Developing crops with high productivity at high
temperature:
A blue sky research initiativeBSR Team
29 Nov 2013
Maximum temperature in the SAT
CriticalTemperature
threshold
Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec0
5
10
15
20
25
30
35
40
45
1983-HQ 1992-HQ
2001-HQ 2012-HQ
1983-ISC 1990-ISC
1998-ISC
Max
imum
T°C
Headquarter
Sahelian Center
T°C rarely crosses critical limits
Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec0
1
2
3
4
5
6
7
8
Max
imum
VPD
Sahelian Center
Headquarter
Vapor pressure deficit (VPD) in the SAT(VPD reflects T°C and Rel. Humidity %)
Prevalent high VPD Effect on plant water balance
VPDthreshold
The basics – Why aquaporins (AQP) ?
Transpiration response to VPD
Transpiration response to AQP inhibition
AQP gene expression
AQP in the bigger picture - Drought
Modified from Murata et al., 2000.
What are aquaporins??
Membrane transporter for waterPassive transport
Terminal drought sensitive
Terminal drought tolerant
0.005
0.01
0.015
0.02
0.025
0.03
0.035
0.04
0.045
0.50 1.00 1.50 2.00 2.50 3.00 3.50
VPD (kPa)
H77/2 833-2PRLT-2/89-33
Tran
spir
atio
n (g
cm
-2 h
-1)
Kholova et al 2010 – J. Exp. Bot
Transpiration restriction at high VPD
How the story began – Water savings in millet
Watersaving
Why such a rapid transpiration response??
Rapid response hydraulic signal
VPD (kPa)
Where is the source of hydraulic limitation??
???We hypothesized roots could be the
source of hydraulic limitation
Is the hydraulic restriction in the roots only ??
09:20 09:40 10:00 10:20 10:40 11:00 11:20 11:40 12:00 12:20 12:40 13:00 13:200.600000000000001
0.700000000000001
0.800000000000001
0.900000000000001
1
1.1
1.2
1.3
1.4
1.5
1.6
Nor
mal
ized
tran
spir
atio
n
VPD change
Low VPD High VPD
No difference in slope No hydraulic limitation in the leaves
Apoplastic Pathway
(Structural)
Symplastic Pathway
(AQP)
Water pathways in the root cylinder
Two pathways have different hydraulic conductance
Hypothesis: Aquaporin control plant water loss ?
????
The basics – Why aquaporins ?
Transpiration response to VPD
Transpiration response to AQP inhibition
AQP gene expression
AQP in the bigger picture - Drought
Terminal drought sensitive
Terminal drought tolerant
0.005
0.01
0.015
0.02
0.025
0.03
0.035
0.04
0.045
0.50 1.00 1.50 2.00 2.50 3.00 3.50
VPD (kPa)
H77/2 833-2PRLT-2/89-33
Tran
spir
atio
n (g
cm
-2 h
-1)
Kholova et al 2010 – J. Exp. Bot
One QTL mapped for low Tr at high VPD
Transpiration response to VPD in pearl millet:Growth chamber
M322M394
M214M321M592 M443M356M73840 cM
1 2 3 4 5 6 7 80
0.005
0.01
0.015
0.02
0.025
0.03
VPD insensitiveVPD sensitive
VPD (kPa)Tr
ansp
irat
ion
(g
cm-2
h-1
)
Xpsmp2237 Xpsmp2072 M13_Xpsmp2066 M13_Xpsmp3056 Xpsmp2206 Xpsmp20590
2
4
6
8
10
12
14
16
low Transpiration Rate in high VPD A - from H77
B - from PRLT
H - heterozygous
markers within LG2 DT-QTL
No
of R
ecom
bina
nts
Two QTL fine-mapped for low Tr at high VPD
Staygreen ILs (Stg3 – Stg B) are VPD-sensitive
9 11 13 15 170.0000
0.0020
0.0040
0.0060
0.0080
0.0100
0.0120
stg1stg3stg4stgB
Time of the day (h)
Tran
