selected methods of measuring drought stress in...

(Selected!) Methods of measuring

drought stress in plantsSilvia B. Kikuta

Department of Integrative Biology

Institute of Botany

University of Natural Resources and

Applied Life Sciences, Vienna

Contents

Definition of stress / strainDefinition of drought stress

Resistance mechanisms of plants

Morpho-anatomical traitsDefinition of water potential (Ψ)

Total water potential (Ψt) and componentsTechniques of measuring plant water status

Pressure chamberThermocouple psychrometry

Plant water content

Pressure-volume (pV) curvesOsmotic adjustment

Elastic adjustmentWater use efficiency

Definition of stress / strain(Larcher 2003)

Stress is considered to be a significant deviation from

optimal conditions of life

Stress causes changes and responses at all functional

levels of the organism

Term stress (stress factor, stressor) indicates the event

Term strain indicates the state (stress response, state

of adaptation) evoked within an organism

Drought stress

Too little water is available in a suitable thermo-

dynamic state

Demand exceeds the supply of water

Reasons:

• Soil dryness

• Inadequate water uptake by plants in shallow

soils

• Osmotic binding in saline soils

• High evaporation

Drought stress develops slowly

Intensity increases with time

Stress level, time scale crucial!

Drought resistance

Capacity of plants to withstand periods of dryness

Serious terminological problems with the term drought

resistance!

Difference of drought resistance in

Natural vegetation

Species conservation, plant survival

Cultivated plants

Sustainable and economically viable plant

production

How plants cope with drought stress

Different survival mechanisms of plants at

dry sites:

1) Drought escape

2) Dehydration avoidance

3) Dehydration tolerance

Drought escapeDrought periods must occur at a predictable time

Important strategy for mediterranean and monsoon climates, not efficient for Central Europe

i) Temporal:

Whole life cycle or physiologically active phase shifted to

periods without stresse.g. winter wheat, winter barley – well suited for their

place of origin (Iraq, Iran; summer drought)Selection of early-ripening genotypes

ii) Spatial:

Development of water-storing belowground organs

e.g. geophytes

Drought (dehydration) avoidanceTissues are sensitive to dehydration →→→→ must maintain high water potentials as long as possible

2 groups of drought avoiders:i) Water savers

Conserve water

ii) Water spenders

Absorb water so fast as to meet transpirational losses

Anatomical and morphological traits help the plant to

increase water uptake

reduce water spending

Morpho-anatomical traits

(A) Water uptake is improved(1) extensive root system with large active surface area(2) shoot/root ratio shifted in favour of the roots

(B) Water loss is reduced(1) transpiration reduced (timely stomatal closure)

(1a) smaller but more densely distributed stomata(2) thick cuticle

(3) epicuticular waxes

(4) leaf colour (yellow, glaucous)

(5) white hairs on leaves

(6) leaf angle

(7) leaf rolling(8) plant senescence(8a) leaf senescence

(9) leaf shedding

Drought (dehydration) tolerance

Species-specific capacity of protoplasma to tolerate severe water loss

Physiological processes proceed even at highdehydration levels

Tolerance mechanisms take over when tissues are no

longer protected by avoidance mechanisms

Drought tolerance usually found in xerophytes

(drought avoidance in mesophytes)

Tolerance aims at plant survival rather than plantgrowth

Definition of water potential (Ψ)

Quantifies the water status in plant systems

Chemical potential of water (µw) indicates the capacity of

water to do work

It is not feasible to measure the chemical potential

absolutely →→→→ potentials are referenced to a standard state (µ°w) set equal to 0, and calculated by difference(J mol-1)

By convention, water in this standard state ispure

at atmospheric pressureat same temperature and vertical height as the

water in the system of interest

Definition of water potential (Ψ)

Conversion of chemical potential of water (µw) to

water potential (Ψ) by dividing µw by the partial

molal volume of water (Vw; m3 mol-1):

W

o

WW

V

µµ −=Ψ

Since J mol-1 = N m mol-1, water potential can be

expressed in terms of pressurePascal (Pa) appropriate SI unit for pressure

1 MPa = 106 pascals = 10 bar

Total water potential (Ψt)

Central parameter of plant water relations

Describes the energy state of water at a given

point in the soil-plant-atmosphere continuum

(SPAC)

Plants may be considered as conduits for water

between humid soil and dry air

Water flows from points where it has more energy

content (higher water potential) to those with less

energy content (lower water potential)

Components of total water potential (Ψt)

2 equations describe the influence of various components on total water potential

