High throughput and low cost phenotyping options for a few drought adaptive traits

Dr. Udayakumar M

Department of Crop Physiology, UAS, GKVK, Bangalore -65

18th Feb, 2014, IPPN

Drought is complex Magnitude and duration of stress is unpredictable

Plants evolved diverse adaptive strategies to sustain growth under

complex stress situations

Opuntia

ABA

A plant character/biochemical mechanisms that facilitates cell turgor, cell survival and improves adaptation under moisture stress

Drought adaptive traits - consensus

2

Major emphasis has been

Identify and validate the relevant trait Is it

relevant?

This is what you have to

look for

Oh ! God NO RAIN

Capture the genetic variability

Accurate imposition of moisture stress is crucial for phenotyping drought adaptive traits

3 Managed drought environment Lysimeter

Transpiration -water uptake- drives the growth

H2O H2O

H2O H2O

H2O

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H2O

H2O H2O

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H2O H2O H2O

H2O

Plants experience stress

When Transpiration exceeds uptake

Resulting in wilting Cell Ψ/ turgor

Affects growth

Plant has to Maintain cell turgor Tolerate the stress effects

4

constitutive traits

Acquired traits

Traits which are inherent to the genotype

Traits/mechanisms which are up regulated

under stress May not substantially increase under

stress

Adaptive mechanisms/traits…. Broadly classified as

Root WUE EW

Associated with water relations, postpones decrease in ψw

Phenology 5

Mainly cellular level tolerance mechanisms

Traits-water relations We developed tools / techniques to assess WUE

Stable isotope technique for WUE – 13C

For CID

Bindumadhava et al., 2005; Current Science, 89(7): 1256 - 1258 Sheshshayee et al., 2005, Journal of Experimental Botany 56: 3033–3039. Sheshshayee et al., 2011, book chapter CIMMYT Udayakumar M et al., 2013, Designer rice

Impa et al., 2005, Crop Science 45: 2517–2522.

y = -0.669x + 22.912 r = 0.831

18.50 19.00 19.50 20.00 20.50 21.00 21.50 22.00

2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 WUE (g.kg-1)

D 13

C

Gravimetry

y = -2,284x + 24,438 R² = 0,646

18,0

19,0

20,0

21,0

22,0

23,0

1,0 1,2 1,4 1,6 1,8 2,0 2,2

∆13 C

(‰)

WUE (g. kg-1) 6

Species

Range in ∆

13C (‰) Rice Germplasm (230) 18.15 – 22.79

Rice Mapping population (100) 19.21 - 22.55

Groundnut Germplasm (246) 16.19 – 22.32

Groundnut RIL (268) 16.68 – 22.96

Root structure

No

of g

enot

ypes

0

10

20

30

40

50

60

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14

y = 0.0002x + 0.998r = 0.64

0.0

2.0

4.0

6.0

8.0

10.0

12.0

14.0

10000 15000 20000 25000 30000 35000 40000 45000

LADxd18O

Root

BM

Phenotyping for roots

Oxygen isotopes- A potential option to quantify transpiration rate and thus root traits

Root weight Sheshshayee et al., 2005 Mohankumar 2011 Udayakumar M et al., 2013

7

Root traits in root structures

Mesophyll efficiency by Ci/gs ratio - 13C/18O ratio

8

Mesophyll efficiency (gm) – Ci/gs Differences in Ci at given gs is reflection of gm

Bindumadhava et al., 2005; Sheshshayee et al., 2011

13C/18O ratio – a surrogate for Ci/gs

Carboxylation efficiency dA/dCi and Ci/gs

Carboxylation efficiency dA/dCi and 13C/18O

Captured the genetic variability under low cost MDE -option to simulate required precipitation levels by sprinklers

13∆C in rice genotypes Wax in rice genotypes

Semi Mechanized ROS Sprinkler system for specific water input

Prathiba, 2012

Sheela, 2012

Sheshayee et al., 2013; Mohankumar, 2011

Managed drought environment facility (MDE)

9

Drought susceptibility index

When, Plant experiences stress

Cell Ψ/ turgor

Expression of stress genes

Brings in altered metabolism for

adaptation

Tolerance to oxidative stress

Protection to macro molecules

Osmotic adjustment

Protein turn over Genes expressed on exposure to stress regulate intrinsic tolerance mechanisms

involved in cell survival at low ψw

Phenomenal progress made in characterizing stress genes

Cellular level tolerance mechanisms - CLT

Acquired tolerance traits

10

Two important groups of traits are emerging as relevant to achieve field level tolerance

Mechanisms regulating water relations

Mechanisms governing the cellular level tolerance

Phenotyping for acquired traits upregulated on exposure to stress is difficult to capture under field conditions and even in big containers

