Cytokinin-induced modifications of source

Transcription

Modifications of source- sink relationships lead to
enhanced crop stress tolerance
Eduardo Blumwald
Dept. of Plant Sciences, University of California@Davis
Observations:
Salt and Drought stress accelerate the senescence of plants,
modifying sink/source relationships.
Hypothesis:
It is possible to enhance the tolerance of plants to abiotic
stress by delaying stress-induced senescence, increasing
source fitness and improving source/sink relationships
Strategy:
Regulated IPT expression by a maturation- and stressinducible promoter, could maintain optimal levels of
cytokinin levels during stress, delaying stress-induced
senescence and improving sink loading. [IPT (isopentenyl
transferase ) is the limiting factor for cytokinin
biosynthesis]
Gene
Promoter
Target
Phenotype
Reference
IPT
HSP70 (maize)
Tobacco
Arabi
Release of auxiliary buds/Reduced stem and leaf
area/Underdeveloped root area
Medford et al (1989) Plant Cell 1:403-413
IPT
HSP70 (Drosophila)
Tobacco
Van Loven et al. (1993) J. Exp. Bot. 44:1671-
IPT
Native (Agrobacterium)
Potato
Decreased Photosynthesis/Increased Photorespiration
Increased CO2-compensation point
Catsky et al. (1993) Biol. Plant. 35:191-198
IPT
promoterless
Tobacco
Hewelt et al. (1994) Plant J. 6:879-891
IPT
Cu-induced
Tobacco
Release of auxiliary buds/Delayed senescence
McKensie et al. (1998) Plant Physiol. 116:969
ZOG1
35S
Tobacco
Higher Photosynthetic rates/Higher WUE
Stomatal conductance inconsistent
Haizel et al. (2004) Biol. Plant. 52:49-58
IPT
SAG13
Tomato
Loss apical dominance/stem thickening
Advanced flowering/delayed senescence
Swartzberg et al. (2006) Plant Biol. 8:579-586
IPT
SAG12
Tobacco
Delayed senescence/No abnormalities
Gan & Amasino (1995) Science 270:1986-88
IPT
SAG12
Tobacco
Delayed senescence/Reduced N-translocation (source to
sink) / N-inversion
Jordi et al. (2000) Plant Cell Environ. 23 :279
IPT
SAG12
Lettuce
Increased senescence of upper leaves and delay flowering
McCabe et al. (2004) Plant Physiol. 127:505-
IPT
SEE1 (maize)
Maize
Delayed leaf senescence, Chlorosis of young leaves at low N
Robson et al. (2004) Plant Biotechnol. J. 2 :10
ZOG1
SAG12
Tobacco
Higher Photosynthetic rates/Higher WUE
Stomatal conductance inconsistent
Haizel et al. (2004) Biol. Plant. 52:49-58
IPT
SAG12
Tobacco
Altered source-sink/Slow adjustment of biomass allocation
(shoot/root) during drought
Cowan et al. (2005) Plant 221:801-814
IPT
SAG12
Tomato
Delayed leaf senescence/Advanced flowering
Slight increase in fruit TSS
Swartzberg et al. (2006) Plant Biol. 8:579-586
IPT
SAG12
Wheat
Delayed senescence, no yield difference
Sykorova et al. (2008) J. Exp. Bot. 59:377-38
