Electrolyte leakage after freezing is an indicator of leaf frost sensitivity and is related to climate, season and plant geographical distribution. Leaves of species from warmer regions and/or growing at warmer sites along a steep regional climatic gradient have shown greater frost sensitivity than those of species from colder regions and/or growing at colder sites within a regional gradient. The technique described here is based on the idea that when a cell or tissue experiences an acute thermal stress, one of the first effects is disruption of membranes, eliminating a cell’s ability to retain solutes such as ions. Ion leakage from a tissue can be easily assessed by measuring changes in electrolyte conductivity of a solution bathing the tissue. The technique is suitable for a wide range of leaf types (from tender to sclerophyllous) and taxa (monocotyledons and dicotyledons), and is not affected by cuticle thickness.
What and how to collect?
Collect young, fully expanded sun leaves with no sign of herbivory or pathogen damage. Deciding when to collect is more complicated. The answer will depend on the question being asked, although in most cases, collection should be standardised across taxa. Depending on the contrast of interest, collect foliage during the peak growing season (see Section 3.1), or preferably near the end of the season (see Special cases and extras below in the present Section) or in winter (for winter evergreen species). If a species grows along a wide environmental gradient, and the objective is an interspecific comparison, collect the leaves from the point of the gradient where the species is most abundant. If many species are considered, try to collect them within the shortest possible time interval, to minimise differences resulting from acclimation to different temperatures in the field. Collect leaves from at least five randomly chosen adult individuals of each species.
Storing and processing
Store the leaf material in a cool container until processed in the laboratory (see Section 3.1). Process the leaves on the day of the harvest, so as to minimise natural senescence processes. For each plant, with a cork borer cut four circular 5-mm-diameter leaf disks (to provide for two treatments using two disks each, see below within the present Protocol), avoiding the main veins. For needle-like leaves, cut fragments of the photosynthetically active tissue that add up to a similar LA. Rinse the samples for 2 h in deionised water on a shaker, then blot dry and submerge two disks (or their equivalent in leaf fragments) in 1 mL of deionised water in each of two Eppendorf tubes. Complete submergence is important. For each treatment (see below within the present Protocol), prepare as many replicates (one replicate being two tubes, each containing two disks or equivalent leaf fragments) as the number of plants sampled.
Apply the following two treatments, without any prior acclimation, to the two leaf disk orfragment samples in the respective tubes: (1) incubation at 20°C (or at ambient temperature, as stable as possible) for the control treatment, and (2) incubation at –8°C in a calibrated freezer, for the freezing treatment. Incubations should be for 14 h in complete darkness, to avoid light-induced reactions.
After applying the treatment, let the samples reach ambient temperature and then measure the conductivity of the solution. Do this by placing a sample of the solution from an Eppendorf tube into a standard previously calibrated conductivity meter (such as the Horiba C-172®, Horiba Ltd., Kyoto, Japan) and by recording the conductivity. Then place the Eppendorf tube in a boiling water bath for 15 min to completely disrupt cell membranes, releasing all solutes into the external solution, then re-measure its conductivity. Prior to immersion, puncture the cap of each Eppendorf tube to allow relief of pressure during boiling.
(1) Percentage of electrolyte leakage (PEL) – separately, for the frost treatment and the control for each individual plant replication, as follows:
PEL = (es / et) × 100,
where es is the conductivity of a sample immediately after the treatment, and et is its conductivity after boiling. High values of PEL indicate significant disruption of membranes, and thus cell injury; the higher the PEL, the greater the frost sensitivity.
(2) Corrected PEL – the PEL of the control treatment can vary among species because of intrinsic differences in membrane permeability, experimental manipulations and differences in injury when leaf disks orfragments are cut. To control for these and other sources of error, subtract the PEL of the control treatment of each replicate from that for the freezing treatment. Corrected PEL is thus
Corrected PEL = PEL in the freezing treatment
– PEL in the control treatment.
For calculating the mean, standard deviation or standard error for a species, the average corrected PEL for each individual plant replicate counts as one statistical observation.
Special cases and extras
(1) Applicability to different plant functional types The technique is not suitable for halophytes and succulents. It is not necessarily applicable to deciduous plants and hemicryptophytes, because their significant frost tolerance involves stems and buds rather than leaves. This tolerance could possibly be tested with sections cut from stems, although the reliability of this has not been investigated, to our knowledge, as it has been for leaves.
(2) Season of collection Because of the recognised wide occurrence of autumnal acclimation in frost tolerance, we recommend normally performing the procedure with leaves collected at or near the end of the growing season.
(3) Incubation with dry ice A different treatment, namely incubation at about –78°C (the temperature of dry ice), with the rest of the protocol the same as described above, can be used if a freezer whose temperature can be controlled at about –8°C is not available, or if one wishes to detect tolerance to the kind of severe frost that can occur at high latitudes or altitudes. It would not detect tolerance to merely mild frost.
(4) Acclimation The occurrence of acclimation to mild frost could be detected using a freezer at –8°C, on leaf samples collected on successive dates in summer and autumn.
(5) Sensitivity to high temperatures The same basic technique, with a modification in the treatment temperature, has been successfully applied to leaf sensitivity to unusually high temperatures (~40°C; see More on Methods below in the present Section).
(6) Chilling sensitivity is a physiological limitation that can be ecologically important in mountains at lower latitudes, and might be detected by this technique. It is usually tested for by incubation for 24 h or more at about +5°C, e.g. in an ordinary refrigerator. Alternatively, 0°C in an distilled water (or rain water) bath could be used, because this will not actually freeze plant tissue. A chilling-sensitive tissue would leak electrolytes after such incubation, whereas a chilling-tolerant tissue should not.
References on theory, significance and large datasets: Levitt (1980); Blum (1988); Earnshaw et al. (1990); Gurvich et al. (2002).
More on methods: Earnshaw et al. (1990); Gurvich et al. (2002).