alter amylopectin structure in pea embryos. The Plant Cell, 10, 413–426.
Critchley JH, Zeeman SC, Takaha T, Smith AM, Smith SM. 2001. A critical role for
disproportionating enzyme in starch breakdown is revealed by a knock-out mutation in
Arabidopsis. The Plant Journal, 26, 89-100.
Crowe TC, Seligman SA, Copeland L. 2000. Inhibition of enzymic digestion of
amylose by free fatty acids in vitro contributes to resistant starch formation. Journal of
Nutrition, 130, 2006–2008.
Czuchajowska Z, Klamczynski A, Paszczynska, B, Baik BK. 1998. Structure and
functionality of barley starches. Cereal Chemistry, 75, 747–754.
Da Silva P, Oliveira J, and Rao M. 1997. The effect of granule size distribution on the
rheological behavior of heated modified and unmodified maize starch dispersion. Journal
of Texture Studies, 28, 123–138.
Dai Z, Yin Y, Zhang W. 2009. Activities of key enzymes involved in starch synthesis in
grains of wheat under different irrigation patterns. Journal of Agricultural Science, 147,
437-444.
Dauvillée D, Chochois V, Steup M, Haebel S, Eckermann N, Ritte G, Ral. JP,
Colleoni C, Hicks G, Wattebled F, Deschamps P, d’Hulst C, Liénard L, Cournac L,
Putaux JL, Dupeyre D, Ball SG. 2006. Plastidial. Phosphorylase is required for normal
starch synthesis in Chlamydomonas reinhardtii. The Plant Journal, 48, 274–285.
Dauvillée D, Dumez S, Wattebled F, Roldän I, Planchot V, Berbezy P, Colonna P,
Vyas D, Chatterjee M, Ball S, Mérida A, D’Hulst C. 2005. Soluble starch synthase I: a
major determinant for the synthesis of amylopectin in Arabidopsis thaliana leaves. The
Plant Journal, 43, 398-412.
Davis JP, Supatcharee N, Khandelwal RL, Chibbar RN. 2003. Synthesis of novel
starches in planta: opportunities and challenges. Starch-Stärke, 55, (3-4), 107–120.
de Rocquigny P. 2011. Aiming for higher wheat yields, crops e-news published by
Manitoba agriculture, food and rural initiatives, Canada.
Deeg R, Kraemer W, Ziegenhorn J. 1980. Kinetic determination of serum glucose by
use of the hexokinase/glucose-6-phosphate dehydrogenase method. Journal of Clinical
Chemistry & Clinical Biochemistry, 18(1), 49-52.
- 219 -
Delatte T, Trevisan M, Parker ML, Zeeman SC. 2005. Arabidopsis mutants Atisa1
and Atisa2 have identical phenotypes and lack the same multimeric isoamylase, which
influences the branch point distribution of amylopectin during starch synthesis. The Plant
Journal, 41, 815–830.
Dengate H., Meredith P. 1984. Variation in size distribution of starch granules from
wheat grain. Journal of Cereal Science, 2, 83-90.
Denyer K, Dunlap F, Thorbjornsen T, Keeling P, Smith AM. 1996. The major form
of ADP-glucose pyrophosphorylase in maize endosperm is extra-plastidial. Plant
Physiology, 112, 779-785.
Denyer K, Johnson P, Zeeman S, Smith AM. 2001. The control of amylose synthesis.
Journal of Plant Physiology, 158, 479–487.
Denyer K, Smith AM. 1992. The purification and characterization of two forms of
soluble starch synthase from developing pea embryos. Planta, 186, 609–617.
Denyer K, Waite D, Edwards A, Martin C, Smith AM. 1999. Interaction with
amylopectin influences the ability of granule-bound starch synthase I to elongate
maltooligosaccharides. Biochemical Journal, 342, 647–653.
Denyer K, Waite D, Motawia S, Møller BL, Smith AM. 1999. Granule-bound starch
synthase I in isolated starch granules elongates malto-oligosaccharides processively.
Biochemical Journal. 340, 183–191
Dian W, Jiang H, Wu P. 2005. Evolution and expression analysis of starch synthase III
and IV in rice. Journal of Experimental Botany, 56, 623–632.
Dinges JR, Colleoni C, James MG, Myers AM. 2003. Purification and molecular
genetic characterization of ZPU1, a pullulanase-type starch-debranching enzyme from
maize. The Plant Cell 15:666–680.
Dinges JR, Colleoni C, Myers AM, James MG. 2001. Molecular structure of three
mutations at the maize sugary1 locus and their allele-specific phenotype effects. Plant
Physiology, 125, 1406–1418.
Edwards A, Fulton DC, Hylton CM, Jobling SA, Gidley M, Rossner U, Martin C,
Smith AM. 1999. A combined reduction in activity of starch synthases II and III of
potato has novel effects on the starch of tubers. The Plant Journal, 17, 251–261.
Eerlingen RC, Delcour A. 1995. Formation, analysis, structure and properties of type III
enzyme resistant starch. Journal of Cereal Science, 22, 129–138.
Emes MJ, Bowsher CG, Hedley C, Burrell MM, Scrase-Field ES, Tetlow IJ. 2003.
Starch synthesis and carbon partitioning in developing endosperm. Journal of
Experimental Botany, 54, 569-575.
- 220 -
Emami S, Perara A, Meda V, Tyler RT. 2012. Effect of microwave treatment on starch
digestibility and physico-chemical properties of three barley types. Food Bioprocess
Technology, 5, 2266–2274.
Englyst H, Wiggins HL, Cummins JH. 1982. Analyst, 107, 307-318.
Englyst HN, Kingman SM, Hudson GJ, Cummings JH. 1996. Measurement of
resistant starch in vitro and in vivo.British Journal of Nutrition, 75, 749-755.
Englyst HN, Kingman SM. 1990. Dietary fiber and resistant starch. A Nutritional
Classification of Plant Polysaccharides. In Dietary Fiber: Chemistry, Physiology, and
Health Effects; Kritchevsky, D., Bonfield, C., Anderson, J. W., Eds.; Plenum Press: New
York.
Englyst HN, Veenstra J, Hudson GJ. 1996. Measurement of rapidly available glucose
(RAG) in plant foods: a potential in-vitro predictor of the glycemic response. British
Journal of Nutrition, 75, 327-37.
Evers AD. 1973. The size distribution among starch granules in wheat endosperm.
Starch/Starke, 25, 303–304.
Fahrenholz C. 1998. Cereal grains and by-products: What’s in them and how are they
processed? Pages 57–70 in Advances in Equine Nutrition. J. D. Pagan, ed. Nottingham
Univ. Press, Nottingham, U.K.
Fannon J, BeMiller J. 1992. Structure of corn starch paste and granule remnants
revealed by low temperature scanning electron microscopy after cryopreparation. Cereal
Chemistry, 69, 456–460.
Fannon JE, Gray JA, Gunawan N, Huber KC, BeMiller JN. 2004. Heterogeneity of
starch granules and the effect of granule channelization on starch modification. Cellulose,
11, 247–254.
Ferguson LR, Tasman-Jones C, Englyst H, Harris PJ. 2000. Comparative effects of
three resistant starch preparations on transit time and short-chain fatty acid production in
rats. Nutrition and Cancer, 36(2), 230-237.
Fontaine T, D’Hulst C, Maddelein ML, Routier F, Pépin TM, Decq A, Wieruszeski
JM, Delrue B, Van den KN, Bossu JP, Fournet B, Ball S. 1993. Toward an
understanding of the biogenesis of the starch granule. Evidence that Chlamydomonas
soluble starch synthase II controls the synthesis of intermediate glucans of amylopectin.
Journal of Biological Chemistry, 268, 16223–16230.
Fordham-Skelton AP, Chilley P, Lumbreras V, Reignoux S, Fenton TR, Dahm CR,
Pages M, Gatehouse JA 2002. A novel higher plant protein tyrosine phosphatase
- 221 -
interacts with SNF1-related protein kinases via a KIS (kinase interaction sequence)
domain. The Plant Journal, 29, 705–715.
French D. 1984. Organization of starch granules. In R, Whistler, J. BeMiller, E.
Paschall, eds., Starch Chemistry and Technology, Ed 2. Academic Press, New York.,
184–242.
Fu Y, Ballicora MA, Leykam JF, Preiss J. 1998. Mechanism of reductive activation of
potato tuber ADP-glucose pyrophosphorylase. The Journal of Biological Chemistry,
273, 25045-25052.
Fujita N, Yoshiko T, Yoshinori U, Toshiyuki H, Isao H, Akira I, Sayuri A, Mayumi
Y, Akiko M, Kotaro I, Rumiko I, Akio M, Hirohiko H, Hikaru S, Yasunori N.
2009. Characterization of pullulanase (PUL)-deficient mutants of rice (Oryza sativa L.)
and the function of PUL on starch biosynthesis in the developing rice endosperm. Journal
of Experimental Botany, 60, 1009–1123.
Fujita N, Kubo A, Suh SD, Wong KS, Jane JL, Ozawa K, Takaiwa F, Inaba Y,
Nakamura Y. 2003. Antisense inhibition of isoamylase alters the structure of
amylopectin and the physicochemical properties of starch in rice endosperm. Plant and
Cell Physiology, 44, 607–618.
Fujita N, Satoh R, Hayashi A, Kodama M, Itoh R, Aihara S, Nakamura Y. 2011.
Starch biosynthesis in rice endosperm requires the presence of either starch synthase I or
IIIa. Journal of Experimental Botany, 62(14), 4819-4831.
Fujita N, Taira T. 1998. A 56-kDa protein is a novel granule-bound starch synthase
existing in the pericarps, aleurone layers, and embryos of immature seed in diploid wheat
(Triticum monococcum L.). Planta, 207, 125-132.
Fujita N, Yoshida M, Asakura N, Ohdan T, Miyao A, Hirochika H, Nakamura Y.
2006. Function and characterization of starch synthase I using mutants in rice. Plant
Physiology, 140, 1070–1084.
Fujita N, Yoshida M, Kondo T, Saito K, Utsumi Y, Tokunaga T, Nishi A, Satoh H,
Park JH, Jane JL, Miyao A, Hirochika H, Nakamura Y. 2007. Characterization of
SSIIIa-deficient mutants of rice: the function of SSIIIa and pleiotropic effects by SSIIIa
deficiency in the rice endosperm. Plant Physiology, 144, 2009-2023.
Fulton DC, Edwards A, Pilling E, Robinson HL, Fahy B, Seale R, Kato L, Donald
AM, Geigenberger P, Martin C, Smith AM. 2002. Role of granule-bound starch
synthase in determination of amylopectin structure and starch granule morphology in
potato. Journal of Biological Chemistry, 277, 10834–10841.
Furukawa K, Tagaya M, Inouye M, Preiss J, Fukui T 1990. Identification of lysine 15
at the active site in Escherichia coli glycogen synthase. Conservation of Lys-X-Gly-Gly
- 222 -
sequence in the bacterial and mammalian enzymes. Journal of Biological Chemistry, 265,
2086-2090.
Furukawa K, Tagaya M, Tanizawa K, Fukui T. 1993. Role of the conserved Lys-XGly-Gly sequence at the ADP-glucose-binding site in Escherichia coli glycogen synthase.
Journal of Biological Chemistry, 268, 23837-23842.
Gaines CS, Raeker MO, Tilley M, Finney PL, Wilson JD, Betchel DB, Martin RJ,
Seib PA, Lookhart GL, Donelson T. 2000. Associations of starch gel hardness, granule
size, waxy allelic expression, thermal pasting, milling quality, and kernel texture of 12
soft wheat cultivars. Cereal Chemistry, 77, 163–186.
Gallant DJ, Bouchet B, Baldwin PM. 1997. Microscopy of starch: evidence of a new
level of granule organization. Carbohydrate Polymers, 32, 177–191.