Problems associated with
PSAG12::IPT expression
(sink)
Flowering inhibition,
low seed set and filling
(source)
sink
Reduced N-flux to
sinks
Source/sink “inversion”
source
Lack of chlorophyll
degradation, inhibition of
protein turnover
PSARK promoter
+16 BIHD1
+21 CAATBOX1
+41 ROOTMOTIFAPOX1
+52 CACT
+59 MYCCONSENSUS
-61 CACT
-63 BIHD1
-73 CACT
-81,-89 CAATBOX1
+97 DOFCORE
+106 CAATBOX1
-814 ROOTMOTIFAPOX1
+805 CAATBOX1
-792 CACT
+785 CACT
-747 DOFCORE
ROOTMOTIFAPOX1
+719 ANAERO3CONSENSUS
-715 DOFCORE
+715 CACT
+706 BIHD1
+683 CACT
+662, -674 CAATBOX1
+657 CACT
-645 CCAATBOX1
+628 CACT
-610 CCAATBOX1
-596 CACT
+594 DOFCORE
+739
+123 CACT
+123 MYCCONSENSUS
-189 DOFCORE
-200 CACT
+247 CACT
-249 DOFCORE
-256 CACT
-265 DOFCORE
+273 ROOTMOTIFAPOX1
+279 CACT
-281 DOFCORE
+320 ROOTMOTIFAPOX1
-358 CCAATBOX1
1
100
200
300
400
+364, +375 ROOTMOTIFAPOX1
+391 CACT
-393 DOFCORE
-394 GT1GMSCAM4
-448 CACT
+454 ROOTMOTIFAPOX1
-466 DOFCORE
+481 MYCCONSENSUS
500
600
700
-579 ANAERO1CONSENSUS
+556,+564,+568 DOFCORE
-522 CACT
+511 ATHB2
+508 CAATBOX1
-502 GT1GMSCAM4
-499 DOFCORE
+497 CACT
-495 CACT
822bp
Supplemental 2. PSARK putative cis-elements potentially involved in environmental cue responses.
Delatorre et al., (2012) Plant Sci.
Senescence-Associated Receptor Kinase (SARK)
10
attached leaf
20
25
30
40
50
60 days
mRNA
3.1 kb
0
detached leaf
15
1
2
3
4
5
6
days
mRNA
3.1 kb
Hajouj, Michelis & Gepstein; Plant Physiol. (2000)
OUR STRATEGY
IPT
SARK PROMOTER
IPT
pSARK
Agrobacterium
tumefaciens
Wild-type and transgenic plants expressing PSARK::IPT plants after
15 days Drought followed by 7 days Re-watering.
Wild Type
Wild-type
PSARK::IPT
PSARK::IPT
Rivero et al., (2007), PNAS
TOBACCO FIELD TRIAL , – Brawley (Imperial Valley, CA, 2009)
OWA
½ OWA
1/3
OWA
1/4
OWA
Effect of restricted watering (% OWA) on yield of WT and pSARK-IPT plants
FW [g/plant]
330
280
230
180
WT
130
1
0.9
0.8
0.7
0.6
0.5
0.4
Treatment [% of OWA]
0.3
0.2
Effect of restricted watering (% OWA) on yield of WT and pSARK-IPT plants
FW [g/plant]
330
12%
280
13%
230
180
WT
47%
T2-36
T4-24
130
1
0.9
0.8
0.7
0.6
0.5
0.4
Treatment [% of OWA]
0.3
0.2
Rice and the timing of drought
Nursery
Vegetative stage
Reproductive stage
Experimental design drought stress:
Reproductive stage
Vegetative stage
Germination
Tillering
Panicle initiation
Heading
Maturation
Harvest
Well-watered, control
Re-watered
Post-anthesis stress
Stop water
Re-watered
Stop water
Pre-anthesis stress
Pre-anthesis : Reduction in spikelet fertility and panicle expansion
Post-anthesis: Reduction in grain size and weight and low grain quality
IPT expression in rice
Peleg et al., (2011) Plant Biotechnol. J.