DEMANDS come from the soil-plant-atmosphere continuum (equation 1):

(-) Ψt = (-) ΨS + (-) ΨG + (-) ΨF

whereΨS = substrate (soil) potential

ΨG = gravitational potential

ΨF = frictional potential

Components of total water potential (Ψt)

RESPONSE mechanisms in the plant adjust total water potential to the value preset by potential losses in the

soil-plant-atmosphere continuum (equation 2):

(-) Ψt = (-) Ψo + (±) Ψp

whereΨo = osmotic potential

Ψp = pressure potential

Measurement of total water potential (Ψt)

A) Pressure Chamber Technique

B) Thermocouple Psychrometer Method


Advantages• Method is

simple

fast

accurate

suitable for use in the field

Disadvantages• Method is

destructive

material-consuming


Measurement procedure

Plant organ (leaf, leaf strip, twig, root) cleanly cut from the plant

Immediately placed in the chamber head with the cut end

protruding through a flexible rubber gasket sealing the

chamber

Compressed air is led slowly into the chamber thus increasing the pressure inside gradually


Measurement procedure (cont.)

Pressure applied until water begins to return to the cut

surface

This 'balance pressure' required to force water back to

the cut end is equal in magnitude but opposite in sign to xylem tension that existed in the intact plant organ prior

to excision

Pressure Chamber (Plant Water Status Console)

3000 Series, SOILMOISTURE, Santa Barbara, California, USA (http://www.soilmoisture.com)

(A) release valve(B) stereo microscope(D) lid (E) inlet valve(F) foil(G) sealing (rubber

stopper)(L) light source(M) manometer (P) sample(S) pressure chamber

made from steel


Precautions:

1)Prevent water loss between sampling and

measurement

(cover the plant organ prior to excision with a

plastic bag or aluminium foil to minimize waterloss)

2)Prevent condensation of water on the sample

before measurement

3)Avoid recutting of petioles, twigs


Precautions (cont.):

4) Prevent evaporative water loss into the pressurechamber by keeping the sample enclosed in a plasticbag during measurement

5) Increase pressure slowly (0.003 to 0.005 MPa s-1) to

prevent large temperature changes in the chamber

6) Identify endpoint accurately

7) Use soft, elastic rubbers to avoid the crushing of

petioles or twigs


Measurement principle:

Total water potential may be determined by

measuring relative vapour pressure (equal to relative humidity) of water in the atmosphere

surrounding and in equilibrium with the sample


Measurement principle (cont.):

Total water potential is related to relative vapour

pressure by following equation:

where

Ψ = water potential (Pa)

R = universal gas constant (8.314 J mol-10 K -1)

T = absolute temperature (K)

Vw = molar volume of water (1.8 x 10-5m3 mol-1)

e/eo = relative humidity expressed as a fraction

oW e

e

V

RT= lnψψψψ


Measurement principle (cont.):Tissue sample is sealed in a small chamber containing a

(Peltier) thermocouple

After an equilibration period a cooling current is applied to the thermocouple in order to condense water on the junction

Amount of condensed water is proportional to tissue water potential

Water is allowed to evaporate causing a change in thermocouple output

Output is calibrated for water potential, using NaCl solutions


Advantages• Method is non-destructive

• Continuous measurements of plant water

status of intact plants or organs are possible

• Long-time observations during plant growth

or tissue dehydration can be done


Disadvantages

Main limitations when applied in field or greenhouse:

• Sensors are extremely sensitive to wind and radiation

• Very (!) careful isolation is a prerequisite to obtain

reliable results

• Method is quite time-consuming (slow equilibration

between sample and air in the thermocouplechamber)

2) Measurement of osmotic potential (ΨO)

A) Direct approach:Freeze-thawed or heat-killed

• plant tissue

(e.g. leaf discs or leaf strips)

• press sap(Attention: Dilution by apoplastic water)

measured with Vapor Pressure Osmometer

B) Indirect approach:

Pressure-volume (pV) curve technique

3) Measurement of pressure potential (Ψp)

Indirect approach:

• Difference between total water potential and

osmotic potential:

(±) Ψp = (-) Ψt - (-) Ψo

• Pressure-volume (pV) curve technique

Plant water content

Described by:

Relative water content (R)

FW: Fresh WeightSW: Saturation Weight

DW: Dry Weight

)(

)(

DWSW

DWFW= R

−−−−

−−−−

Relative water content

Very relevant physiological measure of plant water deficit

Estimates current water content of the sampled leaf

tissue relative to the maximal water content it can hold at full turgidity

Normal values of R range between 98% in turgid and

transpiring leaves to about 40% in severely desiccated and dying leaves

In most crop species typical R at about wilting is around 60 % to 70 %


Measurement protocol

All components of leaf water relations change during

the day as irradiance and temperatures change!