Gao M, Fisher DK, Kim KN, Shannon JC, Guiltinan MJ. 1996. Evolutionary
conservation and expression patterns of maize starch branching enzyme I and IIb genes
suggests isoform specialization. Plant Molecular Biology, 30, 1223–1232.
Gao M, Fisher DK, Kim KN, Shannon JC, Guiltinan MJ. 1997. Independent genetic
control of maize starch-branching enzymes IIa and IIb. Plant Physiology, 114, 69–78.
Gao M, Wanat J, Stinard PS, James MG, Myers AM. 1998. Characterization of dull1,
a maize gene coding for a n ovel starch synthase. The Plant Cell, 10, 399-412.
Gao Z, Keeling P, Shibles R, Guan H 2004. Involvement of lysine-193 of theconserved
‘K-T-G-G’ motif in the catalysis of maize starch synthase IIa. Archives of Biochemistry
and Biophysics, 427, 1-7.
Garwood DL, Shannon JC, Creech RG. 1976. Starches of endosperms possessing
different alleles at the amylose-extender locus in Zea mays L. Cereal Chemistry, 53, 355–
364.
Geera BP, Nelson JE, Souza E, Huber KC. 2006. Composition and properties of Aand B-type starch granules of wild-type, partial waxy, and waxy soft wheat. Cereal
Chemistry, 83, 551–557.
Gerard C, Barron C, Colonna P, Planchot V. 2009. Amylose determination in
genetically modified starches. Carbohydrate Polymers, 44, 19–27.
Gerard C, Colonna P, Buleon A, Planchot V. 2001. Amylolysis of maize mutant
starches. Journal of the Science of Food and Agriculture, 1281-1287.
Ghosh HP, Preiss J. 1966. Adenosine diphosphate glucose pyrophosphorylase: a
regulatory enzyme in the biosynthesis of starch in spinach leaf chloroplasts. The
Journal of Biological Chemistry, 241, 4491-4504.
- 223 -
Giroux MJ, Hannah LC. 1994. ADP-glucose pyrophosphorylase in shrunken-2 and
brittle-2 mutants of maize. Molecular Genetics and Genomics, 243, 400-408.
Gomez-Casati DF, Iglesias AA. 2002. ADP-glucose pyrophosphorylase from wheat
endosperm. Purification and characterization of an enzyme with novel regulatory
properties. Planta, 214, 428-434.
Goni I, Garcia-Alonso A, Saura-Calixto F. 1997. A starch hydrolysis procedure to
estimate glycemic index. Nutrition Research, 17, 427–437.
Goni I, Garcia-Diz E, Manas E, Saura-Calixto F. 1996. Analysis of resistant starch: a
method for foods and food products. Food Chemistry, 56, 445-449.
Grimaud F, Rogniaux H, James MG, Myers AM, Planchot V. 2008. Proteome and
phosphoproteome analysis of starch granule-associated proteins from normal maize and
mutants affected in starch biosynthesis. Journal of Experimental Botany, 59, 3395–3406.
Guan H, Li P, Imparl-Radosevich J, Preiss J, Keeling P. 1997. Comparing the
properties of Escherichia coli branching enzyme and maize branching enzyme. Archives
of Biochemistry and Biophysics, 342, 92–98.
Guan HP, Preiss J. 1993. Differentiation of the properties of the branching isozymes
from maize (Zea mays). Plant Physiology, 102, 1269–1273.
Hall D, Sayre J. 1970. A scanning electron-microscopy study of starches. II. cereal
starches. Journal of Texture Research, 40: 256–266.
Hamada S, Ito H, Ueno H, Takeda Y, Matsui H. 2007. The N-terminal region of the
starch-branching enzyme from Phaseolus vulgaris L. is essential for optimal catalysis and
structural stability. Phytochemistry, 68, 1367–1375.
Hang A, Don OD, Gironella AIN, Charlotte S, Burton CS. 2007. Barley amylose and
β-glucan their relationship to protein agronomic traits and environmental factors. Crop
Science, 47, 1754–1760.
Hannah LC, James MG. 2008. The complexities of starch biosynthesis in cereal
endosperms. Current Opinion in Biotechnology, 19, 160–165.
Hannah LC, Shaw JR, Giroux MJ, Reyss A, Prioul JL, Bae JM, Lee JY. 2001. Maize
genes encoding the small subunit of ADP-glucose pyrophosphorylase. Plant Physiology,
127, 173-183.
Haralampu SG. 2000. Resistant Starch: A review of the physical properties and
biological impact of RS3. Carbohydrate Polymers, 41, 285–292.
- 224 -
Harn C, Knight M, Ramakrishnan A, Guan H, Keeling PL, Wasserman BP. 1998.
Isolation and characterization of the zSSIIa and zSSIIb starch synthase cDNA clones
from maize endosperm. Plant Molecular Biology, 37, 639–649.
Hawker JS, Ozbun JL, Ozaki H, Greenberg E, Preiss J. 1974. Interaction of spinach
leaf adenosine diphosphate glucose a-1, 4-glucan a-4-glucosyl transferase and a-1, 4glucan, a-1, 4-glucan-6-glycosyl transferase in synthesis of branched a-glucan. Archives
of Biochemistry and Biophysics, 160, 530–551.
Hedley CL, Bogracheva TY, Wang TL. 2002. A genetic approach to studying
the morphology, structure and function of starch granules using pea as a model.
Starch-Stärke, 54, 235–242.
Hedman KD, Boyer CD. 1982. Gene dosage at the amylose-extender locus of maize:
Effects on the levels of starch branching enzymes. Biochemical Genetics, 20, 483-492.
Hendriks JHM, Kolbe A, Gibon Y, Stitt M, Geigenberger P. 2003. ADP-glucose
pyrophosphorylase is activated by post-translational redox-modification in response to
light and to sugars in leaves of Arabidopsis and other plant species. Plant Physiology,
133: 1-12.
Hennen-Bierwagen TA, Lin Q, Grimaud F, Planchot V, Keeling PL, James MG,
Myers AM. 2009. Proteins from multiple metabolic pathways associate with starch
biosynthetic enzymes in high molecular weight complexes: a model for regulation of
carbon allocation in maize amyloplasts. Plant Physiology, 149, 1541–1559.
Hennen-Bierwagen TA, Liu F, Marsh RS, Kim S, Gan Q, Tetlow IJ, Emes MJ,
James MG, Myers AM. 2008. Starch biosynthetic enzymes from developing Zea mays
endosperm associate in multisubunit complexes. Plant Physiology, 146:1892–1908.
Herman EM, Larkins BA. 1999. Protein storage bodies and vacuoles. The Plant Cell,
11, 601–614.
Higgins J. 2004. Resistant starch consumption promotes lipid oxidation. Journal of
Nutrition and Metabolism, 1:1-8.
Hilbert GE, MacMasters MM. 1946. Pea starch, a starch of high amylose content.
Journal of Biological Chemistry, 162, 229–238.
Hirose T, Terao T. 2004. A comprehensive expression analysis of the starch synthase
gene family in rice (Oryza sativa L.). Planta, 220, 9-16.
Hizukuri S, Takeda Y, Maruta N. 1989. Molecular structure of rice starches.
Carbohydrate Research, 189, 227–235.
Hizukuri S. 1986. Polymodal distribution of the chain lengths of amylopectin, and its
significance. Carbohydrate Research, 147, 342–347.
- 225 -
Hizukuri S. 1996. Starch: analytical aspects. In: Eliasson AC (ed.) Carbohydrates in
Food. New York: Dekker, pp. 347–429.
Hoecker U, Vasil IK, McCarty DR. 1995. Integrated control of seed maturation and
germination programs by activator and repressor functions of Viviparous-1 of maize.
Genes & Development, 9, 2459–2469.
Holding DR, Otegui MS, Li B, Meeley R, Dam T, Hunter BG, Jung R, Larkins BA.
2007. The maize floury1 gene encodes a novel endoplasmic reticulum protein involved
in zein protein body formation. The Plant Cell, 19, 2569–2582.
Hoover R, Zhou Y. 2003. In vitro and in vivo hydrolysis of legume starches by αamylase and resistant starch formation in legumes –review. Carbohydrate Polymers, 54,
401–417.
Huber KC, BeMiller JN. 2001. Location of sites of reaction within starch granules.
Cereal Chemistry, 78, 173–180.
Hughes EC, Briarty LG. 1976. Stereological analysis of contribution made to mature
wheat endosperm starch by large and small granules. Starch-Stärke, 28(10), 336– 337.
Hussain H, Mant A, Seale R, Zeeman S, Hinchliffe E, Edwards A, Hylton C,
Bornemann S, Smith AM, Martin C, Bustos R. 2003. Three isoforms of isoamylase
contribute different catalytic properties for the debranching of potato glucans. The Plant
Cell, 15, 133–149.
Hylton C, Smith AM. 1992. The rb mutation of peas causes structural and regulatory
changes in ADP glucose pyrophosphorylase from developing embryos. Plant
Physiology, 99, 1626-1634.
Imparl-Radosevich JM, Gameon JR, McKean A, Wetterberg D, Keeling P, Guan H.
2003. Understanding catalytic properties and functions of maize starch synthase
isozymes. Journal of Applied Glycoscience, 50, 177–182.
Imparl-Radosevich JM, Li P, Zhang L, McKean AL, Keeling PL, Guan H. 1998.
Purification and characterization of maize starch synthase I and its truncated forms.
Archives of Biochemistry and Biophysics, 353, 64–72.
Inouchi N, Glover DV, Takaya T, Fuwa H. 1983. Development changes in fine
structure of starches of several endosperm mutants of maize. Starch-Stärke, 35, 371–376.
Izdorczyk MS, Storsley J, Labossiere D, McGregor AW, Rossnagel BG. 2000.
Variations in total and soluble β-glucan content in hulless barley: Effect of thermal,
physical and enzymatic treatments. Journal of Agricultural and Food Chemistry, 48,
982–989.
- 226 -
Jadhav SJ, Lutz SE, Ghorpade VM, Salunkhe DK. 1998. Barley: chemistry and valueadded processing. Critical Reviews in Food Science, 38, 123–171.
James GM, Denyer K, Myers AM. 2003. Starch synthesis in the cereal endosperm.
Current Opinion in Plant Biology, 6, 215–222.
James MG, Robertson DS, Myers AM. 1995. Characterization of the maize gene
sugary1, a determinant of starch composition in kernels. The Plant Cell, 7, 417–429.
Jane J, Chen YY, McPherson AE, Wong KS, Radosavljevic M, Kasemsuwan T.
1999. Effects of amylopectin branch chain length and amylose content on the
gelatinization and pasting properties of starch. Cereal Chemistry, 76, 629–637.
Jane J, Kasemsuwan T, Lees S, Zobel HF, Robyt JF. 1994. Anthology of starch
granule morphology by scanning electron microscopy. Starch-Stärke, 46, 121–129.
Jane JL, Robyt JF. 1984. Structure studies of amylose-V complexes and retrograded
amylose by action of alpha amylases, and a new method for preparing amylo-dextrins.
Carbohydrate Research, 132, 105–118.
Jane JL, Wong KS, McPherson AE. 1997. Branch structure difference in starches of Aand B-type X-ray patterns revealed by their naegeli dextrins. Carbohydrate Research,
300, 219–227.
Jenkins DJA, Wolever TMS, Taylor RH, Barker H, Fielden H, Baldwin JM,
Bowling AC, Newman HC, Jenkins AL, Goff DV. 1981. Glycemic index of foods: A
physiological basis for carbohydrate exchange. The American Journal of Clinical
Nutrition, 34, 362-360.
Jenkins PJ. 1994. X-ray and neutron scattering studies of starch granule structure. PhD
thesis, University of Cambridge, UK
Jeon JS, Nayeon R, Tae-Ryong H, Harkamal W, Yasunori N. 2010. Starch
biosynthesis in cereal endosperm. Plant Physiology and Biochemistry, 48, 383-392.