Vegetative
160
Reproductive
140
a
a
2-(∆∆CT)
120
b
100
b
80
60
40
20
c
d
0
c
c
e
g
g
g
g
g
g
n
tag llerin llerin lerin ootin erin fillin ratio
s
g
B flow ain atu
Ti te til
lin rly ti
d
a
st Gr s m
e
L
Se Ea
po
s
ain
r
ek
e
G
2w
120
2-(∆∆CT)
100
80
60
aa
5T-WW
8T-WW
5T-WS
8T-WS
1200
1000
PSARK::IPT
aa
aa
40
20
1400
abab
bc
aa
bb
2-(∆∆CT)
140
•
bb
bb
800
2DS
3DS
4DS
aa
a
b
aa
aa
600
200
aaaa
0
1DS
5T-WW
8T-WW
5T-WS
8T-WS
400
0
BS
3 days of drought stress
ReW
bb a a bb a a b b
bb
cc
bb
f
eaf
eaf
eaf
icle uncle g lea
l
l
l
n
d
h
d
d
ir
Pa
on
Fla
urt
Pe
Th
Fo
Sec
bb
ot
Ro
aa
s
Well-watered
Water-stress
Re-watering
A
IPT
WT
IPT
WT
IPT
C
F
D
G
E
H
WT
B
YIELD
Peleg et al., (2011) Plant Biotechnol. J. 9:747-758
Auxins
Ethylene Gibberellins
SA
JA
Brassinosteroids
ABA
CK
Ck
induce changes in hormone
homeostasis
Brassinosteroids-related
genes
Sucrose, mg/g dry weight
Sucrose and starch content (flag leaves at pre-anthesis and grains after maturation)
Flag leaves
80
60
WT
5T
WT
5T
a
a
a
20
a
b
15
b
a
a
40
10
20
5
0
1.2
Starch, mg/g dry weight
Grains
25
WT
5T
a
1.0
a
b
a
WT
5T
300
a
250
.8
.6
0
350
30%
b
b
200
150
.4
b
100
.2
50
0.0
0
Well-watered Water stress
Well-watered
Water stress
Starch degradation pathway
6
5T-WW
8T-WW
WT-WS
5T-WS
8T-WS
4
2
0
-2
-4
-6
MAT
AGPase
a-AMI
DEP2
ISA3
PHOH1
Osk1
CWINV
Fructose-6-P
Glu-1-P
UDP-Glucose
13
SP
SPS
2
GPT
5
SuT
12
SS
15
SPP
SBE
PPT
STP
4
ADP-Glucose
14
Sucrose-6-P
3
We can check it
SUSY
Amylose
11
Sucrose
1
AGPsase
Trehalose
Starch
CytINV
Trehalose-6-P
UDP-Glucose
2.4
2.0
1.5
1.1
0.7
0.3
-0.2
-0.6
-1.1
-1.5
-1.9
Fructose
TPS
6
NPP
Glucose
Starch degradation
Glucose
FK
SP
α-AMY
9
β-AMY
8
HK
Glucose-1-P
α-AMY
Glucose
Fructose-6-P
Raffinose
10
Glucose 6-P
Chloroplast
1. SUSY, Sucrose synthase; 2. SPS, sucrose phosphate synthase; 3. SuT1, Sucrose transporter; 4. STP, Sugar transporter; 5. SP, sucrose
posphatase; 6. Trehalose-6-P synthase; 7. Trehalose synthase; 8. Glucose-6-phosphate isomerase; 9. β-amylase; 10. α-Amylase, 11 . 1,4-aglucan branching enzyme; 12. SS, starch synthase 3; 13. Glucose-1-phosphate adenylyl transferase (ADP-glucose pyrophosphorylase); 14 TPT,
Phosphate/phosphoenol pyruvate translocator; SBE: starch branching enzyme; NPP: nucleotide pyrophosphatase; HK: hexokinase;
