| As a typical response to abiotic and biotic stress, most plants up-regulate the expression of dehydrins(DHNs), which are group 2 of late embryogenesis abundant(LEA) proteins. These proteins are considered to participate in plant environmental stress tolerance. All DHNs contain at least one K-segment, which is believed to play a significant role in DHN function by forming an amphipathic helix.WZY2, an YSK2-type DHN gene, was isolated from the wheat cultivar Zhengyin 1; this gene could be induced by low temperature, anoxia, indoleacetic acid, methyl jasmonate, abscisic acid, and gibberellin treatments. K1, K2, Y plus S, or K1 plus K2 were removed from full-length WZY2(YSK2 type) to generate the truncated derivatives ΔK1, ΔK2, ΔYS, and ΔK1K2, respectively. In vivo assays of Escherichia coli(E. coli) viability enhancement, in vitro lactate dehydrogenase(LDH) activity protection and ex vivo protein aggregation prevention assays were used to evaluate function of WZY2 and its truncated derivatives. Comparison of WZY2, DHN14 and WCOR80 was also indicating the importance of K-segment. Furthermore, the relative expression of WZY2 gene and WZY2 protein in shoot and root of Zhengyin 1(winter wheat) and Chinese spring(spring wheat) revealed difference of cold stress tolerance in these subjects. The main results obtained as following:WZY2 was essential for the maintenance of the cell survival rate under freezing, thermal and peroxide stresses; the order of WZY2 metal ion tolerance was Ni2+>Zn2+>Co2+>Cu2+≈Mn2+; this DHN provided an appropriate environment to stabilize LDH enzyme as molecular shield; and protein was localized in the nucleus and cytoplasm, showing the effect of preventing protein aggregation under low temperature stress. The presented data indicate that WZY2 exerts its function as a protectant.The K-segment is a major functional component of this DHN. The proteins with two Ksegments(WZY2 and ΔYS) had the most significant impact on improving stress tolerance in all the experimental systems employed in this study. ΔYS performed in a manner similar to WZY2 in maintaining bacterial growth, enhancing LDH activity and preventing protein aggregation. The recombinant proteins that contained one K-segment(i.e., ΔK1 and ΔK2) had complex effects, and ΔK1 and ΔK2 had similar effects that were smaller than those of WZY2 and ΔYS both in vitro and in E. coli cells. ΔK1 could not be observed in wheat leaf epidermal cells, implied the necessity of its existence. Although the recombinant protein with no Ksegment(ΔK1K2) exhibited higher LDH activity protection efficiency than the controls, this was different with other assays.Under low temperature, WZY2 and WZY2 expression accumulation discrepancy occurred in winter wheat(cold-tolerant) and spring wheat(cold-sensitive). The relative expressions of WZY2 in winter wheat(aboveground and underground parts) reached the peaks with steadily rising trend. In spring wheat WZY2 upregulated expression in 24-48 hours, after which expression decreased rapidly in shoot and slowly in root. The relative expressions of WZY2 in winter wheat was higher than that of spring wheat. WZY2 protein expression in shoots of two cultures was higher than the roots. In addition, winter wheat retained WZY2::GFP for a longer time in leaf epidermal cells as compared to spring wheat. Prolonged retention of DHNs by tolerant cultivar appears to be an adaptive advantage of wheat to withstand cold stress.The procedure for monitoring dynamic changes in the localization of GFP-tagged DHNs in our system was rapid and easy to perform, and showed the natural movements of the DHNs.This work tested the functions of protective agents in ex vivo experimental systems for the first time and further evaluated the results of other experiments in vitro(LDH activity) and in vivo(E. coli viability). |
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