Ji Q, Oomen RJF, Vincken JP, Bolam DN, Gilbert HJ, Suurs L, Visser RGF, 2004.
Reduction of starch granule size by expression of an engineered tandem starch-binding
domain in potato plants. Plant Biotechnology Journal, 2, 251–260.
Jin X, Ballicora MA, Preiss J, Geiger JH. 2005. Crystal structure of potato tuber ADPglucose pyrophosphorylase. European Molecular Biology Organization Journal, 24,
294–704.
Johnson PE, Patron NJ, Bottrill AR, Dinges JR, Fahy BF, Parker ML, Waite DN,
Denyer K. 2003. A low-starch barley mutant, Risø 16, lacking the cytosolic small
- 227 -
subunit of ADP-glucose pyrophosphorylase, reveals the importance of the cytosolic
isoform and the identity of the plastidial small subunit. Plant Physiology, 131, 684–696.
Kang MY, Sugimoto Y, Kato I, Sakamoto S, Fuwa H. 1985. Some properties of
large and small granules of barley (Hordeum Vulgare. L) endosperm. Agricultural and
Biological Chemistry, 49, 1291–1297.
Kawagoe Y, Kubo A, Satoh H, Takaiwa F, Nakamura Y. 2005. Roles of isoamylase
and ADPglucose pyrophosphorylase in starch granule synthesis in rice endosperm. The
Plant Journal, 42, 164–174.
Kawasaki T, Mizuno K, Shimada H, Satoh H, Kishimoto N, Okumura S, Ichikawa
N, Baba T. 1996. Coordinated regulation of the genes participating in starch biosynthesis
by the rice floury-2 locus. Plant Physiology, 110, 89–96.
Keeling LP, Myers AM. 2010. Biochemistry and genetics of starch synthesis. Annual
Review of Food Science and Technology, 1, 271–303.
Keenan JM, Jun Z, Kathleen LM, Anne MR, Gale H B, Emily T, Christina KJ,
Richard TT, Sheri M, Roy JM, Maren H. 2006. Effects of resistant starch, a nondigestible fermentable fiber, on reducing body fat. Obesity, 14(9), 1523-1534.
Kempa S, Rozhon W, Samaj J, Erban A, Baluska F, Becker T, Haselmayer J,
Schleiff E, Kopka J, Hirt H, Jonak C. 2007. A plastid-localized glycogen synthase
kinase 3 modulates stress tolerance and carbohydrate metabolism. The Plant Journal, 49,
1076–1090.
Kerk D, Conley TR, Rodriguez FA, Tran HT, Nimick M, Muench DG, Moorhead
GBG. 2006. Achloroplastlocalized dual-specificity protein phosphatase in Arabidopsis
contains a phylogenetically dispersed and ancient carbohydrate-binding domain, which
binds the polysaccharide starch. The Plant Journal, 46, 400–413.
Kiesselbach TA. 1949. The Structure and reproduction of corn. University of Nebraska
College of Agriculture, Lincoln, NE. Research Bulletin, 161.
Kim HS, Huber KC. 2008. Channels within soft wheat starch A- and B-type granules.
Journal of Cereal Science, 48, 159–172.
Kim KN, Fisher DK, Gao M, Guiltinan MJ. 1998. Molecular cloning and
characterization of the amyloseextender gene encoding starch branching enzyme IIB in
maize. Plant Molecular Biology, 38, 945 -956.
Klucinec JD, Thompson DB. 2002. Structure of amylopectins from ae-containing maize
starches. Cereal Chemistry, 79, 19–23.
Knowles RV, Phillips RL. 1988. Endosperm development in maize. International
- 228 -
Review of Cytology, 12, 97–136.
Kosar-Hashemi B, Li Z, Larroque O, Regina A, Yamamori M, Morell MK,
Rahman S. 2007. Multiple effects of the starch synthase II mutation in developing
wheat endosperm. Functional Plant Biology, 34, 431–438.
Kossman J, Abel GJW, Springer F, Lloyd JR, Willmitzer L. 1999. Cloning and
functional analysis or a cDNA encoding a starch synthase from potato (Solanum
tuberosum L.) that is predominantly expressed in leaf tissue. Planta, 208, 503-511.
Kossmann J, Lloyd J. 2000. Understanding and influencing starch biochemistry.
Critical Reviews in Plant Sciences, 19, 171–226.
Kötting O, Santelia D, Edner C, Eicke S, Marthaler T, Gentry MS, Comparot-Moss
S, Chen J, Smith AM, Steup M, Ritte G, Zeeman SC. 2009. SEX4, a glucan
phosphatase, dephosphorylates amylopectin at the granule surface during starch
breakdown in Arabidopsis leaves. The Plant Cell, 21, 334–346.
Kreis M, Forde BG, Rahman S, Miflin BJ, Shewry PR. 1985. Molecular evolution of
the seed storage proteins of barley, rye and wheat. Journal of Molecular Biology. 183,
499–502.
Kubo A, Akdogan G, Nakaya M, Shojo A, Suzuki S, Satoh H, Kitamura S. 2010.
Structure, physical, and digestive properties of starch from wx ae double-mutant rice.
Journal of Agricultural and Food Chemistry, 58(7), 4463-4469.
Kubo A, Fujita N, Harada K, Matsuda T, Satoh H, Nakamura Y. 1999. The starchdebranching enzymes isoamylase and pullulanase are both involved in amylopectin
biosynthesis in rice endosperm. Plant Physiology, 121, 399–409.
Kubo A, Guray A, Makoto N, Aiko S, Shiho S, Hikaru S, Shinichi K. 2010. Structure,
physical, and digestive properties of starch from wx ae double-mutant rice. Journal of
Agricultural and Food Chemistry, 58, 4463-4469.
Kubo A, Rahman S, Utsumi Y, Li Z, Mukai Y, Yamamoto M, Ugaki M, Harada
K, Satoh H, Konik-Rose C, Morell MK, Nakamura Y.. 2005. Complementation of
sugary-1 phenotype in rice endosperm with the wheat isoamylase1 gene supports a direct
role for isoamylase1 in amylopectin biosynthesis. Plant Physiology, 137, 43–56.
Kugimiya M, Donovan JW, Wong RY. 1980. Phase transitions in amylose-lipid
complexes in starch: a calorimetric study. Starch /Stärke, 32, 265–270.
Kuriki T, Stewart DC, Preiss J. 1997. Construction of chimeric enzymes out of maize
endosperm branching enzymes I and II: Activity and properties. The Journal of
Biological Chemistry, 272, 28999–29004.
- 229 -
La Cognata U, Willmitzer L, Muller BR. 1995. Molecular cloning and
characterization of novel isoforms of potato ADP-glucose pyrophosphorylase.
Molecular Genetics and Genomics, 246, 538-548.
Langeveld SMJ, Van Wijk R, Stuurman N, Kijne JW, De Pater S. 2000. B-type
granule containing protrusions and interconnections between amyloplasts in developing
wheat endosperm revealed by transmission electron microscopy and GFP expression.
Journal of Experimental Botany, 51(349), 1357–1361.
Laudencia-Chingcuanco D, Stamova B, You F, Fowler B, Chibbar R, Anderson O.
2007. Transcriptional profiling of wheat caryopsis development using cDNA
microarrays. Plant Molecular Biology. 63, 651–668.
Lauro M, Forssell P, Suortti T, Hulleman S, Poutamen SK. 1999. αAmylolysis of large barley starch granules. Cereal Chemistry, 76, 925–930.
Leloup VM, Colonna P, Marchis-Mouren G. 1992a. Mechanism of the adsorption of
pancreatic alpha-amylase onto starch crystallites. Carbohydrate Research, 232, 367–374.
Leloup VM, Colonna P, Ring SG. 1992b. Physico-chemical aspects of resistant starch.
Journal of Cereal Science, 16, 253–266.
Lemke H, Burghammer M, Flot D, Rossle M, Riekel C. 2004. Structural processes
during starch granules hydration by synchrotron radiation microdiffraction.
Biomacromolecules, 5, 1316–1324.
Leszczyñski W. 2004. Resistant starch–classification, structure, production. Polish
Journal of Food And Nutrition Sciences, 54(13), 37–50.
Leterrier M, Holappa L, Broglie K, Beckles DM. 2008. Cloning, characterisation and
comparative analysis of a starch synthase IV gene in wheat: functional and evolutionary
implications. BioMed Central Plant Biology. 8, 98.
Li JH, Vasanthan T, Rossnagel B, Hoover R. 2001. Starch from hull-less barley I
granule morphology, composition and amylopectin structure. Food Chemistry, 74, 395–
405.
Li JH, Vasanthan T, Rossnagel B, Hoover R. 2001. Starch from hull-less barley: II.
Thermal, rheological and acid hydrolysis characteristics. Food Chemistry, 74, 407–415.
Li L, Hongxin J, Mark C, Michael B, Jay-lin J. 2008. Characterization of maize
amylose-extender (ae) mutant starches. Part I: Relationship between resistant starch
contents and molecular structures. Carbohydrate Polymers, 74, 396–404.
Li Z, Li D, Du X, Wang H, Larroque O, Jenkins CLD, Jobling SA, Morell MK.
2011. The barley amo1 locus is tightly linked to the starch synthase IIIa gene and
- 230 -
negatively regulates expression of granule-bound starch synthetic genes. Journal of
Experimental Botany, 62(14), 5217-5231.
Li Z, Mouille G, Kosar-Hashemi B, Rahman S, Clarke B, Gale K, Appels R, Morell
MK. 2000. The structure and expression of the wheat starch synthase III gene: Motifs in
the expressed gene define the lineage of the starch synthase III gene family. Plant
Physiology, 123, 613-624.
Li Z, Sun F, Xu S, Chu X, Mukai Y, Yamamoto M, Ali S, Rampling L, KosarHashemi B, Rahman S, Morell MK. 2003. The structural organisation of the gene
encoding class II starch synthase of wheat and barley and the evolution of the genes
encoding starch synthases in plants. Functional & Integrative Genomics, 3, 76–85.
Lin TP, Spilatro SR, Preiss J. 1988. Characterization of D-enzyme 4-alpha
glucanotransferase in Arabidopsis leaf. Plant Physiology, 86, 260–265.
Lindeboom N, Peter R, Chang R, Tyler T. 2004. Analytical, biochemical and
physicochemical aspects of starch granule size, with emphasis on small granule starches a
review. Starch-Starke, 56, 89–99.
Liu F, Makhmoudova A, Lee EA, Wait R, Emes MJ, Tetlow IJ. 2009. The amylose
extender mutant of maize conditions novel protein-protein interactions between starch
biosynthetic enzymes in amyloplasts. Journal of Experimental Botany, 60, 4423-4440.
Liu F, Ahmed Z, Lee EA, Donner E, Liu Q, Regina A, Morell MK, Emes MJ,
Tetlow IJ. 2012a. Allelic variants of the amylose extender mutation of maize
demonstrate phenotypic variation in starch structure resulting from modified protein–
protein interactions. Journal of Experimental Botany, 63(3), 1167–1183.
Liu F, Romanova N, Lee EA, Regina A, Evans M, Gilbert EP, Morell MK, Emes
MJ, Tetlow IJ. 2012b. Glucan affinity of starch synthase IIa determines binding of
starch synthase I and starch-branching enzyme IIb to starch granules. Biochemical
Journal, 448, 373–387.
Liu H, Yu L, Xie F, Chen L. 2006. Gelatinization of corn starch with different
amylose/amylopectin content. Carbohydrate Polymers, 65 (3), 357–363.
Liu Q. 2005. Food Carbohydrates, Chemistry, Physical Properties, and Applications:
Understanding Starches and Their Role in Foods (Steve WC. Eds). New York: Taylor &
Francis Group
Liu Q, Gu Z, Donner E, Tetlow IJ, Emes MJ. 2007. Investigation of digestibility in
vitro and physicochemical properties of A- and B-type starch from soft and hard wheat
flour. Cereal Chemistry. 84(1), 15–21.
