Developing crops with high productivity at high temperature

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 ?



AQP gene expression


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…




AQP gene expression


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

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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…




AQP gene expression


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…




AQP gene expression


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


-300

-200

-100

0

100

200

300

400

0 500 1000 1500 2000 2500 3000 3500


yiel

d ga

in (k

g/ha

)

1 postflowering

2 flowering


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


-300

-200

-100

0

100

200

300

400

0 500 1000 1500 2000 2500 3000 3500


yiel

d ga

in (k

g/ha

)

1 postflowering

2 flowering


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

Government & Nonprofit

Developing crops with high productivity at high temperature