For 2 hours at and after solar noon, the change is very small

Time “window” for leaf sampling, unless a daily curve of R is of interest


Measurement protocol (cont.)

4 to 6 top-most fully expanded leaves taken from

different plants (of one treatment, genotype)

Samples placed in pre-weighed airtight (possibly also oven proof) vials

Vials immediately placed in a picnic cooler (ca. 10 to 15oC)


Measurement protocol (cont.)In the lab vials weighed to obtain sample fresh weight

Then samples

immediately hydrated to full turgidity (saturation)

weighed to obtain saturation weight

oven dried at 80oC for 24h

weighed (after being cooled down in a desiccator) to

determine dry weight

Plant water content

Described by:

Water saturation deficit (WSD)

R 1= WSD −−−−

)(

)(

DWSW

FWSW= WSD

−−−−

−−−−

Relative drought index

Index compares actual water saturation deficit (WSDact) with critical threshold value for water saturation deficit

(WSDcrit):

RDI = WSDact / WSDcrit

RDI = Rcrit / Ract

RDI = Ψact / Ψcrit

Critical threshold may refer to first visible signs of drought injuries

By comparing individuals of the same species in different

locations information on severity of drought can be gained

Pressure-volume (pV) curves

Describe the relationship between total water potential

(Ψt) and relative water content (R) of living organs

Equation

Ψo * V = constant

says that the product of osmotic potential and volume of solution should be a constant for any given amount

of osmotically active solutes in an ideal osmotic system

Decrease in cellular pressure with progressive water

loss is related to decrease in volume

Pressure-volume (pV) curves

Linear relationships may be obtained by converting either

potential or water content to its reciprocal

Ψo = 1 / V * constant

V = 1 / Ψo * constant

Typ I Transformation (ψt vs. R-1)ψt Total Water Potentialψo Osmotic Potentialψo(sat) Osmotic Potential at full saturationψo(tlp) Osmotic Potential at turgor loss point

Typ II Transformation (1/ψt vs. R)1 1 / Osmotic potential at full saturation2 1 / Osmotic potential at turgor loss point3 Relative water content at turgor loss point4 Relative symplast volume5 Total water volume at saturation6 Relative apoplast volume

Day courses of

Total water potential

Turgor potentialRelative water content

Pressure-volume (pV) curves (Typ I transformation) of two water regimes of

Triticum durum (Probstdorfer Grandur). � Controls: Plants were grown at

80% of soil water saturation. � Stress variant: Plants were grown at 33% of

soil water saturation. 63 days of drought stress duration.

Elastic adjustment

Epsilon is the elastic modulus for plant cells

Is a proportionality factor for change in Ψp that occurs when cell volume changes

Can be estimated from pressure-volume relationship:

ε = [∆ Ψp / ∆ R] * R

∆Ψp change in average turgor pressure of the tissueR relative water content

Epsilon is high in tissues with rigid cell walls: greater drop in Ψp per unit change in R occurs than in more elastic tissues

Turgor adjustment as displayed by pressure-volume

curves from control (�) and drought stressed (�) durum leaves. A: area difference caused by osmotic adjustment.

B+C: area difference caused by elastic adjustment.

Water use efficiency of productivity (WUEP)

Informative for ecological, agricultural, forestry purposes

Defined as:

WUEP [g DM . kg–1 H2O] =

Water requirement per unit of dry mass produced varies among species and varieties

Strongly dependent on individual state of plant development, plant density, environmental conditions,

water supply, evaporation

Organic dry matter productionWater consumption

Water use efficiency of productivity (WUEP)

Selection of species and varieties appropriate for growing

conditions in dry areas possible →→→→ amount of irrigation water regulated

Water use efficiency

decreases with increased water use increases with increased dry matter production

A plant adopting to drought by stomatal closure (water

saver) increases its WUE by decreasing transpiration, but

it simultaneously decreases efficiency by lowering its photosynthetic rate

Drought resistance and crop yield

Passioura proposed a general description of yield and water use which is widely accepted by agronomists:

Yield = T x WUE x HI

whereT = total seasonal crop transpiration

WUE = crop water use efficiencyHI = crop harvest index (ratio of economic yield

to total aboveground biomass)

selected methods of measuring drought stress in...

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