- 231 -
Lloyd JR, Landschutze V, Kossmann J. 1999. Simultaneous antisense inhibition of two
starch-synthase isoforms in potato tubers leads to accumulation of grossly modified
amylopectin. Biochemical Journal, 338, 515–521.
Lopes MA, Larkins BA. 1993. Endosperm origin, development, and function. The
Plant Cell, 5, 1383–1399.
Lorenz K, Collins F. 1995. Physicochemical characteristics and functional properties
of starch from a high β-glucan waxy barley. Stärke, 47, 14–18.
MacGregor AW, Ballance DL. 1980. Hydrolysis of large and small starch granules
from normal and waxy barley cultivars by alpha-amylases from barley malt. Cereal
Chemistry, 57, 397–402.
MacGregor AW, Morgan JE. 1984. Structure of amylopectins isolated from large and
small starch granules of normal and waxy barley. Cereal Chemistry, 61, 222-228.
MacGregor AW, Morgan JE. 1986. Hydrolysis of barley starch granules by alphaamylases from barley malt. Cereal Foods World, 31, 688–693.
Maddelein ML, Libessart N, Bellanger F, Delrue B, D'Hulst C, Van den KN,
Fontaine T, Wieruszeski JM, Decq A, Ball S. 1994. Toward an understanding of the
biogenesis of the starch granule. Determination of granule-bound and soluble starch
synthase functions in amylopectin synthesis. The Journal of Biological Chemistry, 269,
25150–25157.
Magnard JL, Lehouque GL, Massonneau AS. 2003. ZmEBE genes show a novel,
continuous expression pattern in the central cell before fertilization and in specific
domains of the resulting endosperm after fertilization. Plant Molecular Biology, 53,
821–836.
Mangalika WHA, Miura H, Yamauchi H, Noda T. 2003. Properties of starches from
near-isogenic wheat lines with different wx protein deficiencies. Cereal Chemistry, 80,
662-666.
Marshall J, Sidebottom C, Debet M, Martin C, Smith AM, Edwards A. 1996.
Identification of the major starch synthase in the soluble fraction of pea embryos. The
Plant Cell, 8, 1121–1135
Martin C, Smith AM. 1995. Starch synthesis. The Plant Cell, 7, 971–985.
McCoy JG, Bitto E, Bingman CA, Wesenberg GE, Bannen RM, Kondrashov DA,
Phillips GN. 2007. Structure and dynamics of UDP-glucose pyrophosphorylase from
Arabidopsis thaliana with bound UDP-glucose and UTP. Journal of Molecular Biology,
366, 830–841.
- 232 -
Mechin V, Thévenot C, Le Guilloux M, Prioul JL, Damerval C. 2007. Developmental
analysis of maize endosperm proteome suggests a pivotal role for pyruvate
orthophosphate dikinase. Plant Physiology, 143, 1203–1219.
Mentschel J, Claus R. 2003. Increased butyrate formation in the pig colon by feeding
raw potato starch leads to a reduction of colonocyte apoptosis and a shift to stem cell
compartment. Metabolism, 52(11), 1400-1405.
Miao M, Jiang B, Zhang T. 2009. Effect of pullulanase debranching and
recrystallization on structure and digestibility of waxy maize starch. Carbohydrate
Polymers, 76, 214–221.
Mitsui T, Kimiko I, Hidetaka H, Hiroyuki I. 2010. Biosynthesis and degradation of
starch. Bulletin of the Faculty of Agriculture, Niigata University, 62(2), 49-73.
Mohlmann T, Tjaden J, Henrichs G, Quick WP, Hausler R, Neuhaus HE. 1997.
ADP-glucose drives starch synthesis in isolated maize endosperm amyloplasts:
characterization of starch synthesis and transport properties across the amyloplast
envelope. Biochemical Journal, 324, 503-509.
Morell MK, Blennow A, Kosar-Hashemi B, Samuel MS. 1997. Differential expression
and properties of starch branching enzyme isoforms in developing wheat endosperm.
Plant Physiology, 113, 201–208.
Morell MK, Kosar-Hashemi B, Cmiel M, Samuel MS, Chandler P, Rahman S,
Buléon A, Batey IL, Li Z. 2003. Barley sex6 mutants lack starch synthase IIa activity
and contain a starch with novel properties. The Plant Journal, 34, 173–185.
Mori H, Tanizawa K, Fukui T. 1993. A chimeric alpha-glucan phosphorylase of plant
type L and H isozymes. Functional role of 78-residue insertion in type L isozyme. The
Journal of Biological Chemistry, 268, 5574–5581.
Morrison WR, Karkalas J. 1990. Starch, in Methods in Plant Biochemistry, P.M. Day,
Ed., Academic Press, San Diego, Calif, USA.
Morrison WR, Tester RF, Snape CE, Law R, Gidley MJ. 1993. Swelling and
gelatinization of cereal starches IV, Some effects of lipid-complexed amylose and free
amylose in waxy and normal barley starches. Cereal Chemistry, 70, 385–391.
Mortz E, Krogh TN, Vorum H, Gorg A. 2001. Improved silver staining protocols for
high sensitivity protein identification using matrix-assisted laser desorption/ionizationtime of flight analysis. Proteomics. 1(11), 1359-1363.
Mouille G, Maddelein ML, Libessart N, Talaga P, Decq A, Delrue B, Ball S. 1996.
Phytoglycogen processing: a mandatory step for starch biosynthesis in plants. The Plant
Cell, 8, 1353–1366.
- 233 -
Mu HH, Yu Y, Wasserman BP, Carman GM. 2001. Purification and characterization
of the maize amyloplast stromal 112-kDa starch phosphorylase. Archives of
Biochemistry and Biophysics, 388, 155-164.
Müller-Röber BT, Kossmann J, Hannah LC, Willmitzer L, Sonnewald U. 1990. One
of two different ADP-glucose pyrophosphorylase genes responds strongly to elevated
levels of sucrose. Molecular Genetics and Genomics, 224, 136–146.
Mu-Forster C, Huang R, Powers JR, Harriman RW, Knight M, Singletary GW,
Keeling PL, Wasserman BP. 1996. Physical association of starch biosynthetic enzymes
with starch granules of maize endosperm. Granule-associated forms of starch synthase I
and starch branching enzyme II. Plant Physiology, 111, 821–829.
Muir JG, Lu ZX, Young GP, Cameron-Smith D, Collier GR, O’Dea K. 1995.
Resistant starch in the diet increases breath hydrogen and serum acetate in human
subjects. The American Journal of Clinical Nutrition, 61(4), 792-799.
Muir JG, Yeow EG, Keogh J, Pizzey C, Bird AR, Sharpe K, O’Dea K, Macrae FA.
2004. Combining wheat bran with resistant starch has more beneficial effects on fecal
indexes than does wheat bran alone. The American Journal of Clinical Nutrition, 79(6),
1020-1028.
Myers AM, Morell MK, James MG, Ball SG. 2000. Recent progress toward
understanding the biosynthesis of the amylopectin crystal. Plant Physiology, 122, 989–
997.
Naka M, Sugimoto Y, Sakamoto S, Fuwa H. 1985. Some properties of large and
small granules of waxy barley Hordeum Vulgare endosperm starch. Journal of
Nutritional Science and Vitaminology, 31, 423–430.
Nakamura T, Tomoya S, Patricia V, Mike S, Junichi Y, Yasuhiro S, Hideyo Y,
Masao T. 2006. Sweet wheat. Genes and Genetic Systems, 81, 361-365.
Nakamura T, Vrinten P, Hayakawa K, Ikeda J. 1998. Characterization of a granulebound starch synthase isoform found in the pericarp of wheat. Plant Physiology, 118,
451–459.
Nakamura Y, Kawaguchi K. 1992. Multiple forms of ADP-glucose pyrophoshorylase
of rice endosperm. Plant Physiology, 84, 336-342.
Nakamura Y, Umemoto T, Ogata N, Kuboki Y, Yano M, Sasaki T. 1996. Starch
debranching enzyme (R-enzyme or pullulanase) from developing rice endosperm:
purification, cDNA and chromosomal localization of the gene. Planta, 199, 209–218.
- 234 -
Nakamura Y. 2002. Towards a better understanding of the metabolic system for
amylopectin biosynthesis in plants: Rice endosperm as a model tissue. Plant Cell
Physiology, 43, 718–725.
Neuhaus HE, Stitt M. 1990. Control analysis of photosynthate partitioning. Impact of
reduced activity of ADP-glucose pyrophosphorylase or plastid phosphoglucomutase on
the fluxes to starch and sucrose in Arabidopsis thaliana (L.) Henyh. Planta, 182, 445454.
Naguyen HN, Sabelli PA, Larkins BA. 2007. Endoreduplication and programmed cell
death in the cereal endosperm. In Endosperm (Olsen OA, ed), Vol 8. Springer-Verlag,
Berlin/Heidelberg, pp 21–43.
Nichols DJ, Keeling PL, Spalding M, Guan H. 2000. Involvement of conserved
aspartate and glutamate residues in the catalysis and substrate binding of maize starch
synthase. Biochemistry, 39, 7820-7825.
Nielsen TH, Krapp A, Röper-Schwarz U, Stitt M. 1998. The sugar-mediated
regulation of genes encoding the small subunit of Rubisco and the regulatory subunit of
ADP glucose pyrophosphorylase is modified by nitrogen and phosphate. Plant, Cell &
Environment, 21, 443–455.
Niittylä T, Comparot-Moss S, Lue WL, Messerli G, Trevisan M, Seymour MD,
Gatehouse JA, Villadsen D, Smith SM, Chen J, Zeeman SC, Smith AM. 2006.
Similar protein phosphatases control starch metabolism in plants and glycogen
metabolism in mammals. Journal of Biological Chemistry, 281, 11815–11818.
Nishi A, Nakamura Y, Tanaka N, Satoh H. 2001. Biochemical and genetic analysis of
the effects of amylose extender mutation in rice endosperm. Plant Physiology, 127, 459–
472.
Noakes M, Clifton PM, Nestel PJ, Le LR, McIntosh G. 1996. Effect of high-amylose
starch and oat bran on metabolic variables and bowel function in subjectswith hyper
triglyceridemia. The American Journal of Clinical Nutrition, 64(6), 944-951.
O‘Dea K, Snow P, Nestel P. 1991. Rate of starch hydrolysis in vitro as a predictor of
metabolic responses to complex carbohydrate in vivo. The American Journal of Clinical
Nutrition, 34, 1991–1993.
O’Dea K, Nestel PJ, Antonoff L. 1980. Physical factors influencing postprandial
glucose and insulin responses to starch. The American Journal of Clinical Nutrition, 33,
760–765.
Olsen OA. 2001. Endosperm development: cellularization and cell fate specification.
Annual Review of Plant Physiology and Plant Molecular Biology, 52, 233–267.
- 235 -
Olsen OA. 2004. Nuclear endosperm development in cereals and Arabidopsis thaliana.
The Plant Cell, 16, S214–S227.
Oscarsson M, Andersson R, Salomonsson AC, Aman P. 1996. Chemical composition
of barley samples focusing on dietary fiber components. Journal of Cereal Science, 24,
161–170.
Östergård K, Björck I, Gunnarsson A. 1998. A study of native and chemically
modified potato starch. Part I: Analysis and enzyme availability in vitro. Starch, 40, 58–
66.
Palopoli N, Busi MV, Fornasari MS, Gomez-Casati D, Ugalde R, Parisi G. 2006.
Starch-synthase III family encodes a tandem of three starch binding domains. Proteins,
65, 27–31.
Patron JN, Smith AM, Fahy BF, Hylton CM, Naldrett MJ, Rossnagel BG, Denyer
K. 2002. The altered pattern of amylose accumulation in the endosperm of low-amylose
barley cultivars is attributable to a single mutant allele of granule-bound starch synthase I
with a deletion in the 5--non-coding region1. Plant Physiology, 130, 190–198.