spira
tion
(g
cm-2
h-1
)Recurrent R16
Stg3StgB
Transpiration response to VPD in Sorghum1 - Introgression lines
0.62 1.05 1.58 2.01 2.43 3.05 3.450.000
0.002
0.004
0.006
0.008
0.010
0.012
0.014
0.016
0.018
VPD (kPa)
Tran
spir
atio
n (g
h-1
cm
-2)
Transpiration response to VPD in Sorghum2 - Germplasm
Germplasm differences in VPD-sensitivity
VPD-Sensitive
VPD-Insensitive
BP= 2.58 R2 =0.68BP= 2.51 R2 =0.76
R2 =0.89 R2 =0.83
Transpiration response to VPD in Chickpea
Transpiration response to VPD in Peanut
Mouride
If VPD < 2.09, TR = 0.0083 (VPD) – 0.002 If VPD ≥ 2.09, TR = 0.0013 (VPD) + 0.015 R² = 0.97
B UC-CB46
TR = 0.0119 (VPD) - 0.0016 R² = 0.97
D
Transpiration response to VPD in cowpea
Tolerant lines are VPD-sensitive(water saving)
Tolerant Sensitive
Belko et al – 2012 (Plant Biology)
VPD
Tran
spira
tion
(g c
m-2 h
-1)
0.0 2.0 4.0
0.0
1.0
Main types of Tr response to VPD
Water Saving
Large pattern variation within/across speciesVPD-sensitivity often link to drought adaptation
Large variation in all species
Often discriminate tolerant from sensitive lines
Now, systematic screening
Exciting results in other crops (rice, maize)
In summary…
The basics – Why aquaporins ?
Transpiration response to VPD
Transpiration response to AQP inhibition
AQP gene expression
AQP in the bigger picture - Drought
Apoplastic pathway
Symplastic Pathway
(AQP)
Sorting out the proportion of apoplastic and symplastic water transport
1 mM K4[Fe(CN)6] for 3 h Then 1 mM CuSO4.
Apoplastic pathway inhibition
Apoplastic pathway
Symplastic Pathway
(AQP)
Symplastic pathway inhibition
AQP inhibitors: AgNO3 – HgCl2 – H2O2
Follow-up of transpiration before/after inhibition
0.600000000000001
0.700000000000001
0.800000000000001
0.900000000000001
1
1.1
1.2
Time of the day
Nor
mal
ized
Tra
nspi
rati
onVPD-sensitive
VPD - insensitive
Less symplastic inhibition in VPD-sensitive
Pearl millet: Symplastic inhibition
Treatment
M322M394
M214M321M592 M443M356M73840 cM
10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 2700.600000000000001
0.700000000000001
0.800000000000001
0.900000000000001
1
1.1
1.2
ICMR1029 1mM
ICMR2042 1mM
H77 1mM
PRLT 1mM
Time (minutes)
Nor
mal
ized
tran
spir
atio
n
Symplastic inhibition in near-isogenic lines(QTL from VPD-sensitive parent)
NILs behave like QTL donor parent
M322M394
M214M321M592 M443M356M73840 cM
TreatmentVPD-sensitive
More apoplastic inhibition in VPD-sensitive
10:10 10:40 11:10 11:40 12:10 12:40 01:10 01:40 02:10 02:40 03:100.00
0.20
0.40
0.60
0.80
1.00
1.20
Time
Nor
mal
ized
Tra
nspi
rati
on
Apoplastic inhibition
Pearl millet: Apoplastic Inhibition
VPD-sensitive
VPD - insensitive
M322M394
M214M321M592 M443M356M73840 cM
0
0.2
0.4
0.6
0.8
1
1.2Apoplast & symplast inhibition at low
VPD
Time
Nor
mal
ized
tra
nspi
rati
on
Apoplastic & Symplastic inhibi-
tion
Symplasticinhibition
Apoplasticinhibition
Apoplastic transport predominant
Low VPD small differences/effects
0
0.2
0.4
0.6
0.8
1
1.2
Time(mins)
Nor
mal
ized
tran
spir
atio
nApoplast & symplast inhibition at high VPD
Symplasticinhibition