INV: invertase;
CWINV
V-INV
Fructose-6-P
Glu-1-P
UDP-Glucose
13
SP
SPS
2
GPT
5
SuT
12
SS
15
SPP
SBE
PPT
STP
4
ADP-Glucose
14
Sucrose-6-P
3
We can check it
SUSY
Amylose
11
Sucrose
1
AGPsase
Trehalose
Starch
CytINV
Trehalose-6-P
UDP-Glucose
2.4
2.0
1.5
1.1
0.7
0.3
-0.2
-0.6
-1.1
-1.5
-1.9
Fructose
TPS
6
NPP
Glucose
Starch degradation
Glucose
FK
SP
α-AMY
9
β-AMY
8
HK
Glucose-1-P
α-AMY
Glucose
Fructose-6-P
Raffinose
10
Glucose 6-P
Chloroplast
INV: invertase;
CWINV
V-INV
Fructose-6-P
Glu-1-P
UDP-Glucose
13
SP
SPS
2
GPT
5
SuT
12
SS
15
SPP
SBE
PPT
STP
4
ADP-Glucose
14
Sucrose-6-P
3
We can check it
SUSY
Amylose
11
Sucrose
1
AGPsase
Trehalose
Starch
CytINV
Trehalose-6-P
UDP-Glucose
2.4
2.0
1.5
1.1
0.7
0.3
-0.2
-0.6
-1.1
-1.5
-1.9
Fructose
TPS
6
NPP
Glucose
Starch degradation
Glucose
FK
SP
α-AMY
9
β-AMY
8
HK
Glucose-1-P
α-AMY
Glucose
Fructose-6-P
Raffinose
10
Glucose 6-P
Chloroplast
INV: invertase;
CWINV
V-INV
Fructose-6-P
Glu-1-P
UDP-Glucose
13
SP
SPS
GPT
5
SuT
ADP-Glucose
12
14
Sucrose-6-P
3
AGPsase
SS
15
SBE
STP
4
PPT
SUSY
11
Sucrose
1
We can check it
Amylose
Trehalose
Starch
CytINVTrehalose-6-P
Starch degradation
UDP-Glucose
2.4
2.0
1.5
1.1
0.7
0.3
-0.2
-0.6
-1.1
-1.5
-1.9
NPP
Glucose
Fructose
FK
GlucoseTPS
6
10
SP
α-AMY
β-AMY
8
HK
Glucose-1-P
α-AMY
Glucose
Raffinose
Chloroplast
INV: invertase;
1 Aminotransferase; 2. Fd-GOGAT; 3- Tyrosine aminotransferase 4. aminotransferase; 5.
phosphate/phosphoenolpyruvate translocator;; 7. Alanine aminotransferase; 8. glyoxalase family protein; 9. Glycerol3-phosphate dehydrogenase; 10. phenylalanine ammonia-lyase; 11. Amino acid kinase; 12. NRT (nitrate transporter); 13.
GDH; 14. glutamine-dependent NAD;15. Isocitrate dehydrogenase 16- Isocitrate dehydrogenase. 17. Citrate
transporter; 18. methylisocitrate lyase 2; 19. Enolase; 20. Aconitase; 21 valyl-tRNA synthetase
1 Aminotransferase; 2. Fd-GOGAT; 3- Tyrosine aminotransferase 4. aminotransferase; 5.
phosphate/phosphoenolpyruvate translocator;; 7. Alanine aminotransferase; 8. glyoxalase family protein; 9. Glycerol3-phosphate dehydrogenase; 10. phenylalanine ammonia-lyase; 11. Amino acid kinase; 12. NRT (nitrate transporter); 13.
