The Cell Membrane Stability (
A major impact of
plant environmental stress is cellular membrane modification, which results in
its perturbed function or total dysfunction. The exact structural and
functional modification caused by stress is not fully understood. However, the
cellular membrane dysfunction due to stress is well expressed in increased
permeability and leakage of ions, which can be readily measured by the efflux
of electrolytes. Hence the estimation of membrane dysfunction under stress by
measuring cellular electrolyte leakage from affected leaf tissue into an
aqueous medium is finding a growing use as a measure of
The protocol
The general protocol involves the application
of stress to the leaf after it has been subjected to hardening, followed by the
measurement of electrolyte leakage using the conductometric method. The most
common application is for heat tolerance and therefore the initial detail is
given for heat stress (see schematic figure).
1. The plant must be
exposed to moderate heat stress for at least 24h before the test in order to
allow for hardening (acclimation). The capacity for hardening is a major
component of the capacity for tolerance. Hardening can be achieved in the
natural field environment, if heat stress occurs, or in the greenhouse or a
programmed chamber. Exposure of intact plants for 24h to 320C (cool
season plants) to 360C (warm season plants) is sufficient, even at
low light.
2. Leaf discs or
pieces of leaf tissue cut with scissors or even whole small leaves are detached
and placed in standard glass vials that can accommodate an conductivity
electrode (see below). The total area of leaf material per vial is about 15 to
25 cm2. The exact area is not important and it does not have to be
the same for all vials. The sample is then washed for 2-3 times with de-ionized
water. The water is drained off but samples remain wet so that they would not
desiccate. In the case of screening, at least 10 vials (samples) are prepared
for each genotype. In that case 5 pairs are taken from five different plants
(replicates). For each pair, one vial is designated as treatment (T in Figure)
and the other as control (C in Figure).
3. The treatment
vials are subjected to the heat stress treatment in vitro. (Treatment can also
constitute of low temperature in the case of chilling tolerance). They are
placed in racks and covered (not stoppered) with ‘Saran’ wrap so as to drying
the samples. Racks are placed in thermostated water bath so that the leaf
samples will be completely below the water surface level. Temperature is set to
a predetermined stress (treatment) temperature and the samples remain in the
bath for 1h. The control vials are placed in a rack, covered with Saran wrap
and placed at root temperature (18-250 C). The treatment temperature
should be such that it will result in average population
4. After treatment
20cc of deionized water is added to each vial making certain that all leaf
materials are submerged. All vials are then placed for incubation at about 100C
(typically, on the lowest refrigerator shelf) for 24h. After incubation samples
are equilibrated for 1h to room temperature and the conductivity of the medium
is measured by inserting a conductivity electrode into each vial. Care is taken
to ascertain that after the electrode is taken out of the vial all leaf samples
remain submerged in the medium and not has been “exported” on the
electrode.
5. All vials covered
with Saran wrap or plastic sheet are placed in an autoclave for 15 min to kill
all tissues. Conductivity of all samples is measured after samples are
equilibrated to room temperature.
6. Calculation: see
Figure - where T1 and T2 are treatment conductivities before and after
autoclaving and C1 and C2 are the respective control conductivities. Calculated
results are often better when each T value is calculated against the average of
all C values for the given entry.
When the method is adopted for
assessing drought resistance as a measure of the desiccation tolerance of cell
membranes, plants of all materials must
be stressed to the same relative water content* (RWC) of about 70%
(depending on crop species) before being sampled (‘treatment’ samples) into
stoppered vials and brought to the lab. Samples should not desiccate between
sampling and washing. Control samples are taken from similar leaves of well
water plants at RWC close to 98%. Once washed, 20 cc of deionized water is
added to all samples and they are directly placed for 24h incubation period (#4
above). The remaining procedure is as described above. An example of such work is given in the
abstract below (our Reference Database ID number 4793).
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