Patron NJ, Greber B, Fahy BF, Laurie DA, Parker ML, Denyer K. 2004. The lys5
mutations of barley reveal the nature and importance of plastidial ADP-Glc transporters
for starch synthesis in cereal endosperm. Plant Physiology, 135, 2088–2097.
Patron NJ, Keeling PJ. 2005. Common evolutionary origin of starch biosynthetic
enzymes in green and red algae. Journal of Phycology, 41, 1131–1141.
Peng M, Gao M, Abdel-Aal ES, Huel P, Chibbar RN. 1999. Separation and
characterisation of A-type and B-type starch granules in wheat endosperm. Cereal
Chemistr, 76, 375–379.
Peng M, Gao M, Baga M, Hucl P, Chibbar RN. 2000. Starch-branching enzymes
preferentially associated with A-type starch granules in wheat endosperm. Plant
Physiology, 124, 265–272.
Keeling PL, Mayers AM. 2010. Biochemistry and genetics of starch synthesis. Annual
Review of Food Science Technology, 1, 271–303.
Phillips J, Muir JG, Birkett A, Lu ZX, Jones GP, O’Dea K, Young GP. 1995. Effect
of resistant starch on fecal bulk and fermentation-dependent events in humans. The
American Journal of Clinical Nutrition, 62(1), 121-130.
Pilling E, Smith AM. 2003. Growth ring formation in the starch granules of potato
tubers. Plant Physiology, 132, 365–371.
Prioul J, Méchin L, Damerval K. 2008. Molecular and biochemical mechanisms in
- 236 -
maize endosperm development: The role of pyruvate-Pi-dikinase and Opaque-2 in the
control of C/N ratio. Comptes Rendus Biologies, 331(10), 772–779.
Putseys JA, Derde LJ, Lamberts L, Ostman E, Bjrck IM, Delcour JA. 2010.
Functionality of short chain amylose-lipid complexes in starch-water systems and their
impact on in vitro starch degradation. Journal of Agricultural and Food Chemistry, 58,
1939–1945.
Radchuk VV, Ludmilla B, Nese S, Kathleen M, Hans-Peter M, Hardy R, Ulrich W,
Winfriede W. 2009. Spatio-temporal profiling of starch biosynthesis and degradation in
the developing barley grain. Plant Physiology 150 (1), 190-204.
Raeker MO, Gaines CS, Finney PL, Donelson T. 1998. Granule size distribution and
chemical composition of starches from 12 soft wheat cultivars. Cereal Chemistry, 75,
721–728.
Rahman S, Kosar-Hashemi B, Samuel MS, Hill A, Abbott DC, Skerritt JH, Preiss J,
Appels R, Morell MK. 1995. The major proteins of wheat endosperm starch granules.
Australian Journal Plant Physiology, 22, 793–803.
Rahman S, Li Z, Beatey I, Cochrane MP, Apples R, Morell MK. 2000. Genetic
alteration of starch functionality in wheat. Journal of Cereal Science, 31, 91–110.
Rahman S, Regina A, Li Z, Mukai Y, Yamamoto M, Kosar-Hashemi B, Abrahams
S, Morell MK. 2001. Comparison of starch-branching enzyme genes reveals
evolutionary relationships among isoforms. Characterization of a gene for starchbranching enzyme IIa from wheat D genome donor Aegilops tauschii. Plant Physiology,
125, 1314–1324.
Rahman S, Bird A, Regina A, Li Z, Ral JP, McMaugh S, Topping D, Morell MK.
2007. Resistant starch in cereals: Exploiting genetic engineering and genetic variation.
Journal of Cereal Science, 46, 251-260.
Ral JP, Colleon C, Wattebled F, Dauvillee D, Nempont C, Deschamps P, Li Z,
Morell M, Chibbar R, d’Hulst C, Ball S. 2006. Circadian clock regulation of starch
metabolism establishes GBSSI as a major contributor to amylopectin synthesis in
Chlamydomonas reinhardtii. Plant Physiology 142, 305-317.
Randolph LF. 1936. Developmental morphology of the caryopsis in maize. Journal of
Agricultural Research, 53, 881–916.
Ratnayake WS, Jackson DS. 2008. Starch gelatinization. Advances in Food and Nutrition
Research, 55, 221-268.
Reddy RK, Ali ZS, Bhattacharya KR. 1993. The fine structure of rice-starch
amylopectin and its relation to the texture of cooked rice. Carbohydr. Polym. 22:267–75
- 237 -
Regina A, Kosar-Hashemi B, Ling S, Li Z, Rahman S, Morell MK. 2010. Control of
starch branching in barley defined through differential RNAi suppression of starch
branching enzyme IIa and IIb. Journal of Experimental Botany, 61(5), 1469–1482.
Regina A, Kosar-Hashemi B, Li Z, Pedler A, Mukai Y, Yamamoto M, Gale K,
Sharp PJ, Morell MK, Rahman S. 2005. Starch branching enzyme IIb in wheat is
expressed at low levels in the endosperm compared to other cereals and encoded at a nonsyntenic locus. Planta, 222, 899–909.
Ring SG, Gee JM, Whittam M, Orford P, Johnson IT. 1988. Resistant starch: its
chemical form in foodstuffs and effect on digestibility in vitro. Food Chemistry, 28, 97–
109.
Ritchie S, Swanson JS, Gilroy S. 2000. Physiology of the aleurone layer and starchy
endosperm during grain development and early seedling growth: new insights from cell
and molecular biology. Seed Science Research, 10, 193–212.
Roldan I, Wattebled F, Lucas MM, Delvallee D, Planchot V, Jimenez S, Perez R,
Ball S, D’Hulst C, Merida A. 2007. The phenotype of soluble starch synthase IV
defective mutants of Arabidopsis thaliana suggests a novel function of elongation
enzymes in the control of starch granule formation. The Plant Journal, 49, 492-504.
Rolletschek H, Koch K, Wobus U, Borisjuk L. 2005. Positional cues for the
starch/lipid balance in maize kernels and resource partitioning to the embryo. The Plant
Journal, 69–83.
Rost TL, Artucio PID, Risley EB. 1984. Transfer cells in the placental pad and
caryopsis coat of Pappophorum subbulbosum Arech. (Poaceae). American Journal of
Botany. 71, 948–957.
Rösti S, Denyer K. 2007. Two paralogous genes encoding small subunits of ADPglucose pyrophosphorylase in maize, Bt2 and L2, replace the single alternatively spliced
gene found in other cereal species. Journal of Molecular Evolution, 65, 316–327.
Rydberg U, Andersson L, Andersson R, Aman P. Larsson H. 2001. Comparison of
starch branching enzyme I and II from potato. European Journal of Biochemistry. 268,
6140–6145.
Ryoo N, Yu C, Park CS, Baik MY, Park IM, Cho MH, Bhoo SH, An G, Hahn TR,
Jeon JS. 2007. Knockout of a starch synthase gene OsSSIIIa/Flo5 causes white-core
floury endosperm in rice (Oryza sativa L.). Plant Cell Reports, 26, 1083–1095.
Sabelli AP, Larkins BA. 2009a. The contribution of cell cycle regulation to endosperm
development. Sex Plant Reproduction. 22, 207–209.
- 238 -
Sabelli PA, Larkins BA. 2008. The endoreduplication cell cycle: regulation and
function. In DPS Verma, Z Hong, eds, Cell Division Control in Plants, Vol 9. Springer,
Berlin/Heidelberg, pp 75–100.
Sabelli PA, Larkins BA. 2009b. The development of endosperm in grasses. Plant
Physiology, 149, 14–26.
Sahlstrom S, Bavre AB, Brathen E. 2003. Impact of starch properties on hearth bread
characteristics. II. Purified A- and B-granule fractions. Journal of Cereal Science, 7,
285–293.
Sahlstrom S, Brathen E, Lea P, Autio K. 1998. Influence of starch granule size
distribution on bread characteristics. Journal of Cereal Science, 28(2) 157–164.
Salanoubat M, Belliard G. 1989. The steady-state level of potato sucrose synthase
mRNA is dependent on wounding, anaerobiosis and sucrose. Gene, 84, 181–185.
Salman H, Blazek J, Lopez-Rubio A, Gilbert EP, Hanley T, Copeland L. 2009.
Structure–function relationships in A- and B- granules from wheat starches of similar
amylose content. Carbohydrate Polymers, 75(3), 420-427.
Sandhu K, Singh N, Kaur M. 2004. Characteristics of the different corn types and their
grain fractions: physiochemical, thermal, morphological, and rheological properties of
starch. Journal of Food Engineering 64, 119–127.
Satoh H, Shibahara K, Tokunaga T, Nishi A, Tasaki M, Hwang SK, Okita TW,
Kaneko N, Fujita N, Yoshida M, Hosaka Y, Sato A, Utsumi Y, Ohdan T, Nakamura
Y. 2008. Mutation of the plastidial alpha-glucan phosphorylase gene in rice affects the
synthesis and structure of starch in the endosperm. The Plant Cell, 20, 1833–1849.
Scheible WR, González-Fontes A, Lauerer M, Müller-Röber B, Caboche M, Stitt M.
1997. Nitrate acts as a signal to induce organic acid metabolism and repress starch
metabolism in tobacco. The Plant Cell, 9, 783–798.
Schulman AH, Ahokas H. 1990. A novel shrunken endosperm mutant of barley. Plant
Physiology, 78, 583–589.
Schulman AH, Tomooka S, Suzuki A, Myllarinen P, Hizukuri S. 1995. Structural
analysis of starch from normal and shx (shrunken endosperm) barley (Hordeum vulgare
L.). Carbohydrate Research, 275, 361-369.
Schupp N, Ziegler P. 2004. The relation of starch phosphorylases to starch metabolism
in wheat. Plant and Cell Physiology, 45, 1471-1484.
- 239 -
Senoura T, Asao A, Takashima Y, Isono N, Hamada S, Ito H, Matsui H. 2007.
Enzymatic characterization of starch synthase III from kidney bean (Phaseolus vulgaris
L.). The Federation of European Biochemical Societies Journal, 274, 4550–4560.
Seo BS, Kim S, Scott MP, Singletary GW, Wong KS, James MG, Myers AM. 2002.
Functional interactions between heterologously expressed starch branching enzymes of
maize and glycogen synthases of Brewer’s yeast. Plant Physiology, 128, 1189–1199.
Shahram E, Venkatesh M, Mark d’P, Robert TT. 2010. Impact of micronization on
rapidly digestible, slowly digestible, and resistant starch concentrations in normal, highamylose, and waxy barley. Journal of Agricultural and Food Chemistry, 58, 9793–9799.
Shannon JC, Pein FM, Cao HP, Liu KC. 1998. Brittle-1, an adenylate translocator,
facilitates transfer of extraplastidial synthesized ADP-glucose into amyloplasts of maize
endosperms. Plant Physiology, 117, 1235-1252.
Shannon JC, Pien FM, Liu KC. 1996. Nucleotides and nucleotide sugars in developing
maize endosperms (synthesis of ADPglucose in brittle-1). Plant Physiology, 110, 835843.
Shapter FM, Lee LS, Henry RJ. 2008. Endosperm and starch granule morphology in
wild cereal relatives. Plant Genetic Resources: Characterization and Utilization, 6(2),
85–97.
Shi YC, Seib PA. 1992. The structure of four waxy starches related to gelatinization and
retrogradation. Carbohydrate Research, 227, 131–145.
Shi YC, Seib PA. 1995. Fine structure of maize starches from four wx-containing
genotypes of the W64A in bread line in relation to gelatinization and retrogradation.
Carbohydrate Polymers, 26, 141–147.
Shinde SV, Nelson JE, Huber KC. 2003. Soft wheat starch pasting behaviour in relation
to A- and B-type granule content and composition. Cereal Chemistry, 80, 91-98.