GDH; 14. glutamine-dependent NAD;15. Isocitrate dehydrogenase 16- Isocitrate dehydrogenase. 17. Citrate
transporter; 18. methylisocitrate lyase 2; 19. Enolase; 20. Aconitase; 21 valyl-tRNA synthetase
—●— Wild-type
—●— PSARK::IPT
Water-stress
Water-stress
Well-watered
**
**
*
**
**
**
*
Protein Carbonylation
WW
Protease activity
135
95
72
52
42
34
26
17
10
135
95
72
52
42
34
26
17
10
WS
PROTEIN TURNOVER
PROTEIN DEGRADATION
PROTEIN SYNTHESIS
Assimilated partitioning between source and sink
PSARK::IPT/WT
Well-watered conditions
Flag leaf
CWInv
SPS
SuSysynthesis
SuSydegradation
Cyt Inv
SUC
GS
NR
NADH-GOGAT
STP
Third leaf
SPS
CWInv SuSy
synthesis
SuSydegradation
Cyt Inv
GS
NR
NADH-GOGAT
Amyl.Activity
NiR
Sucrose
Gluc
Chloroplast
GDHamination GDHdeamination
Fruc
Glu-6-P
NO3-
VInv
Vacuole
NH3
Cytosol
Amyl.Activity
Sucrose
NiR
Gluc
Chloroplast
GDHamination GDHdeamination
VInv
Vacuole
Fruc
Glu-6-P
NO3NH3
Cytosol
Assimilated partitioning between source and sink
Water-stress conditions
PSARK::IPT/WT
Enhanced Suc synthesis
Enhanced primary N-assimilation
Less N-reassimilation
(Glu ↔ 2-Oxoglutarate + NH3)
Reduced Suc degradation
Third leaf
Enhanced Suc synthesis
Enhanced primary N-assimilation
Reduced Suc degradation
Salinity stress at reproductive stage:
Salt treatments started at booting stage and gradually
increased up to 120mM, picture after 28 days of salinity
Control
Wild type
Salinity stress
wild type
PSARK: : IPT
Grain yield
Grain yield (g/plant)
30
25
WT
5T
8T
20T
20
a
a a
15
10
b
5
0
control
Salt stress
Ion analysis – Potassium (K):
increased up to 120mM, samples after 3 weeks of salt
30000
Potassium (ppm)
25000
b
WT-WW
IPT-WW
WT-SS
IPT-SS
ab
20000
a
a
15000
b
10000
5000
a a
a
0
roots
Flag leaf
Ion analysis – Sodium (Na):
Sodium (ppm)
80x103
WT-control
IPT-control
WT-salt stress
IPT-salt stress
ab
b
b
a a
a a
a
0
Roots
Flag leaf
Transcriptome analysis of
salinity stress
WT PSARK::IPT
WW SS WW SS
-2.598
-2.079
-1.559
-1.039
-0.52
0
0.5196
1.0393
1.5589
2.0786
2.5982
Drought and salinity common genes
96 Genes
drought & salinity
Combination of salinity and drought stress
Salt treatments started at 4 leaf stage. Drought was
applied at pre-anthesis stage.
Grain yield salt and drought combination
35
WT
20T
5T
Grain yield, g/plant
30
25
75% 73% 74%
a
20
56%
a 44% 51%
ab
b
15
62% 64%
56% a a
a
a
b
28%
23% c
10
a
29%
b
5
0
Control
Salt
Drought
S+D
8T
Protein content
mg protein / g FW
60
50
40
30
WT
5T
8T
a
a
a
b
a
a
a
b
b
20
10
0
Control Drought Salinity
Treatment
S+D
Wheat-Salinity stress at reproductive stage:
increased up to 100mM
Wild type
Control
Salt
48T
Control
42T
Salt
Control
84T
Salt
Control
Salt
Wheat grain yield salinity (100mM) at reproductive stage
Average yield of control treatment 18-22 g/plant
Grain yield, g/plant
20
a
18
a
16
14
12
10
8
6
b
b
b
4
2
0
WT
48N
48T
84N
Genotype
84T
A
A
WT
3
B
B
WT
3
Fig. 8. Wild-type and transgenic peanut plants in the field and phenotype
of pods from these peanut plants. WT, wild-type; 3, transgenic peanut
plant line No. 3.
Fig. 9. The percentage of 3- and 4-seeded pods
from wild-type and transgenic peanut plants
grown in the field in 2010. WT, wild-type; NT,
non-transgenic line coming from tissue culture;
3, 7, 11, 13, four independent transgenic peanut
plants.
Quin et al., Plant Cell Physiol. (2011)
A. 2009 Field Trial (dryland)
B. 2010 Field Trial (dryland)
70%
57%
57%
53%
38% 37%
C. 2010 Field Trial (irrigated)
Fig. 10. Peanut yields from wild-type, nontransgenic, and transgenic peanut plants in the
field conditions. A. Yields from dryland peanuts in
2009. WT, wild-type; 3, 7, and 11, three
independent transgenic peanut plants. *,
significant at 5%; **, significant at 1%. n = 40 for
each line. B. Yields from dryland peanut in 2010.
NT, non-transgenic line coming from tissue culture;
13, another independent transgenic peanut line. C.
