In this connection, it was interesting to analyze heat stable COR-proteins of maize; to demonstrate similarity of maize COR-polypeptides to those of freezing-tolerant cereals; to correlate heat stable polypeptide composition of studied cereals with their cryotolerance.
Three-day-old seedlings of maize grown at 28 C were transferred to 10 C for three days to induce acclimation. For comparison, three-day-old seedlings of wheat and rye grown at 22 C were used. They were acclimated at 4 C for three days. Unhardened seedlings were used for reference. The tolerance assessment of control and hardened cereals was conducted by electrolyte leakage techniques (Palta, Plant Physiol., 60:393-397, 1977). In order to study changes in synthesis of heat-stable proteins characteristic of cold acclimation state, thermostable proteins were extracted from seedlings and separated by SDS gel electrophoresis (Stupnikova, Russian J. Plant Physiol., 45:744-748, 1998).
The data show that the amount of leaked electrolytes from control and hardened seedlings of all crops do not differ considerably (Fig. 1).
Figure 1. Relative freezing tolerance of maize and other cereals (measured by electrolyte leakage techniques). Relative admittance (% to admittance of killed tissue electrolyte) was assessed in control (1), hardened (2), control and freezing at -6 C (3), hardened and freezing at -6 C seedlings (4). Means and standard errors of the means are shown.
Freezing at -6 C resulted in increased ion leakage. It reflects perturbation of membrane semipermeability by freeze-thaw stress and is indicative of cell viability. As expected, the maize plants showed higher electrolyte leakage (that is, more freezing injury) in comparison with more tolerant cereals. However, all hardened plants were more tolerant than control, and were characterized by lower amounts of leaked electrolytes (Fig. 1). Thus, chilling-sensitive maize, like chilling- and freezing-tolerant cereals, also developed cryotolerance during hardening.
In this connection it was interesting to study proteins related to cold acclimation state of maize and to compare them with other cereals. The total and heat-stable polypeptides were analyzed. It was found that total protein composition of control and hardened seedlings did not differ from each other (data are not presented). At the same time, the difference in heat stable proteins was strong (Fig. 2). The acclimated maize seedlings accumulated thermostable polypeptides with mol. weights 50, 46, 35, 31, 27, 24, 22 kD, from which those with mol. weights 50, 35, 27, 22 kD apparently were synthesized de novo. Conversely, the more tolerant plants accumulated high- and medium molecular proteins.
Figure 2. Heat stable proteins from control (1, 3, 5) and hardened (2, 4, 6) seedlings of maize (1, 2) and other cereals (rye — 3, 4; wheat - 5, 6). Mol. wts of maize proteins are indicated on the left. Electrophoresis was run in 13 % SDS-PAGE.
Thus, the medium- and low molecular COR-proteins were characteristic of hardened state of maize and differed from those of tolerant cereals. The accumulation of medium- and low molecular polypeptides during cold treatment was revealed also in such chilling-sensitive plants as soybean (Boudet, 1993, NATO ASI Series, 116, 725-739). Possibly this trait differentiates the maize (and other chilling-sensitive plants) adaptation mechanism from that of chilling- and freezing tolerant cereals.
The research was funded by the Russian Foundation of Basic Research (project 99-04-48121).
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