Transcript Slide 1

Engineering drought-tolerant crops plants
Eduardo Blumwald
Dept. of Plant Sciences, University of California@Davis
Observations:
(i) Salt and Drought stress accelerate the senescence of plants.
(ii) Stress modifies Sink/source relationships.
(iii) Cytokinins delay leaf senescence.
Hypothesis:
In contrast to the common dogma that senescence is a beneficial process to
plants during stress, it is possible to actually enhance the tolerant of plants to
drought if the drought-induced senescence of leaves is delayed during the
drought episode.
Rationale:
Based on the assumption that senescence is a type of cell death program that
could be unnecessarily activated in different plants during stress and that
suppressing it would enable plants to mount a vigorous acclimation response
Strategy:
Regulated IPT expression by a maturation- and stress-inducible promoter,
could maintain optimal levels of cytokinin levels during stress, delaying stressinduced senescence. [IPT (isopentenyl transferase ) is the limiting factor for cytokinin
biosynthesis]
SARK
IPT
Wild-type and transgenic plants expressing PSARK::IPT plants after
15 days drought Treatment followed by 7 days re-watering.
K
Wild-type
PSARK::IPT
Oxidative Metabolism Conclusions
WT
SOD
H2O2
O2
-
O2
e-
hn
PSII
PSI
GSH
H2O
APX
MDHAR
AsA
MDHA
H2O
NADP+
NADP+
GR
DHAR
NADPH
DHA
GSSG
NADPH
Oxidative Metabolism Conclusions
424
SOD
H2O2
O2
-
O2
e-
hn
PSII
PSI
GSH
H2O
APX
MDHAR
AsA
MDHA
H2O
NADP+
NADP+
GR
DHAR
NADPH
DHA
GSSG
NADPH
WT
During drought
2
1
Before Drought
15 d
13 d
8d
10 d
6d
4d
2d
15 d
13 d
8d
During drought
15 d
13 d
10 d
8d
6d
4d
2d
15 d
13 d
10 d
8d
6d
4d
2d
45 d
43 d
40 d
38 d
36 d
34 d
32 d
0
During Rewat.
Middle Leaves
During drought
Water Use Efficiency (WUE)
BOTTOM LEAVES
WT
WT
pSARK-IPT
3
2
1
Before Drought
During drought
Top Leaves
During Rewat.
15 d
13 d
10 d
8d
6d
4d
2d
15 d
13 d
10 d
8d
6d
4d
2d
45 d
43 d
40 d
38 d
36 d
34 d
32 d
0
30 d
15 d
13 d
10 d
8d
6d
2d
4d
During Rewat.
mmol (CO2) mol (H2O)
pSARK-IPT
15 d
13 d
10 d
8d
6d
4d
2d
45 d
43 d
40 d
38 d
36 d
34 d
10 d
1
Before Drought
35
30
25
20
15
10
5
0
32 d
pSARK-IPT
2
During Rewat.
Photosynthesis Rate
(BOTTOM LEAVES)
30 d
WT
3
30 d
15 d
13 d
10 d
8d
6d
4d
2d
During drought
mmol (CO2 ) mol (H2O)
pSARK-IPT
15 d
13 d
10 d
8d
6d
4d
2d
45 d
43 d
40 d
38 d
36 d
34 d
32 d
30 d
-2 -1
m mol (CO2)m s
During Rewat.
Water Use Efficiency (WUE)
MIDDLE LEAVES
WT
Middle Leaves
-2 -1
m mol (CO2)m s
6d
During drought
Bottom Leaves
40
35
30
25
20
15
10
5
0
Bottom Leaves
4d
0
During Rewat.
Photosynthesis Rate
(MIDDLE LEAVES)
Before Drought
pSARK-IPT
3
Top Leaves
Before Drought
WT
4
30 d
a.g.
32 d
a g.
34 d
a.g.
36 d
a.g.
38 d
a.g.
40 d
a.g.
43 d
a.g.
45 d
a.g.
2d
mmol (CO2) mol (H2O)
15 d
13 d
8d
10 d
6d
4d
2d
15 d
13 d
8d
10 d
6d
4d
2d
45 d
43 d
40 d
38 d
36 d
34 d
32 d
Before Drought
Water Use Efficiency (WUE)
BOTTOM LEAVES
pSARK-IPT
35
30
25
20
15
10
5
0
30 d
-2 -1
m mol (CO2)m s
Photosynthesis Rate
(TOP LEAVES)
RESTRICTED AMOUNT OF WATER EXPERIMENTS
PHOTOSYNTHESIS
120
umol CO2 m -2 s-1
40
30
20
10
APICAL LEAVES
100
80
60
40
20
0
0
7
10
15
20
25
30
35
40
45
50
55
60
65
70
75
7
Days ofLEAVES
growth
MIDDLE
25
100
10
40
20
0
0
10 15 20 25
30 35 40 45 50
55 60 65 70 75
Days of growth
MIDDLE LEAVES
7
60
5
APICAL LEAVES
8
mmol CO2 m -2 s-1
15
10
9
8
7
6
5
4
3
2
1
0
7
10 15 20 25 30 35 40 45 50 55 60 65 70 75
MIDDLE LEAVES
Days of growth
80
20
umol CO2 m -2 s-1
umol CO2 m-2 s-1
WATER USE EFFICIENCY
mmol CO2 m -2 s-1
APICAL LEAVES
50
umol CO2 m-2 s-1
STOMATAL CONDUCTANCE
6
5
4
3
2
1
15
20
25
30
35
40
45
50
55
60
65
70
umol CO2 m -2 s-1
25
20
15
10
0
8
7 10 15 20 25 30 35 7 40 45 50 55 60 65 70 75
Days6of growth
MIDDLE LEAVES
5
4
10
15
20
25
30
35
40
45
50
55
60
65
70
75
7
10 15 20 25 30 35 40 45 50 55 60 65 70 75
Days of growth
100
BASAL LEAVES
5
0
7
75
Days of growth
O2 m -2 s-1
umol CO2 m-2 s-1
30
10
BASAL LEAVES
Days of growth
BASAL
LEAVES
8
7
80
mmol CO2 m -2 s-1
7
60
40
20
MIDDLE LEAVES
0
7 10 15 20 25 30 35 40 45 50 55 60 65 70 75
1000 ml WT
1000 ml IPT
Days of growth
6
5
4
3
2
1000
ml WT
1
0
10007 ml
10 IPT
15 20
300 ml WT
300 ml IPT
25
30 35
40
45 50
Days of growth
55 60
65
70 75
A/Ci CURVES:
1000 ml - 30 days a.g.