Apoplasticinhibition
Apoplastic transport less predominant
High VPD larger differences/effects
Root hydraulic conductance measurement
VPD-Sensitive VPD-Insensitive0.002
0.0025
0.003
0.0035
0.004
0.0045
0.005
0.0055
0.006Ro
ot c
ondu
ctiv
ity
Root hydraulic conductivity
Symplastic transport less predominantIn VPD-sensitive
Apoplast
Symplast (AQP)
Control
Apoplast
Symplast (AQP)
VPD-Sensitive VPD-Insensitive0.002
0.003
0.004
0.005
0.006
0.007Ro
ot c
ondu
ctiv
ity
Root hydraulic conductivity
Apoplastic transport more predominantIn VPD-sensitive
Control
0
0.2
0.4
0.6
0.8
1
1.2
10 30 50 70 90 110
130
150
170
190
210
230
250
270
290
310
330
350
370
390
410
NTR
Time (mn)
Control100 uM HgCl2200 uM HgCl2
Before treatment
ICC 14799
0
0.2
0.4
0.6
0.8
1
1.2
10 30 50 70 90 110
130
150
170
190
210
230
250
270
290
310
330
350
370
390
410
NTR
Time (mn)
Control100 uM HgCl2200 uM HgCl2
Before treatment
ICC 4958
Chickpea: Symplastic Inhibition
Less symplastic inhibition in VPD-sensitive
VPD - sensitive
VPD - insensitive
Apoplastic inhibition of chickpea genotypes
0.00
0.20
0.40
0.60
0.80
1.00
1.20
0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300Time (mins)
NTR
(Nor
mal
ised
Tra
nspi
ratio
n Ra
tio)
ICC 4958 Control ICC 4958 TreatmentICC 8058 Control ICC 8058 TreatmentICC 867 Control ICC 867 TreatmentICC 14799 Control ICC 14799 Treatment
1mM K4[Fe(CN)6] 0.5mM CuSO4
VPD-sensitive
VPD - insensitive
Chickpea: Apoplastic Inhibition
Less apoplastic inhibition in VPD-sensitive
Apoplast
Symplast (AQP)
Apoplast
Symplast (AQP)
More water via apoplast in VPD-sensitive Limited water via symplast in VPD-sensitive
Insensitive
Sensitive
VPD sensitive have apoplastic water transport
They have limited “tuning” via the symplast
More effort to be put on hydraulic measurement
Many more materials to test
In summary…
The basics – Why aquaporins ?
Transpiration response to VPD
Transpiration response to AQP inhibition
AQP gene expression
AQP in the bigger picture - Drought
VPD-insensitive
VPD-sensitive
Any difference in aquaporin expressionIn sorghum contrasting for VPD response??
0.62 1.05 1.58 2.01 2.43 3.05 3.450.000
0.002
0.004
0.006
0.008
0.010
0.012
0.014
0.016
0.018
VPD (kPa)
Tran
spir
atio
n (g
pl-1
cm
-2)
• 14 PIPs in the Sorghum genome (4 PIP1, 10 PIP2)• Comparable to maize and rice• RTqPCR primers designed• Putative reference genes
AQP gene expression in sorghum
• 3 conditions (low VPD am, low VPD pm / High VPD pm)
• RNA then cDNA, ref genes
• TRqPCRFrom Hanna Anderberg
0.62 1.05 1.58 2.01 2.43 3.05 3.450.000
0.002
0.004
0.006
0.008
0.010
0.012
0.014
0.016
0.018
VPD (kPa)
Tran
spir
atio
n (g
pl-1
cm
-2)
Morning (low VPD)
VPD-insensitive
VPD-sensitive
0.62 1.05 1.58 2.01 2.43 3.05 3.450.000
0.002
0.004
0.006
0.008
0.010
0.012
0.014
0.016
0.018
VPD (kPa)
Tran
spir
atio
n (g
pl-1
cm
-2)
VPD-insensitive
VPD-sensitive
Afternoon (low VPD)
0.62 1.05 1.58 2.01 2.43 3.05 3.450.000
0.002
0.004
0.006
0.008
0.010
0.012
0.014
0.016
0.018
VPD (kPa)
Tran
spir
atio
n (g
pl-1
cm
-2)
Afternoon (high VPD)
VPD-insensitive
VPD-sensitive
