CHAPTER 14

DETERMINATION OF RELATIVE WATER

CONTENT

Luís González and Marco González-Vilar

Depto Bioloxía Vexetal e Ciencia do Solo. Universidade de Vigo. Spain

INTRODUCTION

Tissue water content may be expressed in several ways, including the

amount of water per unit dry or fresh weight and per unit weight of water
at full hydration. Fresh weight seems to be the less accurate of them to
measure tissue water content because is highly influenced by changes in
tissue dry weight (Turner, 1981).
The relative water content (RWC) stated by Slatyer in 1967 is 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. Thus there are a relationship between RWC
and water potential (Figure 1). This relation varies significantly according
to nature and age of plant material.
The RWC express the water content in per cent at a given time as
related to the water content at full turgor:

The procedure to get full saturation needs to reach constant weight in

the tissue. Young leaves, which are still suffering expansion, absorb water
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M.J. Reigosa Roger, Handbook of Plant Ecophysiology Techniques, 207–212.
© 2001 Kluwer Academic Publishers. Printed in the Netherlands.
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for a substantially longer period (usually several days). Young tissue is
respiring up and consumes part of its dry weight. This can result in a
significant error. To avoid this problem it is necessary to reduce the time
for saturation of the tissue by reducing the sample size as much as you can
manage it (weigh, dry...) and keep away the sample from physiological
activity by physical inhibition of growth and respiration during
determination. Saturation of the tissue portions at 4°C inhibits
satisfactorily the growth.

PHYSIOLOGICAL ASPECTS

Water makes up most of the mass of plant cells. In each cell, cytoplasm
makes up only 5 to 10% of the cell volume and the remainder is a large
water-filled vacuole.
There is a strong correlation between alterations in leaf protoplast
volume and changes in leaf photosynthetic activity. Sometimes decreases
in tissue water content may be more important than decreases in water
potential or pressure potential in terms of influencing growth. Then, it is
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not enough to study water relations in plants to know only water content
but is often important to measure both, tissue water content and water
potential for plants growing under field conditions. Plants in natural
environments can support different environmental conditions that interact
with its genetics characteristics. Diploid Dactylis glomerata showed a
smaller RWC than tetraploid citotype (González-Vilar et al.,
unpublished). Values for tetraploid plants were close to 80 and diploid
plants presented values round 70 with significant differences at 0.05 level.
This could imply that these plants suffered marked metabolic changes
with ceasing of photosynthetic activity, respiration increment and proline
and abscisic acid accumulation.
Small discs or tissue pieces are used to determine a great variety of
physiological processes in plants (photosynthesis, enzymatic activity,
pigments content...) although it should be taken in account the possible
heterogeneity of the leaf to get a good correlation between RWC and
some physiological processes.
In the process of tissue saturation there are two phases of water
uptake. Initially there is a rapid uptake of water to satisfying the water
deficit of the tissue. Later there is a slower uptake of water caused by
growth and other physiological factors, difficult to define, how it takes
place. Young tissues are more sensible to this second step (Figure 2).
Samples with a long saturation time, like young tissues, need some
kind of data correction for growth.
RWC is a measure of the relative cellular volume that shows the
changes in cellular volume that could be affecting interactions between
macromolecules and organelles. As general rule, a RWC among 100-90 %
is related to closing of the stomata pore in the leaf and a reduction in the
cellular expansion and growth. Contents of 90-80 % are correlated with
changes in the composition of the tissues and some alterations in the
relative rates of photosynthesis and respiration. Levels of RWC below 80
% imply usually water potential of the order of -1.5 MPa or less, and this
would produce changes in the metabolism with ceasing of the
photosynthesis, increment of the respiration and proline and abscisic acid
accumulation.
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MEASUREMENT OF RELATIVE WATER CONTENT

For the previously exposed reasons the ‘piece extrapolation routine’ is the
most suitable method to assess the plant RWC. The following procedure
was modified from Sharper et al. 1990.
1. Weigh and label with a number 0.2 ml Eppendorff tubes and label
with the same numbers 1.5 ml Eppendorff tubes. (Take into account
the number of replicates. It will depend on the species but for this
technique you must use at less five replicates for sample).
2. Fill with cold de-ionised water the 1.5 ml tubes.
3. Remove the youngest developed leaf of the plant. (If the sample is
collected in the field or greenhouse and is not processed immediately
you must place it in sealed plastic bags and transfer it to a cooled
insulated container to prevent growth and reduce evaporation of
water from the cut tissue).
4. Put four sections of a same leaf in a tube (choose
homogeneous pieces and avoid apical and basal parts of the leaf.
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Those fractions would have really different expansion rate). Seal the
tubes and place it in ice to prevent growth and evaporation.
5. Weigh the tubes with tissue sections inside to give a value for tissue
fresh weight (FW).
6. Once every sample has been weighed, transfer the piece of leaf to a
1.5 ml Eppendorff tubes containing de-ionised water with the same
number of the small tube.
7. Tubes should be placed in ice, and left for 4 hours in a fridge to allow
the tissue taking up water.
8. Take away the tissue sections from the de-ionised water. Remove
carefully the excess of water on the leaf surface with tissue paper.
9. Transfer every sample to their original tubes (0.2 ml) and reweigh.
This gives a measure of fully turgid fresh weight (TFW).
10. Open the tubes, and place them in a stove to 70 °C during at less 48
hours.
11. After drying reweigh the tissue to obtain a value for dry weight
(DW).

Relative water content (RWC) can then be calculated using the

following equation:

REFERENCES AND FURTHER READINGS

González-Vilar M., González L., Reigosa M.J. Ecophysiological responses to light

intensity and water stress in two citotypes of cocksfoot (Dactylis glomerata L.) in
Galicia, NW of Spain. Submitted.
Larcher W. Physiological Plant Ecology. Berlin: Springer-Verlag, 1995
Lawlor D.W. "The Effects of Water Deficit on Photosynthesis. In Environment and Plant
Metabolism. Flexibility and Acclimation. N. Smirnoff, ed. Oxford: BIOS Scientific
Publishers, 1995
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Sharp R.E., Hsiao T.C., Silk W.K. Growth of the maize primary root at low water
potentials. II. Role of growth and deposition of hexose and potassium in osmotic
adjustment. Plant Physiol 1990; 93:1337-1346
Taiz L., Zieger E. Plant Physiology. Sunderland: Sinauer Associates, 1998
Turner N.C. Techniques and experimental approaches for the measurement of plant water
status. Plant Soil 1981; 58:339-366