Functional Analysis Of A Salt Stress Responsive Protease Inhibitor Gene TaWRSI5in Wheat

Protease inhibitors (PIs) are widely distributed in plants. It is known that protease inhibitors play important roles in the response to biotic stress, but their roles are not well known in the response to abiotic stress. Previous cDNA microarray studies indicated that the expression of Bowman-Birk type protease inhibitor family can be induced by salt stress in a salinity tolerant wheat introgression cultivar SR3obtained by asymmetric somatic hybridization between common wheat JN177and tall wheatgrass. One of these protease inhibitor gene named TaWRSI5was isolated from SR3by using mRNA differential display technique, this gene showed different expression level between SR3and JN177under salt stress, its heterologous expression in Arabidopsis thaliana improved the salinity tolerance of transgenic plants in seedling stage (Shan Lei, Doctoral thesis). However, it is still unclear whether this gene responsive to other abiotic stresses, the regulatory pattern of its expression under stress and its function in resistance to other abiotic stresses.In this work, we isolated the promoter and coding region of TaWRSI5from both JN177and SR3and sequence alignment indicated that they have four nucleotides differences but only lead to the substitution of one amino acid residue, no sequence changes were found between promoters of TaWRSI5in both JN177and SR3. A few ABA responsive cis-elements and drought related MYB binding site were found in the promoter of TaWRSI5. The cytosine methylation level in the TaWRSI5promoter region was decreased under salinity stress in SR3but not in JN177, no methylation modification was detected in the coding region of TaWRSI5both in SR3and JN177. Moreover, the expression of TaWRSI5was noticeably up-regulated in SR3but not in JN177when exposure to the methyltransferases inhibitor5-azacytidine (5-azaC). The implication was that DNA demethylation was responsible for the salinity stress-induced up-regulation of the gene in SR3, while other regulation mechanism(s) must have been operating in JN177. Further analysis of TaWRSI5by qRT-PCR indicated that it can be induced by salt as well as drought, ABA and H2O2. Subcellular localization analysis shows the ubiquity of the encoded protein in onion epidermal cells. The heterologous overexpression of TaWRSI5in Arabidopsis significantly increased the root length under osmotic stress when compared with Arabidopsis wild-type Col-0, moreover, the germination of transgenic seeds was more rapid and more complete than that of wild-type seeds under both osmotic and salinity stress, which confirmed the promotive effect of the transgene for germination in the presence of osmotic and salinity stress. The expression of P5CS1, which encode the key enzyme for proline synthesis, was up-regulated in transgenic plants, and the transgenic plants also showed higher proline content. These results clearly showed that TaWRSI5is involved in plant salinity and osmotic stress resistance via modulation of proline accumulation.