Low TE High TE Low TE High TE0
2
4
6
8
10
12
14
16
18 PIP1;1PIP1;2PIP1;3PIP1;4PIP2;1PIP2;2PIP2;4PIP2;5PIP2;6PIP2;7PIP2;8PIP2;9PIP2;10
Hig
h VP
D/L
ow V
PD
PIP relative expression (High VPD/Low VPD)
VPD – insensitive line increases expression of PIP2
PIP2;6
PIP2;9
PIP2;7
VPD-Insensitive VPD-Sensitive
Phylogenetic relationships of AQPs across cereals
Tentative annotation based on rice
Tentative annotation based on sorghum
Tentative annotation based on maize
PIP1;1 Pip1;3/4 pip1.1PIP1;2 Pip1;3/4 Pip1;3/4PIP1;3 pip1.5 pip1.5PIP1;4 pip1.6 pip1.6
PIP2;10 pip2.7 pip2.7PIP2;10 pip2.2 pip2.1PIP2;2 hypothetical protein PIP2;2PIP2;3 pip PIP2;4 pip2.3 pip2.4PIP2;5 pip2.5 pip2.5PIP2;6 pip2.6 pip2.5PIP2;7 pip 95%PIP2;1PIP2;8 pip2.6 pip2.6PIP2;9 pip2.6 pip2.3
Tentative annotation in rice, sorghum, maize
VPDinsensitive
High VPDLow VPD
VPD sensitive
VPDinsensitive
VPD sensitive
VPD-sensitive_LVPD VPD-insensitive_LVPD VPD-sensitive_HVPD VPD_insensitive_Hvpd-0.10.00.10.20.30.40.50.60.7
band
inte
nsity
More AQP protein in VPD–insensitive line
AQP protein measurement with maize PIP2;6 antibodies
Total RNA Ist Strand cDNA
Degenerate primer designing using other closely related species as a source
M -Ve 1 2 3 4 5 6 7 8 9
PCR amplifications of Aqp genes from cDNA.
Cloning of PgAqp genes into pCR8/GW/TOPO vector Plasmid DNA isolated for positive Aqp clones
Sequencing and analysis
Cloning of Aquaporin genes in pearl millet (homology based cloning strategy) .
Phylogentic relationships between pearl millet, maize and rice Aquaporin proteins
L R L R L R L R1122 1086 1152 1078
0.1
1
10
100
PIP1.1 PIP1.2 PIP2.1 PIP2.3 PIP2.6 TIP1.1 TIP2.2Re
lati
ve e
xpre
ssio
n
Genotype and Stress conditions
VPD - Insensitive VPD - Sensitive
PIP relative expression (Low VPD)
High AQP expression in VPD-insensitive line
L R L R L R L R1122 1086 1152 1078
0.1
1
10
100PIP1.1 PIP1.2 PIP2.1 PIP2.3 PIP2.6 TIP1.1 TIP2.2
Genotype and Stress conditions
PIP relative expression (High VPD/Low VPD)Re
lati
ve e
xpre
ssio
n
VPD - Insensitive VPD - Sensitive
Even Higher AQP expression in VPD-insensitive line under high VPD
Increase in AQP expression in VPD-insensitive under high VPD
Transcript abundance & protein agree
Up regulation in both leaves and roots
On-going work in other species
Comparative genomics
In Summary…
The basics – Why aquaporins ?
Transpiration response to VPD
Transpiration response to AQP inhibition
AQP gene expression
AQP in the bigger picture - Drought
0.62 1.05 1.58 2.01 2.43 3.05 3.450.000
0.002
0.004
0.006
0.008
0.010
0.012
VPD (kPa)
Tran
spira
tion
(g p
l-1 cm
-2)
Why are VPD-sensitive sorghum so interesting?
VPD-insensitive
VPD-sensitive
VPD-sensitive have high transpiration efficiency
2.0
3.0
4.0
5.0
6.0
7.0
152 Germplasm tested
TE
10 lowest TE are VPD-Insensitive
10 highest TE are VPD-sensitive
High TE lines limit transpiration at high VPD
3.0
3.2
3.4
3.6
3.8
4.0
4.2
4.4
4.6
TE
1 2 3 4 5 6 7 80
0.005
0.01
0.015
0.02
0.025
0.03
VPD (kPa)
Tran
spir
atio
n (g
cm
-2 h
-1)
Same result in sorghum and pearl millet
What about pearl millet?
