Developing crops with high productivity at high temperature A blue sky research initiative BSR Team: 29 Nov 2013

1. Developing crops with high
productivity at high
temperature:
A blue sky research initiative
BSR Team
29 Nov 2013

2. Maximum temperature in the SAT
Critical
Temperature
threshold
0
5
10
15
20
25
30
35
40
45
Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec
MaximumT°C
1983-HQ 1992-HQ
2001-HQ 2012-HQ
1983-ISC 1990-ISC
1998-ISC
Headquarter
Sahelian Center
T°C rarely crosses critical limits

3. 0
1
2
3
4
5
6
7
8
Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec
MaximumVPD 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
VPD
threshold

4. The basics – Why aquaporins (AQP) ?
Transpiration response to VPD
Transpiration response to AQP inhibition
AQP gene expression
AQP in the bigger picture - Drought

5. Modified from Murata et al., 2000.
What are aquaporins??
Membrane transporter for water
Passive transport

6. 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)
H77/2 833-2
PRLT-2/89-33
Transpiration(gcm-2h-1)
Kholova et al 2010 – J. Exp. Bot
Transpiration restriction at high VPD
How the story began – Water savings in millet
Water
saving

7. Why such a rapid transpiration response??
Rapid response hydraulic signal
VPD (kPa)

8. Where is the source of hydraulic limitation??
???
We hypothesized roots could be the
source of hydraulic limitation

9. Is the hydraulic restriction in the roots only ??

10. 0.6
0.7
0.8
0.9
1
1.1
1.2
1.3
1.4
1.5
1.6
09:20 09:40 10:00 10:20 10:40 11:00 11:20 11:40 12:00 12:20 12:40 13:00 13:20
Normalizedtranspiration
VPD
change
Low VPD High VPD
No difference in slope No hydraulic
limitation in the leaves

11. Apoplastic
Pathway
(Structural)
Symplastic
Pathway
(AQP)
Water pathways in the root cylinder
Two pathways have different hydraulic conductance
Hypothesis: Aquaporin control plant water
loss ?
????

12. The basics – Why aquaporins ?
Transpiration response to VPD
Transpiration response to AQP inhibition
AQP gene expression
AQP in the bigger picture - Drought

13. 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)
H77/2 833-2
PRLT-2/89-33
Transpiration(gcm-2h-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
M322
M394
M214
M321
M592
M443
M356
M738
40cM

14. 0
0.005
0.01
0.015
0.02
0.025
0.03
1 2 3 4 5 6 7 8Transpiration
(gcm-2h-1) VPD (kPa)
VPD insensitive
VPD sensitive
0
2
4
6
8
10
12
14
16
Xpsmp2237 Xpsmp2072 M13_Xpsmp2066 M13_Xpsmp3056 Xpsmp2206 Xpsmp2059
NoofRecombinants
markers within LG2 DT-QTL
low Transpiration Rate in high VPD
A - from H77
B - from PRLT
H - heterozygous
Two QTL fine-mapped for low Tr at high VPD

15. Staygreen ILs (Stg3 – Stg B) are VPD-sensitive
0.0000
0.0020
0.0040
0.0060
0.0080
0.0100
0.0120
9 11 13 15 17
Transpiration(gcm-2h-1)
Time of the day (h)
stg1
stg3
stg4
stgB
R16
B35
Recurrent R16
Stg3
StgB
Transpiration response to VPD in Sorghum
1 - Introgression lines

16. 0.000
0.002
0.004
0.006
0.008
0.010
0.012
0.014
0.016
0.018
0.62 1.05 1.58 2.01 2.43 3.05 3.45
Transpiration(gh-1cm-2)
VPD (kPa)
Transpiration response to VPD in Sorghum
2 - Germplasm
Germplasm differences in VPD-sensitivity
VPD-Sensitive
VPD-
Insensitive

17. BP= 2.58 R2 =0.68BP= 2.51 R2 =0.76
R2 =0.89
R2 =0.83
Transpiration response to VPD in Chickpea

18. Transpiration response to VPD in Peanut

19. Mouride
IfVPD<2.09,TR=0.0083(VPD)–0.002
IfVPD≥ 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)

20. VPD
Transpiration(gcm-2h-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 species
VPD-sensitivity often link to drought adaptation

21. Large variation in all species
Often discriminate tolerant from sensitive lines
Now, systematic screening
Exciting results in other crops (rice, maize)
In summary…

22. The basics – Why aquaporins ?
Transpiration response to VPD
Transpiration response to AQP inhibition
AQP gene expression
AQP in the bigger picture - Drought

23. 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.

