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1/19/2016
Outline
Soybean Transporter Database (SoyTD):
Genome-Wide Identification and
Exploration of Natural Variants in Soybean
Transporter Genes
o Introduction
o Methods
o Results
Gunvant Patil
o Comparative analysis of transporters in plants
(Henry Nguyen Lab)
o Classification of Soybean transporters
National Center for Soybean Biotechnology &
Division of Plant Sciences
University of Missouri, Columbia
o Insights through – Transcriptome profiling and Whole genome resequencing
o Example: soybean SWEET and SALT transporters, and wheat boron transporter
o Database design
o Summary
Soybean Genomics
January 12, 2016
Introduction
Classification based on
driving energy
Transport proteins (transporter) are membrane channels/pumps and facilitate
exchange of selective molecules, ions from the external and internal environment.
Extracellular space
o Active Transport (Primary
and Secondary)
Intracellular space
o Channel (passive diffusion)
Functional Role
o Carrier-type facilitators
(facilitated diffusion)
- Transporter are selective barrier and exchange wide variety of molecules
ATP
Selective
Channels
Carriers
Active
Transport
- Nutrient uptake and transport to sink tissue.
- Pump/exclude toxic ions, metals
- Signal transduction
Uniport
Classed based on driving
energy
o Active Transport (Primary
and Secondary)
Symport
Facilitated Diffusion
- Maintain osmotic balance in different cell types (aquaporin).
Typical transmembrane
protein
Antiport
Secondary Active Transport
Primary active transport
Channels
Facilitated diffusion
Secondary active transport (Co-transporters & Exchange)
Energy derived from hydrolysis of ATP to
ADP liberating energy from high energy
phosphate bond
Transport water or specific ions by conc.
Gradients.
Open in response to signal
Passive transport is powered by the potential
energy of a concentration gradient and does
not require the expenditure of metabolic
energy
No direct coupling of ATP; instead, the electrochemical
potential difference created by pumping ions out of the cell is
used.
Milton Saier et al. (http://tcdb.ucsd.edu/tcdb/)
Transporter gene resources and database
Extracellular space
Publically available Resources
Intracellular space
N
o Channel (passive diffusion)
o Carrier-type facilitators
(facilitated diffusion)
Binding Domain
ATP
Selective
Channels
Carriers
Active
Transport
Uniport
TMD
Symport
Facilitated Diffusion
TCDB
(Transporter Classification)
Saier et. al. 2014
http://www.tcdb.org/
TransportDB
(Relational database)
Ren et. al. 2007
http://www.membranetransport.org/
Human
Genome wide identification
Ye et. al. 2014
http://htd.cbi.pku.edu.cn/
intracellular
Arabidopsis
Schwacke et. al.
2003
http://aramemnon.botanik.uni-koeln.de/
C
Medicago
Miao et. al. 2012
http://bioinformatics.cau.edu.cn/MtTrans
porter/
TOPCON
(membrane topology prediction)
Tsirigos et. al. 2015
http://topcons.cbr.su.se/
TMHMM
(membrane topology prediction)
Krogh et. al. 2001
http://www.cbs.dtu.dk/services/TMHMM
/
extracellular
Antiport
Secondary Active Transport
Primary active transport
Channels
Facilitated diffusion
Secondary active transport (Co-transporters & Exchange)
Energy derived from hydrolysis of ATP to
ADP liberating energy from high energy
phosphate bond
Transport water or specific ions by conc.
Gradients.
Open in response to signal
Passive transport is powered by the potential
energy of a concentration gradient and does
not require the expenditure of metabolic
energy
No direct coupling of ATP; instead, the electrochemical
potential difference created by pumping ions out of the cell is
used.
