selected methods of measuring drought stress in...
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(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
A) Pressure Chamber Technique
Advantages• Method is
simple
fast
accurate
suitable for use in the field
Disadvantages• Method is
destructive
material-consuming
A) Pressure Chamber Technique
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
A) Pressure Chamber Technique
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
A) Pressure Chamber Technique
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
A) Pressure Chamber Technique
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
B) Thermocouple Psychrometer Method
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
B) Thermocouple Psychrometer Method
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
B) Thermocouple Psychrometer Method
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
B) Thermocouple Psychrometer Method
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
B) Thermocouple Psychrometer Method
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 %
Relative water content
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
Relative water content
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)
Relative water content
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
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)
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