Sikka VK, Choi S, Kavakli IH, Sakulsingharoj C, Gupta S, Ito H, and Okita TW.
2001. Subcellular compartmentation and allosteric regulation of the rice endosperm
ADP-glucose pyrophosphorylase. Plant Science, 161, 461–468.
Singh N, and Kaur L. 2004. Morphological, thermal, rheological and retrogradation
properties of potato starch fractions varying in granule size. Journal of the Science of
Food and Agricultur, 84, 1241–1252.
Singletary GW, Banisadr R, Keeling PL. 1997. Influence of gene dosage on
carbohydrate synthesis and enzymatic activities in endosperm of starch-deficient mutants
of maize. Plant Physiology, 113, 293–304.
- 240 -
Smith AM, Denyer K, Martin C. 1997. The synthesis of the starch granule. Annual
Review of Plant Physiology and Plant Molecular Biology, 48, 67-87.
Smith AM. 2010. Starch and starch granules. in: encyclopedia of life sciences
(ELS). John Wiley & Sons, Ltd: Chichester.pub, pp.1–4.
Sokolov LN, Dejardin A, Kleczkowski LA. 1998. Sugars and light/dark exposure
trigger differential regulation of ADP-glucose pyrophosphorylase genes in Arabidopsis
thaliana (thale cress). Biochemical Journal, 336, 681–687.
Sokolov LN, Dominguez-Solis JR, Allary AL, Buchannan BB, Luan S. 2006. A
redox-regulated protein phosphatase binds to starch diurnally and functions in its
accumulation. Proceedings of the National Academy of Sciences, 103, 9732–9737.
Song Y, Jane J. 2000. Characterization of barley starches of waxy, normal and high
amylose varieties. Carbohydrate Polymers, 41, 365–377.
Sonnewald U, Basner A, Greve B, Steup M. 1995. A second L-type isozyme of potato
glucan phosphorylase: cloning, antisense inhibition and expression analysis. Plant
Molecular Biology, 27, 567–576.
Soulaka AB, Morrison WR. 1985. The amylose and lipid content, dimensions, and
gelatinisation characteristics of some wheat starches and their A-granule and B-granule
fraction. Journal of the Science of Food and Agriculture, 36, 709-718.
Stamova BS, Laudencia-Chingcuanco D, Beckles DM. 2009. Transcriptomic analysis
of starch biosynthesis in the developing grain of hexaploid wheat. International Journal
of Plant Genomics, 1–23.
Stinard PS, Robertson DS, Schnable PS. 1993. Genetic isolation, cloning and analysis
of a mutator-induced, dominant antimorph of the maize amylose extender1 locus. The
Plant Cell, 5, 1555–1566.
Stoddard FL. 2003. Genetics of starch granule size distribution in tetraploid and hexa
ploid wheats. Australian Journal of Agricultural Research, 54(7), 637-648.
Streb S, Zeeman SC. 2012. Starch metabolism in Arabidopsis. The Arabidopsis Book.
10, 160.
Sullivan TD. 1995. The maize brittle1 gene encodes amyloplast membrane polypeptides.
Planta, 196, 477-484.
Sun C, Sathish P, Ahlandsberg S, Jansson C. 1998. The two genes encoding starchbranching enzymes IIa and IIb are differentially expressed in barley. Plant Physiology,
118, 37–49.
- 241 -
Sun Z, Henson CA. 1991. A quantitative assessment of the importance of barley seed αamylase, β-amylase, debranching enzyme, and α-glucosidase in starch degradation.
Archives of Biochemistry and Biophysics, 284, 298–305.
Szydlowski N, Ragel P, Hennen-Bierwagen TA, Planchot V, Myers AM, Merida A,
d’Hulst C, Wattebled F. 2011. Integrated functions among multiple starch synthases
determine both amylopectin chain length and branch linkage location in Arabidopsis leaf
starch. Journal of Experimental Botany, 62, 4547–4559.
Szydlowski N, Ragel P, Raynaud S, Lucas MM, Roldan I, Montero M, Muñoz FJ,
Ovecka M, Bahaji A, Planchot V, Pozueta-Romero J, D'Hulst C, Mérida A. 2009.
Starch granule initiation in Arabidopsis requires the presence of either class IV or class
III starch synthases. The Plant Cell, 21, 2442–2457.
Takaha T, Yanase M, Okada S, Smith SM. 1993. Disproportionating enzyme (4-a
glucanotransferase: EC 2.4.1.25) of potato. Journal of Biological Chemistry, 268, 1391–
1396.
Takeda Y, Guan HP, Preiss J. 1993. Branching of amylose by the branching
isoenzymes of maize endosperm. Carbohydrate Research, 240, 253–263.
Takeda Y, Preiss J. 1993. Structures of B90 (sugary) and W64A (normal) maize
starches. Carbohydrate Research. 240, 265–275.
Takeda Y, Takeda C, Mizukami H, Hanashiro I. 1999. Structures of large, medium
and small starch granules of barley grain. Carbohydrate Polymers, 38, 109-114.
Tanaka N, Fujita N, Nishi A, Satoh H, Hosaka Y, Ugaki M, Kawasaki S, Nakamura
Y. 2004. The structure of starch can be manipulated by changing the expression levels of
starch branching enzyme IIb in rice endosperm. Plant Biotechnology Journal, 2, 507-516.
Tang H, Ando H, Watanaba K, Takeda Y, Mitsunaga T. 2001. Physicochemical
properties and structure of large, medium and small granule starches in fractions of
normal barley endosperm. Carbohydrate Research, 330, 241–248.
Tang H, Ando H, Watanabe K, Takeda Y, Mitrunga T. 2000. Some physicochemical
properties of small-, medium-, and large-granule starches in fractions of waxy barley
grain. Cereal Chemistry, 77, 27–31.
Tauberger E, Fernie AR, Emmermann M, Renz A, Kossmann J,
Willmitzer L, Trethewey RN. 2000. Antisense inhibition of plastidial
phosphoglucomutase provides compelling evidence that potato tuber
amyloplasts import carbon from the cytosol in the form of glucose-6phosphate. The plant journal, 23(1), 43–53.
- 242 -
Tester FR, John K, Xin Q. 2004. Starch-composition, fine structure and
architecture. Journal of Cereal Science, 39(2), 151–165.
Tester RF, Morrison WR, Schulman AH. 1993. Swelling and gelatinization of cereal
starches. V. Risø mutants of Bomi and Carlsberg II barley cultivars. Journal of Cereal
Science, 17, 1–9.
Tester RF, Morrison WR. 1990. Swelling and gelatinization of cereal starches. III.
Some properties of waxy and non waxy barley starches. Cereal Chemistry, 69, 654–658.
Tetlow IJ. 2011 . Starch biosynthesis in developing seeds. Seed Science Research 21, 532.
Tetlow IJ, Beisel KG, Cameron S, Makhmoudova A, Liu F, Bresolin NS, Wait R,
Morell MK, Emes MJ. 2008. Analysis of protein complexes in wheat amyloplasts
reveals functional interactions among starch biosynthetic enzymes. Plant Physiology,
146, 1878-1889.
Tetlow IJ, Davies EJ, Vardy KA, Bowsher CG, Burrell MM, Emes MJ. 2003b.
Subcellular localization of AD-Pglucose pyrophosphorylase in developing wheat
endosperm and analysis of a plastidial isoform. Journal of Experimental Botany, 54,
715-725.
Tetlow IJ, Kerry JB, Emes MJ. 1994. Starch synthesis and carbohydrate oxidation in
amyloplasts from developing wheat endosperm. Planta, 194, 454–560.
Tetlow IJ, Liu F, Emes MJ. 2008. Functional interactions between starch synthases and
branching enzymes of cereal endosperms. Comparative Biochemistry and Physiology,
150(3), S194–S195.
Tetlow IJ, Morell MK, Emes MJ. 2004. Recent developments in understanding the
regulation of starch metabolism in higher plants. Journal of Experimental Botany,
55, 2131-2145.
Tetlow IJ, Wait R, Lu ZX, Akkasaeng R, Bowsher CG, Esposito S, Kosar-Hashemi
B, Morell MK, Emes MJ. 2004. Protein phosphorylation in amyloplasts regulates starch
branching enzyme activity and protein-protein interactions. The Plant Cell, 16(3), 694–
708.
Tetlow IJ. 2006. Understanding storage starch biosynthesis in plants: a means to
quality improvement. The Canadian Journal of Botany, 84, 1167–1185.
Tetlow IJ. 2011. Starch biosynthesis in developing seeds. Seed Science
Research. 21, 5–32.
- 243 -
Tetlow, IJ, Bowsher CG, Scrase-Field EFAL, Davies EJ, Emes MJ. 2003a. The
synthesis and transport of ADP-glucose in cereal endosperms. Journal of Applied
Glycoscience, 50, 231-236. The Journal of Experimental Botany, 62(14), 4819-4831.
Thorbjørnsen T, Villand P, Denyer K, Olsen OA, Smith AM. 1996. Distinct isoforms
of ADPglucose pyrophosphorylase occur inside and outside the amyloplasts in barley
endosperm. The Plant Journal, 10, 243-250.
Thorbjornsen T, Villand P, Kleczkowski LA, Olsen OA. 1996. A single gene encodes
two different transcripts for the ADP-glucose pyrophosphorylase small subunit from
barley (Hordeum vulgare). Biochemical Journal, 313, 149-154.
Tiessen A, Hendriks JHM, Stitt M, Branscheid A, Gibon Y, Farre EM,
Geigenberger P. 2002. Starch synthesis in potato tuber is regulated by posttranslational redox modification of ADP-glucose pyrophosphorylase. The Plant Cell,
14, 2191-2213.
Tomlinson K, Denyer K. 2003. Starch synthesis in cereal grain. Advances in Botanical
Research, 40, 1–61.
Topping D, Clifton P. 2001. Short chain fatty acids and human colonic function – roles
of resistant starch and non starch polysaccharides. Physiological Reviews, 81, 1031-64.
Tsai CY, Nelson OE. 1969. Mutations at the shrunken-4 locus in maize that produce
three altered phosphorylases. Genetics, 61, 813-821.
Tyynelä J, Schulman AH. 1993. An analysis of soluble starch synthase isozymes from
the developing grains of normal and shx cv. Bomi barley (Hordeum vulgare). Plant
Physiology, 89, 835–841.
Tyynelä J, Stitt M, Lönneborg A, Smeekens S, Schulman AH. 1995. Metabolism of
starch synthesis in developing grains of the shx shrunken mutant of barley (Hordeum
vulgare). Plant Physiology, 93, 77–84.
Umemoto T, Aoki N. 2005. Single-nucleotide polymorphisms in rice starch synthase IIa
that alter starch gelatinization and starch association of the enzyme. Functional Plant
Biology, 32, 763-768.
Umemoto T, Yano M, Satoh H, Short JM, Shamura A, Nakamura, Y. 2002. Mapping
of a gene responsible for the difference in amylopectin structure between japonica-type
and indica-type rice varieties. Theoretical and Applied Genetics, 104, 1–8
Utsumi Y, Nakamura Y. 2006. Structural and enzymatic characterization of the
isoamylase1 homooligomer and the isoamylase1-isoamylase2 hetro-oligomer from rice
endosperm. Planta, 225, 75–87.
- 244 -
Valdez HA, Busi MV, Wayllace NZ, Parisi G, Ugalde RA, Gomez-Casati DF. 2008.
Role of the N-terminal starch-binding domains in the kinetic properties of starch synthase
III from Arabidopsis thaliana. Biochemistry, 47, 3026–3032.
Van Hung P, Morita N. 2005. Physicochemical properties of hydroxypropylated and
cross-linked starches from A-type and B-type wheat starch granules. Carbohydrate
Polymer, 59, 239–246.
Vasanthan T, Bhatty RS. 1996. Physicochemical properties of small and large-granule
starches of waxy, regular, and high-amylose barley. Cereal Chemistry, 73, 199–207.