24. Apoplastic pathway inhibition

25. Apoplastic
pathway
Symplastic
Pathway
(AQP)
Symplastic pathway inhibition
AQP inhibitors: AgNO3 – HgCl2 – H2O2

26. Follow-up of transpiration before/after inhibition

27. 0.6
0.7
0.8
0.9
1
1.1
1.2
NormalizedTranspiration
Time of the day
VPD-sensitive
VPD -
insensitive
Less symplastic inhibition in VPD-sensitive
Pearl millet: Symplastic inhibition
Treatment
M322
M394
M214
M321
M592
M443
M356
M738
40cM

28. 0.6
0.7
0.8
0.9
1
1.1
1.2
10 30 50 70 90 110 130 150 170 190 210 230 250 270
Normalizedtranspiration
Time (minutes)
ICMR1029 1mM
ICMR2042 1mM
H77 1mM
PRLT 1mM
Symplastic inhibition in near-isogenic lines
(QTL from VPD-sensitive parent)
NILs behave like QTL donor parent
M322
M394
M214
M321
M592
M443
M356
M738
40cM
Treatment
VPD-sensitive

29. More apoplastic inhibition in VPD-sensitive
0.00
0.20
0.40
0.60
0.80
1.00
1.20
10:10 10:40 11:10 11:40 12:10 12:40 01:10 01:40 02:10 02:40 03:10
NormalizedTranspiration
Time
Apoplastic
inhibition
Pearl millet: Apoplastic Inhibition
VPD-sensitive
VPD -
insensitive
M322
M394
M214
M321
M592
M443
M356
M738
40cM

30. 0
0.2
0.4
0.6
0.8
1
1.2
Normalizedtranspiration
Time
Apoplast & symplast inhibition at low VPD
Apoplastic &
Symplastic
inhibition
Symplastic
inhibition
Apoplastic
inhibition
Apoplastic transport predominant
Low VPD small differences/effects

31. 0
0.2
0.4
0.6
0.8
1
1.2
Normalizedtranspiration
Time(mins)
Apoplast & symplast inhibition at high VPD
Symplastic
inhibition
Apoplastic
inhibition
Apoplastic transport less predominant
High VPD larger differences/effects

32. Root hydraulic conductance measurement

33. 0.002
0.0025
0.003
0.0035
0.004
0.0045
0.005
0.0055
0.006
VPD-Sensitive VPD-Insensitive
Rootconductivity
Root hydraulic conductivity
Symplastic transport less predominant
In VPD-sensitive
Apoplast
Symplast
(AQP)
Control

34. Apoplast
Symplast
(AQP)
0.002
0.003
0.004
0.005
0.006
0.007
VPD-Sensitive VPD-Insensitive
Rootconductivity
Root hydraulic conductivity
Apoplastic transport more predominant
In VPD-sensitive
Control

35. 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)
Control
100 uM HgCl2
200 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)
Control
100 uM HgCl2
200 uM HgCl2
Before treatment
ICC 4958
Chickpea: Symplastic Inhibition
Less symplastic inhibition in VPD-sensitive
VPD -
sensitive
VPD -
insensitive

36. Apoplastic inhibition of chickpea genotypes
0.00
0.20
0.40
0.60
0.80
1.00
1.20
0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300
Time (mins)
NTR(NormalisedTranspirationRatio)
ICC 4958 Control ICC 4958 T reatment
ICC 8058 Control ICC 8058 T reatment
ICC 867 Control ICC 867 T reatment
ICC 14799 Control ICC 14799 T reatment
1mM K4[Fe(CN)6] 0.5mM CuSO4
VPD-sensitive
VPD -
insensitive
Chickpea: Apoplastic Inhibition
Less apoplastic inhibition in VPD-sensitive