0
10
20
30
40
50
60
300 ml - 40 days a.g.
40
30
20
10
0
0
20
40
60
300 ml - 60 days a.g.
40
30
20
10
0
0
20
40
Internal leaf [CO2] (Pa)
60
70
CO2 assimilation rate [μmol CO2.m-2s-1
CO2 assimilation rate [μmol CO2.m-2s-1
pSARK-IPT
40
30
20
10
0
300 ml - 30 days a.g.
WT
WT
pSARK-IPT
40
30
20
10
0
0
10
20
30
40
50
60
70
50
60
70
50
60
70
300 ml - 50 days a.g.
40
30
20
10
0
0
10
20
30
40
300 ml - 70 days a.g.
40
30
20
10
0
0
10
20
30
40
Internal leaf [CO2] (Pa)
“Photosyn Assistant” Program to determine factors limiting CO2 assimilation
(Farquhar et al., (1980), Sharkey (1985), Harley and Sharkey (1991) and Harley et al. (1992)
Vcmax = maximum rate
of carboxylation by Rubisco
Jmax = PAR-saturated rate of
electron transport (based in NADPH
requirements for RuBP regeneration)
TPU = the rate of Triose Phosphate
Utilization, indicating the availability
of inorganic Pi for the Calvin Cycle
WT
umol CO2 m
-2
s
-1
Vcmax (maximum carboxylation rate of Rubisco)
150
pSARK-IPT
100
50
0
30 days a.g. 30 days a.g. 40 days a.g 50 days a.g. 60 days a.g. 70 days a.g.
1000 ml
300 ml
TPU (Triose Phosphate Use)
(availability of inorganic P for Calvin Cycle)
Jmax (RuBP regeneration)
500
15
WT
pSARK-IPT
-2
s
pSARK-IPT
-1
400
300
umol m
umol m
-2
s
-1
WT
200
100
10
5
0
0
30 days a.g. 30 days a.g.
1000 ml
40 days a.g 50 days a.g. 60 days a.g. 70 days a.g.
300 ml
30 days a.g. 30 days a.g.
1000 ml
40 days a.g 50 days a.g. 60 days a.g. 70 days a.g.
300 ml
Vcmax (maximum carboxylation rate of Rubisco)
500
15
WT
WT
400
100
50
WT
pSARK-IPT
pSARK-IPT
umol m -2 s-1
pSARK-IPT
umol m -2 s-1
umol CO2 m -2 s-1
TPU (Triose Phosphate Use)
(availability of inorganic P for Calvin Cycle)
Jmax (RuBP regeneration)
150
300
200
10
5
100
0
0
0
30 days a.g. 30 days a.g. 40 days a.g 50 days a.g. 60 days a.g. 70 days a.g.
1000 ml
30 days a.g. 30 days a.g. 40 days a.g 50 days a.g. 60 days a.g. 70 days a.g.
300 ml
1000 ml
300 ml
30 days a.g. 30 days a.g.
40 days a.g
1000 ml
50 days a.g. 60 days a.g. 70 days a.g.
300 ml
CALVIN
CYCLE
-1.35
1.75
1.96
0.91
1.96
0.91
2.16
pSARK-IPT 300 ml vs WT 300 ml
[Log2 (Ratio)]
PHOTORESPIRATION
C
M
P
M
M
C
Amino acids/flavonoids
GA/IAA/etc.
sugars
GSH
FIELD TRIALS – Brawley (Imperial Valley, CA)
OWA
½ OWA
1/3
OWA
1/4
OWA
Effect of restricted watering (% OWA) on yield of WT plants
FW [g/plant]
330
a
ab
280
b
230
c
180
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
Effect of restricted watering (% OWA) on yield of WT and pSARK-IPT plants
DW [g/plant]
75
65
55
45
WT
35
T2-36
T4-24
25
1
0,8
0,6
0,4
Treatment [% of OWA]
0,2
Transgenic lines
Collaborations:
Cassava – Willi Gruissem (ETH Zurich)
Cotton – Hong Zhang (Texas Tech Univ)
Flowers – Ryohei Nakano (Okayama University, JAPAN)
Alfalfa – INTA (Castelar, Argentina)
Sugar cane – Judy Zhu (Hawaii Ag. Center)
Dr. Rosa M. Rivero
Ellen Tumimbang
Rosa Jauregui
Dr. Lianhai Fu
UC Davis Genome Center
Prof. Shimon Gepstein (Technion,Israel)
Prof. Ron Mittler (Univ. of Nevada@Reno)
Prof. H. Sakakibara (RIKEN, Tsurumi, Yokohama, Japan)
Dr. Ann Blechl (USDA, Albany, CA)
Will W. Lester Endowment