Yields from irrigated peanuts in 2010.
Quin et al., Plant Cell Physiol. (2011)
Field trials - Cotton
Stomatal cond. (mol H2O/m2/s)
18
16
14
12
10
8
6
4
2
0
WT
SNT IPT2 IPT5 IPT6 IPT9
Transpiration rate (mol H2O/m2/s)
Photosynthesis (µmol CO2/m2/s)
PSARK::IPT Cotton; Photosynthesis; Low-irrigation Field
0.25
0.20
0.15
0.10
0.05
0.00
WT
5
4
3
2
1
0
WT
80
20
18
16
14
12
10
8
6
4
2
0
Fiber yield (g/plant)
Boll number (per plant)
PSARK::IPT Cotton; boll No and fiber yield; Low-irrigation Field
WT
60
40
20
0
WT
Stomatal cond. (mol H2O/m2/s)
18
16
14
12
10
8
6
4
2
0
WT
Transpiration rate (mol H2O/m2/s)
Photosynthesis (µmol CO2/m2/s)
PSARK::IPT Cotton; Photosynthesis; Dry-land Field
0.25
0.20
0.15
0.10
0.05
0.00
WT
3.0
2.5
2.0
1.5
1.0
0.5
0.0
WT
PSARK::IPT Cotton; boll No and fiber yield; Dry-land Field
70
16
60
14
Fiber yield (g/plant)
Boll number (per plant)
18
12
10
8
6
4
50
40
30
20
2
10
0
0
WT
WT
Water stress, 25 days
WELL WATERED
WT
Walnut
pSARK::IPT
WT
pSARK::IPT
Leptadenia pyrotechnica
Greenhouse trials: Corn, Walnut, Leptadenia, Conocarpus erectus,
turf grass, Brachypodium,
Field trials: Canola, wheat, alfalfa, rice, peanut, cotton
Strategy for traits pyramid
35S
AtNHX1
UBI
HSR
35S
Hyg
LB
pSARK
IPT
RB
<trees>
pCAMBIA
pSARK
IPT
UBI
AtNHX1
UBI
HSR
NOS
Hyg
LB
RB
<Brachypodium; wheat; rice>
pH7m24GW
NOS
Km
35S
HSR
35S
AtNHX1
pZK2B
pSARK
IPT
RB
LB
<alfalfa>
Dr. Zvi Peleg (Hebrew University of Jerusalem, Israel)
Dr. Maria Reguera
Dr. Rosa M. Rivero (CEBAS-CSIC-Murcia, Spain)
Dr. Harkamal Walia (Univ Nebraska, Lincoln)
Dr. Songhu Wang
Dr. Ellen Tumimbang
Dr. Yasser Abdeltawab
Zain Baharuddin
Alison Hutmacher
UCD Genome Center
Prof. Ron Mittler (Univ. of Nevada@Reno)
Prof. Vladimir Shulaev (VBI, Virgina Tech Univ.)
Dr. H. Sakakibara (RIKEN, Yokohama, Japan)
Prof. Hong Zhang, Texas Tech University)
Prof. Juan Manuel Ruiz (Univ. of Granada, Spain)
Dr. Paxton Payton, (USDA-ARS, Lubbock, USA)
Dr. Chuck Leslie (UC Davis)
Dr. Ann Blechl (USDA, Albany, CA)
Ings. D. Lewi; E. Pagano, A. Diaz-Paleo, INTA, Castelar, Argentina.
Dr. Maris Apse (Arcadia Biosciences, USA)
Prof. Abdelhouabab Zaid (UAE University)
Dr. Abdel Ismail (IRRI, Philippines)
Will W. Lester Endowment
Changes in JA and SA affect the response of the transgenic
plants to pathogens?
•
WT and PSARK::IPT transgenic plant (6 weeks old) were inoculated with
Gram-negative bacterium Xanthomonas oryzae pv. oryzae . Infection
levels were tested after 20 days (as lesion length).

Cytokinin-induced modifications of source

Transcription

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