37. Apoplast
Symplast
(AQP)
Apoplast
Symplast
(AQP)
More water via apoplast in VPD-sensitive
Limited water via symplast in VPD-sensitive
Insensitive
Sensitive

38. 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…

39. The basics – Why aquaporins ?
Transpiration response to VPD
Transpiration response to AQP inhibition
AQP gene expression
AQP in the bigger picture - Drought

40. VPD-insensitive
VPD-sensitive
Any difference in aquaporin expression
In sorghum contrasting for VPD response??
0.000
0.002
0.004
0.006
0.008
0.010
0.012
0.014
0.016
0.018
0.62 1.05 1.58 2.01 2.43 3.05 3.45
Transpiration(gpl-1cm-2)
VPD (kPa)

41. • 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
• TRqPCR
From Hanna Anderberg

42. 0.000
0.002
0.004
0.006
0.008
0.010
0.012
0.014
0.016
0.018
0.62 1.05 1.58 2.01 2.43 3.05 3.45
Transpiration
(gpl-1cm-2)
VPD (kPa)
Morning (low VPD)
VPD-insensitive
VPD-sensitive

43. 0.000
0.002
0.004
0.006
0.008
0.010
0.012
0.014
0.016
0.018
0.62 1.05 1.58 2.01 2.43 3.05 3.45
Transpiration
(gpl-1cm-2)
VPD (kPa)
VPD-insensitive
VPD-sensitive
Afternoon (low VPD)

44. 0.000
0.002
0.004
0.006
0.008
0.010
0.012
0.014
0.016
0.018
0.62 1.05 1.58 2.01 2.43 3.05 3.45
Transpiration
(gpl-1cm-2)
VPD (kPa)
Afternoon (high VPD)
VPD-insensitive
VPD-sensitive

45. 0
2
4
6
8
10
12
14
16
18
Low TE High TE
HighVPD/LowVPD PIP1;1
PIP1;2
PIP1;3
PIP1;4
PIP2;1
PIP2;2
PIP2;4
PIP2;5
PIP2;6
PIP2;7
PIP2;8
PIP2;9
PIP2;10
PIP relative expression (High VPD/Low VPD)
VPD – insensitive line
increases expression of PIP2
PIP2;6
PIP2;9
PIP2;7
VPD-Insensitive VPD-Sensitive

46. Phylogenetic relationships of AQPs
across cereals

47. Tentative annotation
based on rice
Tentative annotation
based on sorghum
Tentative annotation
based on maize
PIP1;1 Pip1;3/4 pip1.1
PIP1;2 Pip1;3/4 Pip1;3/4
PIP1;3 pip1.5 pip1.5
PIP1;4 pip1.6 pip1.6
PIP2;10 pip2.7 pip2.7
PIP2;10 pip2.2 pip2.1
PIP2;2 hypothetical protein PIP2;2
PIP2;3 pip
PIP2;4 pip2.3 pip2.4
PIP2;5 pip2.5 pip2.5
PIP2;6 pip2.6 pip2.5
PIP2;7 pip 95%PIP2;1
PIP2;8 pip2.6 pip2.6
PIP2;9 pip2.6 pip2.3
Tentative annotation in rice, sorghum, maize

48. VPD
insensitive
High VPDLow VPD
VPD
sensitive
VPD
insensitive
VPD
sensitive
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
VPD-sensitive_LVPD VPD-insensitive_LVPD VPD-sensitive_HVPD VPD_insensitive_Hvpd
bandintensity
More AQP protein in VPD–insensitive line
AQP protein measurement
with maize PIP2;6 antibodies

49. 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 vectorPlasmid DNA isolated for positive Aqp clones
Sequencing
and analysis
Cloning of Aquaporin genes in pearl millet
(homology based cloning strategy) .