Milton Saier et al. (http://tcdb.ucsd.edu/tcdb/)
1
1/19/2016
Workflow
TCDB
database
G.max
protein
blastp
Comparative analysis of putative transporters in 47 plant species
56,044 G.max (a2) protein
TransporterDB
blastp
TM Proteins
TMHMM
TOPCONs
~ 7-12% genes code for
transporter protein
in
filtered
plants
Data integration and filtering
(>2 TMD)
Classification
(TCDB)
Pfam
annotation
3306 SoyTD
NGS workflow
Sequence
Variants (SNPs)
Transcriptome
analysis
e.g. Salinity tolerance in soybean
e.g. Soybean SWEET gene family
Will be available @
www.soykb.org/SoyTD
Relationship between total genes and transporter gene numbers (>2 TMD)
Primary transporters
10000
Lineage specific gain and loss of transporter domains
T. aestivum
y = 0.0831x + 563.65
R² = 0.8948
Dicot
Monocot
Algae
Moss
9000
8000
G.max
7000
6000
Description
Total
L.ja
M.tr
G.ma
M.do
F.ve
M.es
R.co
P.tr
A.th
A.ly
C.pa
T.ca
V.vi
O.sa
O.Sa
indica
B.di
S.bi
Z.ma
Smo
P.pa
O.lu
O.ta
M.rcc
V.ca
C.re
IPR014047
Chromate transporter
6
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
1
1
1
1
IPR011866
Sulphate ABC transporter,
permease protein
2
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
GO:0046715
Boron transmembrane
transporter activity
58
2
3
4
5
2
2
2
3
6
6
2
2
2
3
4
3
2
5
0
0
0
0
0
0
0
IPR004648
GO/InterPro
5000
4000
Loss
Arabidopsis
3000
2000
1000
0
0
20000
40000
60000
80000
Tetrapeptide transporter,
OPT1/isp4
163
4
12
11
13
8
9
7
12
9
9
6
10
10
7
8
6
8
6
6
2
0
0
0
0
0
GO:0005385
zinc ion transmembrane
transporter activity
267
11
11
21
24
13
12
9
15
22
25
10
14
18
11
12
11
11
10
4
3
0
0
0
0
0
IPR015521
MATE family transporter
related protein
311
27
13
28
10
11
10
16
22
16
17
9
17
18
15
17
15
16
11
15
8
0
0
0
0
0
252
19
11
Gain
100000
IPR015606
No. of genes in genome
Estimation of all transporter (min_2) in plant genome (47
species). Best-fit linear regression (r2 = 0.89)
Cationic amino acid
transporter
secondary active sulfate
transmembrane
transporter
7
19
21
9
15
13
21
9
12
8
8
11
12
11
11
14
6
1
1
2
2
2
257
7
9
27
19
12
16
12
16
12
12
10
10
28
11
14
10
11
9
4
4
0
0
1
1
2
IPR003663
Sugar/inositol transporter 1210
56
64
99
110
55
67
52
70
53
54
42
50
48
65
69
57
63
67
32
20
6
2
6
2
1
IPR000109
Oligopeptide transporter 1484
87
77
125
140
62
73
55
82
53
55
56
62
53
85
99
79
89
78
49
20
1
1
1
1
1
GO:0008271
Expansion
Soybean Transporters classification
Classification of soybean Transporters
• Soybean encodes 3306 (2-TMD) transporters or transporter-related proteins.
1800
• Average 164 transporter gene per chromosomes.
1600
• Transporter genes were classified according to Transporter Classification (TC) system (Saier et. al.