11

Amenable to capture genetic variability even under field conditions

H2O H2O

H2O

H2O

H2O

H2O

H2O

H2O

H2O

H2O H2O

H2O

H2O H2O

H2O

H2O H2O H2O

H2O

H2O H2O

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H2O

H2O

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H2O H2O

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H2O H2O

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H2O H2O

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H2O

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H2O H2O

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H2O H2O H2O

H2O H2O

H2O

H2O H2O

H2O

H2O

H2O

H2O H2O H2O

H2O

H2O

H2O

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H2O

H2O

H2O H2O

H2O

H2O

H2O

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H2O H2O

H2O

H2O H2O

H2O

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H2O H2O

H2O

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H2O H2O

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H2O H2O

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H2O H2O

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H2O H2O

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H2O H2O

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H2O H2O H2O

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H2O H2O

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H2O H2O

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H2O H2O

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H2O H2O

H2O

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H2O H2O

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H2O H2O

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H2O

H2O

H2O

H2O H2O H2O

H2O

H2O H2O

H2O

H2O

H2O

H2O

H2O

H2O H2O

H2O

H2O H2O H2O

H2O

H2O

H2O

H2O

H2O H2O

H2O

H2O H2O H2O

H2O H2O

H2O

H2O

H2O

H2O H2O

H2O H2O

H2O H2O

H2O

H2O H2O

H2O H2O

H2O

H2O

H2O H2O

H2O H2O H2O

H2O H2O

H2O

H2O H2O

H2O H2O H2O

H2O H2O

H2O

12

Plant water status differs in the genotypes under stress

Genetic variability in water status affects the expression of acquired traits

Difference in Ψw – alters the expression of acquired traits

Genetic variability in acquired traits has to be seen -at comparable water status

Genetic variability in acquired traits

Are often assessed in

Seedlings

Excised leaves

As a compromise

13

Temperature induction response (TIR)

Acquired tolerant traits

UASB

Should be assessed only on exposure to induction stress

Cotton

Sunflower

Rice

Rice

Desiccation stress response

Induced Non-Induced

Induction response seen even at plant level Induced Non-Induced

Temperature induction response (TIR)

C I NI

C I NI

Salinity induction response (SIR)

Induction response technique – an empirical assessment of survival and recovery

Babitha, 2012

Ehab et al., 2012, Journal of Cotton Science

Babitha, 2012 Sreekanthbabu et al., 2002, J. Plant Physiol

14

Senthil-Kumar et al., 2003, JXB Senthil-Kumar et al., 2007, J. pl. physiol. Ganeshkumar et al., 1999, Theor Appl Gen;99:359–67.

T - Tolerant S - Susceptible

Rice

Cotton

C I NI C I NI

0

1020

30

40

5060

70

8090

100

10.0-20.0

20.0-30.0

30.0-40.0

40.0-50.0

50.0-60.0

60.0-70.0

70.0-80.0

80.0-90.0

90.0-100.0.

Range of thermotolerance

Frequ

ency Temperature

induction response in pea

T S T S

Genetic variability is seen only upon induction stress

Higher expression of Hsps in tolerant genotype

Stress transcriptome analysis should be done only after exposing to induction stress

Narendra, 2012

Ehab, 2012, Journal of Cotton Science

Senthilkumar et al., 2007, J. pl. physiol. Sreekanthbabu et al.,2002, J. Plant Physiol

Phenotype for acquired traits - only after induction UASB 15

Seeds exposed to 45 ⁰C at 100 % RH for 6/8 days

control 6 days of AA

MAS

AC35313

AC39018

IET16348

AC35310

AC39020

TELAHAMSA

KMP-175

Control

6 days of AA

wt AKR1-2

Range of root +shoot length (cm)

No.

of a

cces

sions

AKR1-2 transgenic tolerant to AA Nisarga, 2012

Seed aging is a stress – seedling vigour decreases on aging

Accelerated aging – a surrogate to phenotype for seedling vigour under stress

UASB 16

Genetic variability in Rice for oxidative stress

Untreated

Oxidative stress (Methyl viologen)

Phenotype for tolerance mechanisms – metabolic inhibitors (ROS inducers )

Frequency distribution of rice genotypes for MV induced oxidative stress

05

1015202530

Percentage reduction in growth (cm) N

umbe

r of g

enot

ypes

Class interval

UASB 17

WT M86 M97

Endoplasmic reticulum stress response

Metabolic inhibitors – to screen for specific tolerance mechanism

02468

101214

<WT 40-50 51-60 61-70 71-80 81-90 91-100

No.

of e

ntri

es

DTT induced ER stress

Survival percentage – DTT induced ER stress Rice

Groundnut

Babitha, 2012

UASB 18

Precise imposition of moisture stress is crucial

How to Phenotype acquired tolerant traits at whole plant level

By gravimetry - Capture the change in soil moisture due to ET in

mini lysimeters and replenish to maintain specified

Soil moisture status

Motorised lifting Manual lifting

Gravimetry – is an option

UASB 19

Weigh the pots to measure ET

Even in automated gravimetry systems

Water lost by ET quantified gravimetrically once a day

Limitation - frequency at which water lost by ET is replenished

Plants undergo daily cycles of stress- which again depends on leaf area

Change in the soil water status (% FC) during a 24hr cycle

Soil

moi

stur

e st

atus

(% F

C)