Vermeylen R, Goderis B, Reynaers H, Delcour JA. 2005. Gelatinisation related
structural aspects of small and large wheat starch granules. Carbohydrate Polymers, 62,
170-181.
Villand P, Aalen R, Olsen OA, Lonneborg A, Luthi E, Kleczkowski LA. 1992. PCRamplification and sequence of cDNA clones for the small and large subunits of ADPglucose pyrophosphorylase from barley tissues. Plant Molecular Biology, 19, 381389.
Vorster HH. 2009. Introduction to Human Nutrition, 2nd Edition: A Global Perspective
on Food and Nutrition. (MJ. Gibney, SA. Lanham-New, A. Cassidy, HH, Vorster, Eds).
Singapore: John Wiley & Sons
Vrinten P, Nakamura T. 2000. Wheat granule-bound starch synthase I and II are
encoded by separate genes that are expressed in different tissues. Plant Physiology, 122,
255–263.
Wang SM, Fu-Mee Y, Ai-Hsiang C. 2004. β-amylase is not involved in degradation of
endosperm starch during seed germination of maize. Taiwania, 49(4), 263-272.
Wang X, Brown IL, Evans AJ, Conway PL. 1999. The protective effects of high
amylose maize (amylomaize) starch granules on the survival of Bifidobacterium spp. in
the mouse intestinal tract. Journal of Applied Microbiology, 87, 631–639.
Wang YJ, White P, Pollak L. 1993. Characterization of starch properties of maize
mutants from oh43 inbred line background. Cereal Chemistry, 70, 199-203.
Wattebled F, Dong Y, Dumez S, Delvalle D, Planchot V, Berbezy P, Vyas D,
Colonna P, Chatterjee M, Ball S, D'Hulst C. 2005. Mutants of Arabidopsis lacking a
chloroplastic isoamylase accumulate phytoglycogen and an abnormal form of
amylopectin. Plant Physiology, 138, 184–195.
Wei C, Zhang J, Chen Y, Zhou W, Xu B, Wang Y, Chen J. 2010. Physicochemical
properties and development of wheat large and small starch granules during endosperm
development. Acta Physiologiae Plantarum, 32, 905–916.
- 245 -
Wei C, Qin F, Zhu L, Zhou W, Chen Y, Wang Y, Gu M, Liu Q. 2010. Microstructure
and ultra structure of high-amylose rice resistant starch granules modified by antisense
RNA inhibition of starch branching enzyme. Journal of Agricultural and Food
Chemistry, 58, 1224–1232.
Wilson JD, Bechtel DB, Todd TC, Seib PA. 2006. Measurement of wheat starch
granule size distribution using image analysis and laser diffraction technology. Cereal
Chemistry, 83(3), 259–268.
Yamamori M. 2000. Genetic elimination of a starch granule protein, SGP-1, of wheat
generates an altered starch with apparent high amylose. Theoretical and Applied
Genetics, 101, 21–29.
Yamanouchi H, Nakamura Y. 1992. Organ specificity of isoforms of starch branching
enzyme (Q-enzyme) in rice. Plant and Cell Physiology, 33, 985–991.
Yao Y, Guiltinan MJ, Shannon JC, Thompson DB. 2002. Single kernel sampling
method for maize starch analysis while maintaining kernel vitality. Cereal Chemistry, 79,
757–762.
Yao Y, Thompson DB, Guiltinan MJ. 2004. Maize starch-branching enzyme isoforms
and amylopectin structure. In the absence of starch-branching enzyme IIb, the further
absence of starch-branching enzyme IIa leads to increased branching. Plant Physiology,
136, 3515–3523.
Yeh JY, Garwood DL, Shannon J. 1981. Characterisation of starch from maize
endosperm mutants. Starch-Stärke, 33, 222–230.
Yonemoto PG, Calori-Domingues MA, Franco CML. 2007. Effect of granule size on
the structural and physicochemical characteristics of wheat starch. Ciência e Tecnologia
de Alimentos, 27(4), 761–771.
Yoshimoto Y, Tadahiro T, Yasuhito T. 2002. Molecular structure and some
physicochemical properties of waxy and low-amylose barley starches. Carbohydrate
Polymers 47, 159-167.
Yoshimoto Y, Tashiro J, Takenouchi T, Takeda Y. 2000. Molecular structure and
some physicochemical properties of high amylose barley starches. Cereal Chemistry,
77, 279–285.
You S, Izydorczyk MS. 2007. Comparison of the physicochemical properties of barley
starches after partial-amylolysis and acid/alcohol hydrolysis. Carbohydrate Polymers
(69), 489–502.
Young TE, Gallie DR. 2000a. Programmed cell death during endosperm development.
Plant Molecular Biology, 44, 283–301.
- 246 -
Young TE, Gallie DR. 2000b. Regulation of programmed cell death in maize endosperm
by abscisic acid. Plant Molecular Biology, 42, 397–414.
Yu Y, Mu HH, Mu-Forster C, Wasserman BP, George MC. 2001. Identification of
the maize amyloplast stromal 112-kD protein as a plastidic starch phosphorylase. Plant
Physiology, 125, 351-359.
Yuan RC, Thompson DB, Boyer CD 1993. Fine structure of amylopectin in relation to
gelatinization and retrogradation behavior of maize starches from three wax-containing
genotypes in two inbred lines. Cereal Chemistry, 70, 81–89.
Yun SH, Matheson MK. 1990. Estimation of amylose content of starches after
precipitation of amylopectin by concanavalin A. Starch, 42, 302-305.
Zeeman SC, Kossmann J, Smith AM. 2010. Starch: its metabolism, evolution, and
biotechnological modification in plants. Annual Review of Plant Biology, 61, 209–234.
Zeeman SC, Northrop F, Smith AM, Rees T. 1998. A starch-accumulating mutant of
Arabidopsis thaliana deficient in a starch-hydrolyzing enzyme. The Plant Journal, 15,
357–365.
Zeeman SC, Smith SM, Smith AM. 2007. The diurnal metabolism of leaf starch.
Biochemical Journal, 401, 13–28,
Zeeman SC, Thorneycroft D, Schupp N, Chapple A, Weck M, Dunstan H,
Haldimann P, Bechtold N, Smith AM, Smith SM. 2004. Plastidial α-glucan
phosphorylase is not required for starch degradation in Arabidopsis leaves but has a role
in the tolerance of abiotic stress. Plant Physiology, 135, 849-858.
Zeeman SC, Umemoto T, Lue Wl, Au-Yeung P, Martin C, Smith AM, Chen J. 1998.
A mutant of Arabidopsis lacking a chloroplastic isoamylase accumulates both starch and
phytoglycogen. The Plant Cell, 10, 1699–1711.
Zhang C, Jiang D, Liu F, Cai J, Dai T, Cao W. 2010. Starch granules size distribution
in superior and inferior grains of wheat is related to enzyme activities and their gene
expressions during grain filling. Journal of Cereal Science, 51(2), 226-233.
Zhang G, Ao Z, Hamaker BR. 2006. Slow digestion property of native cereal starches.
Biomacromolecules, 7, 3252–3258.
Zhang X, Colleoni C, Ratushna V, Sirghie-Colleoni M, James MG, Myers AM.
2004. Molecular characterization demonstrates that the Zea mays gene sugary2 codes for
the starch synthase isoform SSIIa. Plant Molecular Biology, 54, 865–879.
- 247 -
Zhang X, Myers AM, James MG. 2005. Mutations affecting starch synthase III in
Arabidopsis alter leaf starch structure and increase the rate of starch synthesis. Plant
Physioogy, 138, 663–674
Zhang X, Szydlowski N, Delvalle D, D’Hulst C, James MG, Myers AM. 2008.
Overlapping functions of the starch synthases SSII and SSIII in amylopectin biosynthesis
in Arabidopsis. BioMed Central Plant Biology, 8, 96.
Zheng GH, Han HL, Bhatty RS. 1998. Physiochemical properties of zero amylose
hulless barley starch. Cereal Chemistry, 75, 520–524.
Zheng Y, Wang W. 2010. Structural character of sorghum endosperm transfer cells and
their relationship with embryo and endosperm. International Journal of Plant Biology, 1,
75–77.
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Appendices
- 249 -
Appendix 1: Description of genotypes used in the study.
S. No. Genotypes
Type
Row source
OAC Baxter
Covered
1
6 1. University of Guelph
GB992033
Covered
2
2 1. University of Guelph
sbeiiaCovered
3
2 2. CSIRO, Canberra
sbeiibCovered
4
2 2. CSIRO, Canberra
HAG amo1
Covered
5
6 2. CSIRO, Canberra
McGwuire
Hulless
6
2 3. CDC, Saskatoon
Sloop
Covered
7
2 2. CSIRO, Canberra
Neopolis
Hulless
8
2 3. CDC, Saskatoon
CDC-Fibar
Hulless
9
2 3. CDC, Saskatoon
CDC-Rattan
Hulless
10
2 3. CDC, Saskatoon
Soft Barley
Hulless
11
2 1. University of Guelph
Hard Barley
Hulless
12
2 1. University of Guelph
SB94983 amo1
Hulless
13
2 3. CDC, Saskatoon
AC
Metcalfe
Covered
14
2 1. University of Guelph
083211-120
Covered
15
2 1. University of Guelph
083211-122
Covered
16
2 1. University of Guelph
083311-104
Covered
17
6 1. University of Guelph
083411-113
Hulless
18
6 1. University of Guelph
AC Alberte
Hulless
19
2 1. University of Guelph
AC Bacon
Hulless
20
6 1. University of Guelph
Emperor
Hulless
21
2 1. University of Guelph
Sunderland
Covered
22
2 1. University of Guelph
083511-109
Hulless
23
2 1. University of Guelph
083511-118
Hulless
24
2 1. University of Guelph
OAC Kawartha
Covered
25
6 1. University of Guelph
083611-118 sex1 Covered
26
6 1. University of Guelph
083611-124 sex1 Hulless
27
2 1. University of Guelph
081011-928
Hulless
28
2 1. University of Guelph
081011-929
Hulless
29
2 1. University of Guelph
081011-930
Hulless
30
2 1. University of Guelph
081011-931
Covered
31
2 1. University of Guelph
081011-932
Hulless
32
2 1. University of Guelph
020113-385 sex1 Covered
33
6 1. University of Guelph
comments
'normal' reference genotype
amo1 reference genotype
wx1
wx1
wx1
wx1
amo1
rob (orange lemma)
rob (orange lemma)
sex1
sex1
amo1
amo1
amo1
amo1
amo1
sex1 rob (orange lemma)
1. Dr. Duane Falk, Cereal Breeder, Department of Plant Agriculture, University of
Guelph, Guelph, Ontario
2. Dr. Mathew Morell, Cereal Chemist, CSIRO, Black Mountain, Canberra, ACT,
Australia
3. Dr. Brian Rossnagel, Cereal Breeder, Crop Development Centre, University of
Saskatchewan, Saskatoon, Saskatchewan
- 250 -
Appendix 2: Comparison of three methods used for amylose determination.
Genotypes
OAC Baxter
sbeiiasbeiib083611-118 sex1
083611-124 sex1
020113-385 sex1
HAG amo1
SB94983 amo1
081011-928
081011-929
081011-930
081011-931
081011-932
Megazyme
25
30
30
35
35
35
40
30
45
46
39
29
44
Iodine
24.8
28.8
27.7
34.5
34.4
32.3
37.0
36.6
43.2
47.4
41.1
33.8
45.5
GPC
24.3
30.63
30.99
40.23
32.54
34.84
35.58
36.59
43.59
45.45
40.45
33.07
42.99
Amylose content measured by three different methods; Megazyme amylose/
amylopectin assay kit, iodine binding and GPC.
- 251 -
Appendix 3: PCA analysis shows association of three methods used for amylose
determination.