2014)
1400
Major Classes (According to TC system)
7
1709
1200
1000
• 1:Channels/Pores
> Facilitate diffusion (energy independent)
800
• 2:Electrochemical Potential-driven
> Uniporter, symporters and antiporters
600
• 3:Primary Active Transporters
> Energy dependent/drive against concentrations
• 4:Group Translocators
> Substrate modified during the transport process
693
427
290
400
200
• 5: Transmembrane Electron Carriers
> Transport electron flow
• 8: Accessary Factor
> Complexes with known transport proteins
• 9: Incompletely Characterized
> Unknown classification
30
47
11
99
0
1:
2:
3: Primary
Channels/Pores Electrochemical
Active
Potential-driven Transporters
Transporters
4:Group
5:
8: Accessary
Translocators Transmembrane
Factor
Electron
Carriers
9: Incompletely Unclassified in
Characterized
TCDB
• Unclassified in TCDB
(TCDB, IUBMB system)
2
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Nodule
Root
Seed_42
Seed_28
Seed_25
Seed_21
Seed_14
Expression profiling of transporters in soybean tissues
Seed_10
Pd_shell_14d
Voltage-Gated Ion Channel (VIC)
1cm_Pd
3.0
Y_Leaf
Top 15 families:
Soy Transporter
Enrichment
Monovalent Cation:Proton Antiporter-2 (CPA2)
Flower
1.0
Mitochondrial Carrier (MC)
Pd_shell_10d
0.0
Sulfate Permease (Sulp)
Auxin Efflux Carrier (AFC)
Sweet Pq-Loop Saliva MtN3 (SWEET)
Flower Specific
Enrichment
Aquaporins
Proton-Dependent Oligopeptide Transporter (POT)
Root Specific
Amino Acid/Auxin Permease (AAA)
ABC Transporter
Major Facilitator (MFS)
Mechanosensitive: Involved in
osmotic stress response
(Hamilton et. al. 2015, Annu.
Rev. Plant Biol.)
Metabolite Transporter (DMT)
Mechanosensitive Calcium Channel
0
50
100
150
200
250
300
350
400
450
Nodule
Specific
No. of Gene
300
Enrichment
Enrichment
Aquaporin, ABC and AA transporters
Ammonium transporters, NRT family
800
700
No. of Gene
250
200
No. of Gene
PQ loop (SWEET), MIPs and metabolite transporters
P-ATPase
Oligosaccharidyl-Lipid/Polysaccharide (MATE)
150
100
685
600
 Overall, majority of transporters were expressed in flower, roots,
500
425
followed by pod shell and seed tissues.
400
300
 339 transporters expressed constitutively in all 14 tissues.
200
50
 Highly abundant transporters (top 100) were enriched for intrinsic
100
0
0
Water
Nitrogen
Metal
Ion
Sugar
Amino acid
Hormones
2
Classification based on solute transport
3
4
5
6
7
8
9
proteins (MIPs) and metabolite transporters.
10 11 12 13 14 15 16 17
Classification based on No. of TMD
RNAseq atlas (Severin et. al 2010)
Identification of genomic variants using WGRS data
• 106 diverse soybean lines sequenced at 15X coverage (Valliyodan et. al. 2016, in press)
• 58 transporter genes showed copy number variation
Examples:
Transporter genes and
trait discovery
• Out of 10 M SNPs about 159,464 (1.6%) SNPs were identified in transporter genes.
20000
100000
G. soja
G. max
15000
G. soja
G. max
80000
60000
10000
40000
5000
20000
0
1. Soybean sugar effluxer (SWEETs)
2. Soybean Na+ transporter
3. Maize and wheat boron transporter
0
Distribution of large Effect SNPs
Distribution SNPs in genic region
1. Soybean SWEET effluxers proteins
2. Soybean salinity tolerance associated with Na+ transporter gene
Tandem repeats 3TM unit
• GmCHX1 (Glyma03g32900) encodes Na+/H+ exchange protein
• GmCHX1 was identified as the causal gene for salt tolerance (Qi et al 2014 Nat Comm)
FPKM
• Metabolite transport influences photosynthetic productivity and plant vigor by controlling source/sink relationships
and biomass partitioning.
• SWEETs are uniporter and facilitate diffusion of sucrose across cell membrane
• Involved in sugar efflux, phloem loading, plant-pathogen interaction and reproductive tissue development
Analysis of 106
Soybean Lines
RNAseq data1
RNAseq data2
GmCHX1 express in roots
and has mechanism to
limits salt transport in
shoot
Soybean SWEETs are highly expressed during seed
development
Patil et al. 2015 BMC Genomics
Patil et al. 2016 Scientific Reports (Accepted)
SPAD ratio and Leaf scorch score
3
1/19/2016
GWAS confirmed GmCHX1 as major transporter gene associated with salinity tolerance
3. Boron transporter in higher plants
• BORON: involve in cross-linking the pectic in primary cell walls, gives physical
strength (Miwa 2010 Ann Botany).