6.00 pm 6.00 am Percent Soil Moisture

Option to replenish water lost by ET at frequent intervals to maintain specific FC %

98 % FC 95 % FC

11.00 am 2.00 pm

93 % FC 89 % FC 85 % FC 80 % FC 78 % FC

Loadcell mini lysimeter assembly for Continuous monitoring of ET and soil moisture status (% FC)

21

DATA COLLECTION & STORAGE HUB

Data + Power cable

Temperature Sensor

Relative Humidity Sensor

Data Acquisition System

Uninterrupted Power Supply

Data

Salient features of the minilysimeter assembly

Continuous measurement Fixed intervals Networked sensors Centralized Master Control Centralized data collection

Master Control Lysimeter Water Status

Load cell – minilysimeter assembly 22

Load Cell devices for continuous measurement of change in weight

in lysimeters

Load cell Lysimeter assembly

Lysimeter facility for real time monitoring of evaporation and transpiration

The mini-lysimeter facility

Open field conditions

Minilysimeters under ROS

24

25

Cumulative transpiration of two Maize plants differing in leaf area

Soil

moi

stur

e %

FC

The system captures accurate changes in ET and soil moisture status

60

65

70

75

80

85

90

95

100

0 10 20 30 40 50 60

Maize-LLA-1700cm2

Maize-HLA-3700cm2

Evap

otra

nspi

ratio

n (K

g)

12% drop

15% drop Irrigation

0

20

40

60

80

100

120

0 3 6 9 12 15 18 21 24

Diurnal changes in transpiration rate in plants differing in leaf area

Empty Pot (E)

Maize-HLA (T)

Maize-LLA (T)

12 am 3 pm 6 pm 9 pm 12 pm 9 am 6am 3 am 6 am

Time of the day

Tran

spira

tion

Rate

(g/h

r)

26

Total transpiration- A function of leaf area

A significant positive correlation signifies accurate capturing of changing water loss by the lysimeter system

Leaf Area (cm2)

Tran

spira

tion

(g) /

plan

t/da

y y = 0,2764x + 125,83 R² = 0,9392

0200400600800

100012001400

1500 2500 3500 4500

Leaf Area (cm2)

Tran

spira

tion

(g) /

plan

t/da

y y = 0,2259x + 83,803 R² = 0,965

0100200300400500600700

0 500 1000 1500 2000 2500

Maize

Soybean

27

Maintenance of soil moisture status by continuous replenishment of the water loss by evapotranspiration

The system provides an option to replenish within 5% change in FC

11 am 2pm 5 pm 8 pm 12 pm Overnight 8 am 5am

98 95 90 80

Continuous monitoring of ET and replenishment of water in the stationary lysimeter assembly

28

Soil

moi

stur

e st

atus

(%FC

)

Can be programmed to attain required soil moisture status at desired pace

Option to program for gradual imposition of moisture stress in lysimeter grown plants

40

50

60

70

80

90

100

0 16 32 48 64 80 96 112 128 144 160 176 1 2 3 4 5 6 7 8 9 10

Reduction of 5% per day

50% FC

Soil

Moi

stur

e (%

)

Days

29

Proposed lysimeter phenomics facility for growing germplasm accessions at desired soil moisture status

A pictorial outlay of the mini lysimeter facility

ROS

Each bay has 450 Mini Lysimeters

High throughput amenability

Load cell mini-lysimeter assembly

Facility under development

30

Phenotyping of acquired tolerant traits at plant level

- Needs to be assessed at comparable water status

- Maintaining precise moisture status is crucial

A conceptual mini lysimeter facility developed

a) Continuous monitoring of E, T and ET in plants grown under natural conditions

b) Precise imposition of stress c) Amenable to screen large number of accessions d) Genetic variability assessment for many physiological traits

Precision high throughput phenotyping technioques for a few drought traits developed

- Water relation traits - Acquired traits at seedling level

In summary

High throughput facility is under development

31

32

Thank you

Acknowledgement: Jalendra kumar H G Mahesh Salimath Sheshshayee M S Mohan Raju B R Arul Selvan K Bobji M

Other students and staff members

Funding from NAE, ICAR COE-program support, DBT DST-DBT National Facility

Cumulative transpiration of two Maize plants differing in leaf area Ev

apot

rans

pira

tion

(Kg)

0,0

0,5

1,0

1,5

2,0

2,5

0 10 20 30 40 50 60

Night Day

Irrigation

Time

Day-1 Day-2 Day-3

Maize-LLA-1700cm2

Maize-HLA-3700cm2

33 The system captures accurate changes in ET and soil moisture status

Emphasis We developed tools / techniques for assess

Stable isotope technique for WUE – 13C

Transpiration 18O (indirectly roots)

Mesophyll efficiency by Ci/gs ratio 13C/18O ratio

Gravimetry

Root structure

For CID

Bindumadhava et al., 1005; Sheshshayee et al., 2011

Rice

Endoplasmic reticulum stress response Genetic variability – Reactive Oxygen Scavenging

Oxidative stress

Untreated

(MV)

Rice

Groundnut