Chapter 5. Leaf relative water content
Daniel Mullan and Julian Pietragalla
The relative water content (RWC; or ‘relative turgidity’)
of a leaf is a measurement of its hydration status (actual
water content) relative to its maximal water holding
capacity at full turgidity. RWC provides a measurement
of the ‘water deficit’ of the leaf, and may indicate a
degree of stress expressed under drought and heat
stress. RWC integrates leaf water potential (ψ; another
useful estimate of plant water status) with the effect of
osmotic adjustment (a powerful mechanism of conserving
cellular hydration) as a measurement of plant water
status. A genotype with the ability to minimize stress by
maintaining turgid leaves in stressed environments will
have physiological advantages (e.g., this allows turgor
dependent processes such as growth and stomatal activity,
and to protect and maintain the photosystem complex).
Leaf RWC is easily and simply measured, without the
need for expensive specialized instruments. Fresh leaf
samples of field grown crops are first weighed then
placed in water, chilled overnight, and re-weighed
before being oven dried and weighed a final time. The
relative difference in the water content of the leaf
samples provides a quantitative measure of their infield hydration status. Trials can be rapidly screened for
genotypes which maintain high leaf RWC values during
water deficit stress, and vice-versa. Sources of error in
the estimation of RWC can be summarized as: (i) change
in dry weight (mainly due to respiratory losses), (ii)
increases in water content in excess of full turgidity, and
(iii) water accumulation in intercellular spaces (Barrs and
Weatherley, 1962).
heat trials sampling is performed at early grain-filling as
an assessment of stress adaptation.
Note that in severely stressed conditions plants will
senesce quickly and measurements should be taken
earlier. Sequential measurements throughout this period
will allow assessment of changing leaf RWC.
Number of samples per plot
Take six leaf samples from different plants in each plot.
Procedure
The following procedure describes a whole leaf
technique (modified from Stocker, 1929). Alternatively, a
leaf disc technique may be used.
Take the following equipment to the field:
• Scissors
• Labeled sample tubes (one per plot)
• Cool box
And, required in the laboratory:
•
•
•
•
Semi-analytical balance (to 3 d.p.)
Distilled water
Blotting paper
Oven
Advice on taking measurements
Samples can be taken under most environmental
conditions. However, it is important that the plant
surfaces are not wet from dew, irrigation or rain.
Select the top-most fully expanded leaf receiving
sunlight, typically the flag leaf, or select leaves down
the canopy profile. Leaf sampling should be achieved
as quickly and efficiently as possible, and use the shade
of the sampler’s body when cutting and holding the
samples. A field assistant is often useful.
Time of day
All weights should be recorded to the nearest milligram
(3 d.p.).
Site and environmental conditions
The optimal time for sampling is at solar noon ±2 hours;
as this is the most stable time of day with respect to
irradiance and temperature and their effect on leaf water
relations. A daily curve of leaf RWC can be obtained by
taking measurements throughout the day.
Plant developmental stage
Samples can be taken at any developmental stage and/or
at regular intervals from the start of tillering to late grainfilling, depending on the experimental objectives/timing
of peak stress. For instance, in terminal drought and/or
Preparations
1. Number and weigh empty sample tubes (tubeW;
Figure 5.1A).
Field measurements
2. Select and cut six fully expanded flag leaves from
randomly chosen plants in each plot (Figure 5.1B).
3. Cut off the top and bottom of all the leaves
together, and any dead or dying tissue (Figure 5.1C),
Canopy temperature, stomatal conductance and water relation traits
25
to leave a 5 cm mid-section, and immediately place
into the pre-weighed tubes and seal the lid (so that
there is no moisture loss/gain from the system).
9. Take the leaf samples out of the tube, and quickly
and carefully blot dry with paper towel (Figure 5.1F).
4. Immediately place the tube into a cooled, insulated
container (at around 10°C–15°C; but not frozen).
11. Place the leaf samples in a labeled envelope and dry
at 70°C for 24h, or until constant mass (Figure 5.1G).
5. Take the tubes to the laboratory as soon as possible.
12. Reweigh the leaf samples (DW; dry weight).
Laboratory measurements
Data and calculations
6. Weigh all sample tubes (tubeW+FW).
First, obtain the fresh weight (FW) of the leaf samples:
7. Add 1 cm of distilled water to each tube (Figure
5.1D).
FW = tubeW+FW - tubeW
8. Place the sample tubes in a refrigerator (at 4°C in
darkness) for 24h (for leaves to reach full turgor)
(Figure 5.1E).
(A)
(D)
10. Weigh the leaf sample (TW; turgid weight).
Then calculate the leaf RWC:
Leaf RWC (%) = ((FW-DW) / (TW-DW)) × 100
Equation 5.2
Where: FW = fresh weight; TW = turgid weight;
DW = dry weight.
(B)
(E)
Equation 5.1
(C)
(F)
(G)
Figure 5.1. Measuring the leaf relative water content: (A) weighing empty tubes; (B) select and cut leaves in the field; (C)
cut the top and bottom of all leaves together; (D) tube containing leaf samples filled with 1 cm of distilled water; (E) sample
tubes in the refrigerator; (F) carefully blot dry the turgid leaf samples; and, (G) dried leaf samples.
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Physiological Breeding II: A Field Guide to Wheat Phenotyping
Worked example
Table 5.1. Calculating the leaf relative water content of severely droughted flag leaves during early grain-filling.
Plot
1
2
3
Tube
weight (g)
12.065
12.111
12.022
Tube weight +
fresh weight (g)
12.730
12.920
12.833
Fresh weight (g)
0.665
0.809
0.811
Turgid weight (g)
0.985
1.322
1.086
Dry weight (g)
0.292
0.350
0.345
Leaf RWC (%)
53.8
47.2
62.9
Typical values of RWC range between 98% in turgid and transpiring leaves to about 40% in severely
desiccated and senescing leaves; leaf RWC at wilting is around 60–70%.
Troubleshooting
Problem
Solution
Lower than expected fresh weight values.
The transfer of cut leaf samples to the sample tube is too
slow, causing the leaves to dehydrate in the air. Leaf sampling
should be achieved as quickly and efficiently as possible,
and use the shade of the sampler’s body when cutting and
holding the samples.
Higher than expected turgid weight values.
Blot drying of leaf samples after soaking is not
sufficient: ensure to dry samples thoroughly of all
surface moisture using dry absorbent tissue.
Do not completely fill the tubes with water as this will overestimate the turgid weight by filling the inter-cellular
spaces with water.
References
Useful references
Barrs, HD. and Weatherley, PE. (1962) A re-examination of the
relative turgidity technique for estimating water deficit in leaves.
Australian Journal of Biological Sciences 15, 413–428.
Stocker, O. (1929) Das Wasserdefizit von Gefässpflanzen in
verschiedenen Klimazonen. Planta 7, 382–387.
Hewlett, JD. and Kramer, PJ. (1963) The measurement of water
deficits in broadleaf plants. Protoplasma 57, 381–391.
Smart, RE. and Bingham, GE. (1974) Rapid estimates of relative water
content. Plant Physiology 53, 258–260.
Turner, NC. and Jones, MM. (1980) Turgor maintenance by osmotic
adjustment: a review and evaluation In: Turner, NC. and Kramer,
PJ. (Eds.) Adaptation of plants to water and high temperature
stress, pp: 87–103.
Weatherley, PE. (1950) Studies in the water relations of the cotton
plant. I. The field measurement of water deficits in leaves. New
Phytologist 49, 81–97.
Canopy temperature, stomatal conductance and water relation traits
27