• High level are toxic to plant growth.
5 M SNP (after filtering)
from 106 lines were
used for GWAS
Str variant identification
Qi et al 2014
Mut (Tolerant)
HTP maker development
WT (Sensitive)
Patil et al. 2016 Scientific Reports (Accepted)
Over 94% accuracy
Mutant in maize : shows reduced tassel
development and defects in inflorescence.
Natural variants in wheat B transporter (Bot-B5b allele) associated
with B toxicity.
Pallotta 2014 Nature
Mach 2014 Plant Cell
www.Soykb.org/soyTD
Database Design
Browse
Transporter Gene Card
Solute
TOPCONs Prediction
Transporter
card
4
1/19/2016
Transporter Gene Card….
Chromosome Distribution
Synonymous
Non_Synonymous
Search
Blast Search
Blast against transporter proteins
S u m m ar y
• Comparative analysis of 47 plant species showed presence of 7 – 12% transporter genes per
genome.
HN001 HN002 HN003 HN004 HN005 HN006 HN007 HN008 HN009 HN010 HN011 HN012 HN013 HN014 HN015 HN016
HN001
HN003
HN004
HN005
HN006
HN007
HN008
HN009
HN017
HN018 HN002
HN019 HN020
HN021
HN022 HN023
HN024
HN025
HN026 HN027
HN028
HN029 HN030 HN031 HN032
HN097 HN098 HN099 HN100 HN101 HN102 HN103 HN104 HN105 HN106 HN107 HN108
• Genome size (duplication/ploidy) positively correlated with number of transporter genes.
• Soybean contains 3306 (>2 TMD) transporter genes and majority of soybean transporter encodes
for ‘Electrochemical Potential-driven’ (Uniporter, symporters and antiporters).
Auxin Efflux
Line
Identification of
HN001
sequence variants in
HN002
1000 soybean genomes
under progress…..
HN003
HN004
HN005
HN006
HN007
HN008
PI
PI518664
(Hutcheson)
PI548402
(Peking)
PI089772
PI090763
PI404166
PI407788A
PI424298
PI437655
Amino acid Transport
Gm09_22030189_Glyma. Gm09_45198657_Glyma.09G
09G251600_+_373:0
050600_+_438:2
CAG<->CAG;Q<->Q
TAC<->TAC;Y<->Y
CAG<->CAG;Q<->Q
TAC<->TAG;Y<->U
CAG<->TAG;Q<->U
CAG<->TAG;Q<->U
CAG<->CAG;Q<->Q
CAG<->CAG;Q<->Q
CAG<->CAG;Q<->Q
CAG<->CAG;Q<->Q
TAC<->TAC;Y<->Y
TAC<->TAC;Y<->Y
TAC<->TAG;Y<->U
TAC<->TAC;Y<->Y
TAC<->TAC;Y<->Y
TAC<->TAC;Y<->Y
• Based on transcriptome profiling, majority of transporters expressed in flower and root tissues.
• Highly abundant transporters were enriched for intrinsic proteins (MIPs) and metabolite
transport.
• Total 159,464 SNPs (1.6% compared to genome wide SNPs) were identified in 106 soybean lines.
• Knowledge-based web resource that integrates information of soybean transporters has been
developed.
5
1/19/2016
Acknow ledgements
Nguyen Lab (MU)
Babu Valliyodan
Rupesh Deshmukh
Division of Plant Sciences
Bioinformatics team @ MU
Trupti Joshi
Yang Liu
Shuai Zeng
Dong Xu
ICRISAT, India
Gaurav Agrawal
Rajeev Varshney
Thank you !
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