LEAF RELATIVE WATER CONTENT (RWC)
Relative water content (RWC) is the appropriate measure of plant water status in terms
of the physiological consequence of cellular water deficit. While water potential as an
estimate of plant water status is useful in dealing with water transport in the soil-plantatmosphere continuum, it does not account for osmotic adjustment (OA). OA is a
powerful mechanism of conserving cellular hydration under drought stress and RWC
expresses the effect of OA in this respect. Hence RWC is an appropriate estimate of plant
water status in terms of cellular hydration under the possible effect of both leaf water
potential and OA.
The method has long been in use, even before it re-examination (Barrs and
Weatherley, 1962), when it was also termed ‘relative turgidity’. Recently it gained
increasing appreciation. Some exemplary uses and discussion of RWC in physiological as
well breeding research can be found in this web site ‘Reference Database’ ID numbers
1903, 2181, 3418, 3813, 3883, 4793 and especially a nice analysis in 3940. Searching
for keyword RWC will provide many more studies where this parameter has been used.
The method is simple. It estimates the current water content of the sampled leaf tissue
relative the maximal water content it can hold at full turgidity. It is a measure of water
deficit in the leaf. Normal values of RWC range between 98% in turgid and transpiring
leaves to about 40% in severely desiccated and dying leaves. In most crop species the
typical RWC at about wilting is around 60% to 70%, with exceptions.
The protocol
All components of leaf water relations change during the day as irradiance and
temperatures change. For no more than two hours at and after solar noon, the change is
very small. This is the time “window” for leaf sampling, unless a daily curve of RWC is of
interest.
Usually, between 4 to 6 samples (replications) are taken from a single treatment or
genotype. Each sample represents a different plant, if possible. Top-most fully expanded
leaves are sampled, unless interested in profiling leaves on the plant.
1.
In large broad-leaves (sunflower, cotton, etc) leaf discs are cut from the leaves, to
obtain about 5-10 cm2/sample. Sample size does not have to be the same for all
samples. Avoid large veins. Leaf discs should be large enough (around 1.5 cm in
diameter) so as to reduce the area of cut leaf surface/sample. Various leaf disc cutters
were designed by laboratories and might be available commercially. Alternatively a sharp
cork borer may be used, cutting the leaf over a piece of dense rubber or a large rubber
stopper. It is important that sampling will proceed quickly.
2.
In smaller composite leaves (groundnuts, alfalfa, clover, chickpeas) several leaflets
make up a fast and convenient sample.
3.
In cereals, a sample may constitute of a mid-leaf section of about 5-10 cm2 cut with
scissors. With larger leaves such as maize or sorghum a section measuring, say, about
1x7 cm can be cut with scissors from the area between the mid-vein and the edge.
Each sample is placed in a pre-weighed airtight (possibly also oven proof) vial. Cereal
leaf sample should be placed in a vial slightly longer than the sample, with its basal part
to the bottom. Vials should be immediately placed in a picnic cooler (around 100C-150C)
but not frozen on ice. . Samples should reach the lab as soon as possible. This is why leaf
sampling should be done quickly and it is important to enlist as much help as possible for
the job.
In the Lab vials are weighed to obtain leaf sample weight (W), after which the sample
is immediately hydrated to full turgidity for 4h under normal room light and temperature.
Some prefer to hydrate samples on the lower shelf of a lab refrigerator (about 10 0C).
Samples 1 and 2 above are reydrated by floating on de-ionized water in a close petri
dish. Sample 3 above receives water into the vial to a level of 1-2 cm after which the vial
is capped.
After 4 hours the samples are taken out of water and are well dried of any surface
moisture quickly and lightly with filter paper and immediately weighed to obtain fully
turgid weight (TW). Samples are then oven dried at 800C for 24h and weighed (after
being cooled down in a desiccator) to determine dry weight (DW). All weighing is done to
the nearest mg. Calculation:
RWC (%) = [(W-DW) / (TW-DW)] x 100,
Where,
W – sample fresh weight
TW – sample turgid weight
DW – sample dry weight.
With good and careful work the method should normally result in about 2% to 3% of
RWC being a statistically significant difference between treatments.
Reference to the original method paper
Barr, H.D. and Weatherley, P.E. 1962. A re-examination of the relative turgidity
technique for estimating water deficit in leaves. Aust. J. Biol. Sci. 15:413-428.
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Relative water content (RWC)
RWC represents a useful indicator of the state of water balance of a plant,
essentially because it expresses the absolute amount of water, which the plant requires
to reach artificial full saturation (González and González-Vilar, 2001).
The RWC, stated by Slatyer in 1967, express the in percentage the water content
at a given time and tissue as related to the water content at full turgor:
RWC = (FW-DW)/(TW-DW)
The following procedure was modified from Sharper et al. (1990):
FW = fresh weight
- collect leaf pieces from 3-4 leaves
- wrap immediately in saranwrap to minimize evaporation, store in dark
- weight the leaf pieces to give a value for tissue FW
- immerse the leaf pieces in de-ionised water overnight and keep away the samples from
physiological activity by physical inhibition of growth and respiration (place in fridge at
darkness)
TW = turgid weight
- next day
- blot leaf pieces to dryness
- reweight and place in a dryer at 60C (24hrs)
DW = dry weight
- reweight and calculate RWC
Small discs or tissue pieces are used to determine a great variety of physiological
processes in plants (photosynthesis, enzymatic activity, pigments content, etc.),
although it should be taken in account the possible heterogeneity of the leaf and canopy
to obtain a good correlation between RWC and some physiological processes.