With this objective, seedlings of maize were germinated at 27 C, seedlings of wheat and rye at 22 C for 3d in darkness. To determine whether exogenous application ABA affected cryotolerance, seedlings were exposed to 1000 µM ABA with the addition of Tween-20 (0.1% solution) for one day. The tolerance assessment of control and ABA treated cereals was conducted by electrolyte leakage technique (Palta, Plant Physiol., 60:393-397, 1977). In order to study changes in heat-stable protein synthesis during ABA exposure, proteins were labeled in vivo with 14C-leucine separated by SDS gel electrophoresis and the derived fluorograms were studied.
The study of response to freezing by electrolyte leakage techniques revealed that freezing at -6 C resulted in an increase of ion leakage from tissues of all cereals (Fig. 1). This points to perturbation of membrane integrity that usually results in decreased survival. Exogenous application of ABA enhanced plant cryotolerance based on the ion leakage levels. As expected the maize plants showed higher electrolyte leakage during freezing (that is more freezing injury) in comparison with more cold-tolerant cereals. It should be noted that the amount of leaked electrolytes from unexposed control and ABA treatment seedlings of all crops are essentially identical (Fig. 1).
Figure 1. Relative freezing tolerance of maize and other cereals (measured by electrolyte leakage techniques). Relative admittance (% admittance of killed tissue electrolyte) was assessed in control, ABA treatment, control and freezing at -6 C (control, freezing), ABA treatment and freezing at -6 C seedlings (ABA, freezing). Means and standard errors of the means are shown.
The ABA augmentation of maize cryotolerance (as is the case with tolerant cereals) was accompanied by alteration of heat stable protein synthesis (Fig. 2). It is interesting that ABA treatment produced more proteins than low temperature acclimation (Stupnikova et al., in the same issue). ABA treated seedlings of maize synthesized a number of new polypeptides with mol. weights 219, 214, 178, 66, 37, 30, 28 and 26 kD; and accumulated proteins with mol. weights 54, 46, 42, 41 and 32 kD. The concentration of all polypeptides detected was greatly increased during hormone application (Fig. 2). Thus, whereas ABA treated seedlings accumulated 13 ABA-inducible proteins, cold adapted plants accumulated only seven. This also applies to more tolerant cereals (for wheat and rye). Moreover, hormone exposed seedlings of maize, unlike cold acclimated ones, synthesized high molecular weight polypeptides, different from those of wheat and rye.
Figure 2. Heat stable ABA-inducible proteins from control (K) and ABA-treatment (ABA) seedlings of maize and other cereals (rye and wheat). Mol. wts of maize proteins are indicated on the right. Electrophoresis was run using 13 % SDS-PAGE.
It appears that increase of maize cryotolerance through exogenous ABA (as is the case with wheat and rye) is associated with both increasing the amount of protein (not only cold-regulated polypeptides) and accumulation of high molecular weight protein.
The research was funded by the Russian
Foundation of Basic Research (project 99-04-48121).
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