50. Phylogentic relationships
between pearl millet, maize
and rice Aquaporin proteins

51. 0.25
0.5
1
2
4
8
16
L R L R L R L R
1122 1086 1152 1078
PIP1.1 PIP1.2 PIP2.1 PIP2.3 PIP2.6 TIP1.1 TIP2.2
Relativeexpression
Genotype and Stress conditions
VPD - Insensitive VPD - Sensitive
PIP relative expression (Low VPD)
High AQP expression in VPD-insensitive line

52. 0.25
0.5
1
2
4
8
16
L R L R L R L R
1122 1086 1152 1078
PIP1.1 PIP1.2 PIP2.1 PIP2.3 PIP2.6 TIP1.1 TIP2.2
Genotype and Stress conditions
PIP relative expression (High VPD/Low VPD)
Relativeexpression
VPD - Insensitive VPD - Sensitive
Even Higher AQP expression
in VPD-insensitive line under high VPD

53. 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…

54. The basics – Why aquaporins ?
Transpiration response to VPD
Transpiration response to AQP inhibition
AQP gene expression
AQP in the bigger picture - Drought

55. 0.000
0.002
0.004
0.006
0.008
0.010
0.012
0.014
0.62 1.05 1.58 2.01 2.43 3.05 3.45
Transpiration(gpl-1cm-2)
VPD (kPa)
Why are VPD-sensitive sorghum so interesting?
VPD-insensitive
VPD-sensitive

56. 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

57. 3.0
3.2
3.4
3.6
3.8
4.0
4.2
4.4
4.6
TE
0
0.005
0.01
0.015
0.02
0.025
0.03
1 2 3 4 5 6 7 8
Transpiration
(gcm-2h-1)
VPD (kPa)
Same result in sorghum and pearl millet
What about pearl millet?
Low TE
(VPD-Insensitive)
High TE
(VPD-Sensitive)

58. 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)
yieldgain(kg/ha)
1 postflowering
2 flowering
3 postflowering-relieved
4 no stress
5 preflowering
Original yield (kg ha-1)
0
Yield increase (kg/ha) with transpiration
sensitivity to high VPD: Rabi sorghum
Yieldincrease

59. -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

60. Lysimetric evaluation
Transpiration in pots
0.000
0.004
0.008
0.012
0.016
0.020
0.62 1.05 1.58 2.01 2.43 3.05 3.45
Transpiration
(gcm-2h-1)
VPD
Low TE
High TE
0
1
2
3
4
5
6
7
Low TE High TE
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)
yieldgain(kg/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

61. 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…

62. Thank you
Collaborators:
F. Chaumont (Univ. Louvain)
H. Anderberg (Lund Univ.)Donors:
ICRISAT
ACIAR
DFID
B&MGF
Technicians / Data analyst:
Srikanth Malayee
Rekha Badham
Students:
M Tharanya
S Sakthi
T Rajini
S Medina
K 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

63. Lysimetric evaluation
Transpiration in pots
0.000
0.004
0.008
0.012
0.016
0.020
0.62 1.05 1.58 2.01 2.43 3.05 3.45
Transpiration
(gcm-2h-1)
VPD
Low TE
High TE
0
1
2
3
4
5
6
7
Low TE High TE
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)
yieldgain(kg/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

64. Xpsmp2237
Xpsmp2072
17.1 cM
Xpsmp2066
12.0 cM
Xpsmp3056
19.1 cM
Xpsmp2206
14.2 cM
Xpsmp2059
2.5 cM
Grain Yld
Flowering time
Grain
Yld
Allele effect
Xibmsp44
Xibmsp4
Xibmsp7
Xibmsp60 Xibmsp34
Xibmsp14 Xibmsp24
Xibmsp31 Xibmsp11
Xibmsp62
Xibmsp27
Xibmsp9
Xibmsp12
Xibmsp15
Xibmsp23
PRLT 2/89-33 H 77/833-2
Increased decreased
decreased Increased
Flowering time
Increased decreased
decreased Increased
Stay green
decreased Increased
Increased decreased
Tr rate Increased decreased
Tr rate
decreased Increased
Stay green
Candidate genes
Zn finger CCCH-type, serine/threonine protein kinase, MADS-box, acetyl CoA carboxylase
Stay green