Figure S1: The name of each character is given in the PCA while different genotypes are
presented by (+). Characters present in close vicinity are associated. Genotypes have
been clustered into different groups depending upon their association with different
characters and each cluster is presented by a different circle: black, normal barley; Light
green, experimental lines; red, waxy; blue, shrunken (sex1); Dark green, high amylose.
- 252 -
Appendix 4: Determination of amylose with 6CL-B column.
1.2
OAC Baxter
sbeiiasbeiib083611-118 sex1
083611-124 sex1
020113-385 sex1
1.0
Absorbance
0.8
0.6
0.4
0.2
0.0
40
60
80
100
120
140
160
180
140
160
180
Fraction number
1.2
OAC Baxter
HAG amo1
SB94983 amo1
081011-928
081011-929
081011-930
081011-931
081011-932
1.0
Absorbance
0.8
0.6
0.4
0.2
0.0
40
60
80
100
120
Fraction number
Amylose eluded in early fractions and amylopectin in late fractions. Each genotype with
different symbol has been presented in the legends.
- 253 -
Appendix 5: Iodine-staining of barley grains (cross section).
To see the internal structure and starch packing with in seed from each genotype, cross
sections along the length of seed were made and incubated in the Lugol’s solution.
Name of each genotype is given under each section of figure.
- 254 -
Appendix 6: DSC data of different genotypes.
Initial Heating Summary
Genotype
Melting of amylose-lipid
Starch Gelatinization
complex
To
Tp
Tc
∆H
To
Tp
Tc
∆H
(Co) (Co) (Co) (J/g) (Co) (Co)
(Co)
(J/g)
SB94983 amo1 58.2 65.9 78.2 10.8 95.9 103.5 111.3 2.0
081011-929
55.7 68.4 81.5 8.19 91.9 102.2 109.9 3.1
081011-931
56.7 62.3 73.3 12.5 96.3 103.6 108.9 1.3
081011-932
55.5 68.1 80.4 9.16 93.0 102.5 109.6 2.5
Re-Heating Summary
Genotype
Melting of retrograded
melting of amylose-lipid
starch
complex
To
Tp
Tc
∆H
To
Tp
Tc
∆H
(Co) (Co) (Co) (J/g) (Co) (Co)
(Co)
(J/g)
SB94983 amo1 40.1 53.1 66.4 4.6
94.0 104.2 110.3 1.4
081011-929
40.6 52.3 64.5 3.2
86.5 99.7
107.3 2.7
081011-931
40.9 52.3 65.3 2.7
97.1 104.5 111.2 1.1
081011-932
42.6 53.3 63.6 2.7
87.8 100.9 107.8 2.2
Thermal analyses of starch from different genotypes were performed as described by
(Liu et al., 2007) using a differential scanning calorimeter (2920 Modulated DSC, TA
Instruments, New Castle, DE).
- 255 -
Appendix 7: Immunological characterization of endosperm amyloplast lysates from
different barley mutants.
Amyloplast lysates (~1.3 mg/ml) were prepared from developing barley endosperms at
18–25 DAP. Aliquots of soluble (stromal) proteins were separated on 10 %
polyacrylamide gels and electroblotted onto nitrocellulose membranes. Immunoblots
were developed with peptide-specific anti-wheat antibodies. Left hand side indicate
cross-reactions of each of the antibodies with its corresponding target protein, and the
name of each genotype is given below. The approximate molecular mass for each
protein, based on its SDS–PAGE migration, is given on right hand side.
- 256 -
Appendix 8: Detection of SS activity and protein.
Non-denaturing electrophoresis was performed in 5% (w/v) polyacrylamide gels
containing 0.3% (w/v) amylopectin. Approximately 300 µg protein from whole cell
extracts was loaded onto each lane. Following electrophoresis, gels were incubated for
48–72 h at 30oC. Activity of SS was visualized by staining gels with I2–KI (A). Arrows
indicate activity band corresponding to different SS protein in zymogram (A). The
identity of SS has been made by coupling non-denaturing PAGE with immunoblotting,
and proteins were detected by using specific antibody (B, C, D, E).
- 257 -
Appendix 9: Detection of SBE activity and protein.
Non-denaturing electrophoresis was performed in 5% (w/v) polyacrylamide gels
containing 0.2% (w/v) maltoheptaose and 1.4 U of rabbit muscle extracted
phosphorylase a. Approximately 300 µg protein from whole cell extracts was loaded
onto each lane. Following electrophoresis, gels were incubated for 3 - 3.5 h at 28oC.
Activity of SBE was visualized by staining gels with I2-IK. Activity of SP is detected as dark
blue bands (A). Migration of SBE isoforms was determined by immunoblotting with
specific antibodies (B, SBEIIa; C, SBEIIb; D, SBEI and SBEIc). Name of each genotype
shown. Arrows indicate specific activity band in zymogram and presence of respective
proteins in immunoblots incubated with different antibodies.
- 258 -
Appendix 10: Detection of SP protein and activity in different genotypes.
(A) Activity of SP (Pho1) is visualized as a dark blue band. (B) Immunodetection of SP
(Pho1 and Pho2).
- 259 -
Appendix 11: Co-immunoprecipitation of stromal proteins from amyloplasts of
different genotypes with SSI, SBEIIa and SBEIIb antibodies.
- 260 -
- 261 -
Aliquots (0.75-1 ml) of amyloplast lysates (0.8–1.3 mg protein ml/ml) prepared from
endosperm of different genotypes at 18–25 DAP were incubated with specific anti-SSI
(A), anti-SBEIIa (B) and anti-SBEIIb (C) antibodies at 25oC for 1h, and then
immunoprecipitated with protein A–Sepharose beads. The protein A–Sepharose–
antibody–antigen complexes were washed several times to remove non-specifically
bound proteins, boiled in 200 µl of SDS loading buffer, and 25 µl was loaded in each lane
of 10 % polyacrylamide gels. Following electrophoresis gels were electroblotted onto
nitrocellulose, and developed with various anti-barley antibodies including (SSI, SSIIa,
SSIII, SSIV, SBEI, SBEIc, SBEIIa, SBEIIb, SP and ISO). However immunoblot incubated with
SSI, SSIIa, SBEI, SBEIIa, SBEIIb and SP antibodies are presented.
- 262 -
Appendix 12: Summary of novel protein–protein interactions formed between
amylopectin-synthesizing enzymes in barley endosperm following either loss of
single gene (SSIIa, SBEIIa and SBEIIb) or alteration in single gene (ssiii-, amo1
mutant).
In reference genotype (A), the major form of SBEII (SBEIIa and SBEIIb) form
phosphorylation-dependent protein complex with SSI and SSIIa independently. In the
- 263 -
branching enzyme mutants (B & C), possibly two distinct protein complexes consisting of
SSI/SSIIa and expressed SBEII, and SP/SBEI and expressed SBEII were purified. In which
protein complex, SP/SBEI/ expressed SBEII partitioned to A-granules not B-granules. Loss
of SSIIa was more significant (D) where due to absence of SSII no protein complex was
formed and consequently no protein was found within the starch granule. In case of
amo1 mutant protein complex consisted of SSI, SSIIa, SBEIIa and SBEIIb were purified.
However, there is a possibility of existing of other complexes in different combinations
such as, SSI/SSIIa/SBEIIa and SSI/SSIIa/SBEIIb. For each of the genotype, the components
of the protein complexes found in the plastid stroma are also found in the respective A
and B starch granules but in sex6- mutant where no protein complex was formed, no
protein was found in the starch granules. Inverted arrows between A and B granules
show possible involvement of same protein complex in A and B granules synthesis.
- 264 -
Appendix 13: Starch, A- and B-granules bound proteins.
- 265 -
- 266 -
Starch granules were isolated from mature barley grains by grinding seeds and
preparing starch granules as described Materials and Methods. The purified, acetone
washed starch was used to separate large A and small B granules with Percoll gradient
centrifugation method from all genotypes. The separated A, B granules and starch were
washed extensively to remove proteins loosely bound to the granule surface. A 50 mg
aliquot of purified starch, A and B granules were boiled in 1 ml of SDS loading buffer,
and 40 µl of the supernatant from the boiled sample loaded onto 4–12 % acrylamide
gradient gels and 10 % acrylamide SDS gels. Following electrophoresis, gels were
electroblotted to nitrocellulose membrane and developed with various specific antiwheat antibodies (SSI, SSIIa, SSIII, SSIV, SBEI, SBEIIa, SBEIIb, SP, ISO1 and GBSS). Each
section is labelled with respective antibody and type of granules used either starch, Aor B-.
- 267 -
Appendix 14: Granule-bound phospho-proteome analysis and starch, A- and Bgranules bound proteins detected by silver staining.
To determine granule-bound phospho-proteome equal amounts of starch from all
genotypes were run onto 4–12 % acrylamide gradient gels, and stained with Pro-Q
Diamond. Following Pro-Q Diamond staining the same gel (Phospho- proteome) was
subject to silver staining (total starch) to visualize all bands. The bands visualized with
silver stain are numbered as (1, SSIII; 2, SBEIc; 3, SP/unknown; 4, 5, 6, SSIIa; SBEIIa;
SBEIIb; 7, SSI; 8, unknown; and 9, GBSS). In A-granules two additional bands, 10 and 11
present degraded GBSS and SS respectively. However all these bands were not found to
be phosphorylated and among above mentioned bands only GBSS1, SSI, SSIIa and SBEIIb
were found to be phosphorylated in their granule-bound state (Phospho-proteome).
- 268 -
Appendix 15: Analysis of starch composition and granule morphology by light
microscope (A, B & C) and electron microscope (D & E).
Separation of A- and B-/C- granules was performed according to method described
earlier (Peng et al., 1999). Total starch containing, A-, B- and C- granules (A). Purified Agranules (B). Purified B-/C- granules (C). Electron micrograph of total starch,
predominant with A- and C- granules (D). Purified B-/C- granules (E). The reference
genotype, OAC Baxter is presented as an illustration for A- and B-/C- granules purity
confirmation in all genotypes studied here. The scale bar on panels A, B and C
represents 20 µm. Scale bar for electron micrograph is given under each panel. Each
type of granule is presented by an arrow.
- 269 -
Appendix 16: Single nucleotide polymorphisms (SNPs) of barley ssIIIa genomic
DNAs from different barley genotypes.
- 270 -
- 271 -
- 272 -
The Names of barley genotypes are labeled on the left hand side. The number of
nucleotide residues is labeled above. The dots indicate the same nucleotide residues as
the first line. The substituted nucleotide residues are indicated at position 330, 2101 (for
083611-124 sex1), 2693, 5610, 8338 and 860 (for HAG amo1, SB94983 amo1, 081011928, 081011-929, 081011-930, 081011-931 and 081011-932) and 3273 and 6323 (for
HAG amo1, SB94983 amo1, 081011-928, 081011-929, 081011-930 and 081011-932).
- 273 -
Appendix 17: SNPs of cDNA sequences of barley ssIIIa genomic DNAs from
different barley genotypes.
- 274 -
The Names of barley genotypes are labeled on the left hand side. The number of
nucleotide residues is labeled above. The dots indicate the same nucleotide residues as
the first line. The substituted nucleotide residues are indicated at position 1084 (for
083611-124 sex1), 1676 and 4439 (for HAG amo1, SB94983 amo1, 081011-928, 081011929, 081011-930, 081011-931 and 081011-932) and 2256 (for HAG amo1, SB94983
amo1, 081011-928, 081011-929, 081011-930 and 081011-932).
- 275 -
Appendix 18: Changes of polypeptide sequences of barley SSIIIa protein from
different barley genotypes.
- 276 -
The Names of barley genotypes are labeled on the left hand side. The number of
nucleotide residues is labeled above. The dots indicate the same nucleotide residues as
the first line. The substituted amino acid residues are indicated at position 362 (for
083611-124 sex1) and 559 and 1480 (for HAG amo1, SB94983 amo1, 081011-928,
081011-929, 081011-930, 081011-931 and 081011-932).
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