Low TE(VPD-Insensitive)
High TE(VPD-Sensitive)
grain yield gain (low TR)
-300
-200
-100
0
100
200
300
400
0 500 1000 1500 2000 2500 3000 3500
original yield (kg/ha)
yiel
d ga
in (k
g/ha
)
1 postflowering
2 flowering
3 postflowering-relieved
4 no stress
5 prefloweringOriginal yield (kg ha-1)
0
Yield increase (kg/ha) with transpiration sensitivity to high VPD: Rabi sorghum
Yiel
d in
crea
se
-1 0 +33
Crop modelling used to predict trait effects
15-30% yield increase at high latitudes
% yield increase with transpiration sensitivity to high VPD: Peanut
Lysimetric evaluation
Transpiration in pots
0.62 1.05 1.58 2.01 2.43 3.05 3.450.000
0.004
0.008
0.012
0.016
0.020
Low TEHigh TE
VPD
Tran
spira
tion
(g c
m-2
h-1
)
Low TE High TE01234567
TE
grain yield gain (low TR)
-300
-200
-100
0
100
200
300
400
0 500 1000 1500 2000 2500 3000 3500
original yield (kg/ha)
yiel
d ga
in (k
g/ha
)
1 postflowering
2 flowering
3 postflowering-relieved
4 no stress
5 preflowering
Original yield (kg ha-1)
0
AQP gene expression
Modeling of Tr restriction effect on yield
The VPD response lead to higher TE
It is itself related to differences in AQP gene expression
Major yield increase possible across crops
Breeding (donors identified)
Harness genetics – Phenotyping (new platform)
In Summary…
Thank you
Collaborators:F. Chaumont (Univ. Louvain)H. Anderberg (Lund Univ.)Donors:
ICRISATACIARDFIDB&MGF
Technicians / Data analyst:Srikanth MalayeeRekha Badham
Students:M TharanyaS SakthiT RajiniS MedinaK Aparna
Colleagues:J Kholova / P Suddhakar Reddy / G Barzana / JM Devi/ KK Sharma / T Shah / P Bhatnagar / Hima Bindhu / RK Varshney / R Srivastava / SP Deshpande
Lysimetric evaluation
Transpiration in pots
0.62 1.05 1.58 2.01 2.43 3.05 3.450.000
0.004
0.008
0.012
0.016
0.020
Low TEHigh TE
VPD
Tran
spira
tion
(g c
m-2
h-1
)
Low TE High TE01234567
TE
grain yield gain (low TR)
-300
-200
-100
0
100
200
300
400
0 500 1000 1500 2000 2500 3000 3500
original yield (kg/ha)
yiel
d ga
in (k
g/ha
)
1 postflowering
2 flowering
3 postflowering-relieved
4 no stress
5 preflowering
Original yield (kg ha-1)
0
AQP gene expression
Modeling of Tr restriction effect on yield
Xpsmp2237
Xpsmp2072
17.1 cM
Xpsmp2066
12.0 cM
Xpsmp3056
19.1 cM
Xpsmp220614.2 cM
Xpsmp20592.5 cM
Grain YldFlowering time
Grain Yld
Allele effect
Xibmsp44
Xibmsp4Xibmsp7
Xibmsp60 Xibmsp34 Xibmsp14 Xibmsp24 Xibmsp31 Xibmsp11
Xibmsp62Xibmsp27Xibmsp9Xibmsp12Xibmsp15
Xibmsp23
PRLT 2/89-33 H 77/833-2
Increased decreased decreased Increased
Flowering time
Increased decreased decreased Increased
Stay greendecreased Increased
Increased decreased
Tr rate Increased decreased
Tr rate decreased Increased
Stay green
Candidate genesZn finger CCCH-type, serine/threonine protein kinase, MADS-box, acetyl CoA carboxylase
Stay green