Studies of post-stress repair in plants showed that the most clear-cut distinctions in cultivars' resistance levels are manifested when the extreme factor is so severe that it decreases the productivity of a sensitive cultivar ca. twofold (Udovenko 1979). This principle of choosing strpless conditions for the wheat cultivars used in this work was applied by Demchenko (1994), who showed that the nearly twofold decrease in the leaf length of the susceptible cultivar occurs after a 3.7-MPa osmotic-stress treatment for 2 days.
Those conditions, however, did not permit us to find any distinct differences between Saratovskaya 29 and Opal with respect to post-stress repair (Fig. 1). The normal content of PAI present in the stem meristem cells (non-stress conditions) was the same in both cultivars; as a result, only one cultivar, Saratovskaya 29, was used as a control (Fig. 1). Further work investigated the effects of various times of the 3.7-MPa treatment (4,6, and 8 days). Increasing the time of osmotic stress stopped leaf growth; the upper part of the lamina and the wheat-root coleoptile began to wilt. On the eighth day of stress, the coleoptile and the leaves dried out; only the shoot apical meristem and the adjacent basal leaf meristems remained viable. At first the seedlings looked lifeless, but at post-stress repair the leaves renewed their growth, indicating the preserved viability of the apex. The longer was the stress time, the later commenced the re-growth of the youngest leaf under these conditions and the lower was the re-growth rate. The cuitivars did not differ in the re-growth dynamics of the youngest leaf (Fig. 2).
The differences in the content of PAI during repair were greatest after a 3.7-MPa osmotic-stress treatment for 8 days (Fig. 3). In particular, we noted that the normal content of PAl in both cultivars increased monotonically during ontogenesis (Fig. 3). The content of PAI in the stem meristems at 2 h after osmotic stress increased ca. fourfold (Saratovskaya 29) and ca. eightfold (Opal), as compared with the control. During repair. PAI content decreased to the control value in Saratovskaya 29 and below the control value in Opal (Fig. 3).
Our results show that the stem apical cells have a higher degree of resistance than do the cells of differentiated wheat tissues. As Gudkov (1985) notes, the reliability resources of the apical meristem are determined not by resistance of single cells but by cooperative interaction of individual meristem cell populations at various cell cycle phases. As with all eukaryotes, the cell cycle in plants is regulated through the G1/S and G2/M transitions. The transitions serve as control points at which cell division is synchronized under the action of adverse factors (Doerner 1994; Dewitte and Murray 2003). In particular, the synchronizing action of osmotic stress on the cell population of the plant apical meristem is associated with a change in the phytohormone balance (Bray 1993), leading to a change in the activity of cyclin-dependent kinases (CDKs) (Schuppler et al. 1998). One can assume that PAl's function is similar to that of cdc2-activating kinases, among which is the protein kinase p34cdc2 , regulating the entry of eukaryotic cells into mitosis (John et al. 1989; Dudits et al. 1998). A sharp increase in the PAI content immediately after stress seems to reflect a synchronous entry of meristematic cells into mitosis (Figs. 1 and 3). We note that the content of PAl depends on the severity and duration of stress, possibly reflecting the degree of cell division synchronization and the genotypic differences between resistant and sensitive cultivars.
At repair, Saratovskaya 29, having a higher degree of reliability of the cell homeostasis in the stem apex, showed a near-normal PAI content, whereas in Opal the PAI content decreased twofold as compared with the control. This possibly reflects a disturbance in the mechanisms regulating the entry of individual meristem cell subpopulations into proliferation (Fig. 3). Thus, determination of the content of PAI in the stem apex enabled us not only to assess the functional state of cells of the stem apical meristem, but also to reveal the degree of their reliability in the post-stress regeneration of plant tissues and organs.
Our data suggest that the resilience of wheat cultivars to extreme factors can be predicted and assessed at the cellular level with our immunochemical test system for PAI. More work to explain the nature of PAI and the molecular mechanisms of its expression will aid in obtaining a more penetrating insight into the regulatory mechanisms underlying cell division and further cellular differentiation in plant apical meristems under the influence of various environmental factors.
Acknowledgments
We thank Dmitry N. Tychinin (IBPPM RAS) for the English translation of the manuscript.