Expression Profling Analysis Of Rice Genes Under Cold Stress And Characterization Of Stress-Related TIFY Genes In Rice

Rice is not only the most important crop in the world, but also a promising model plant for plant molecular biological research. Cold is one of the major abiotic stresses and frequently influences the growth, development, and agricultural yield of rice. So, it becomes more and more urgent to improve rice tolerance to cold stress. In this research, by cold stress (4℃) screening of 172 rice accessions, part of the core collection representing rice germplasm bank, and five cold-tolerant germplasms were identified. Genomic gene expression profiles of five cold-tolerant materials and four cold-sensitive conventional cultivated rice varieties under cold stress were then compared by using Affymetrix DNA chip. We found that many members of the TIFY family were prominently induced by cold treatment. This research identifies 20 rice TIFY genes including a subfamily named JAZ with 12 members. Recently, JAZ family has been intensively investigated for their roles as key regulators of jasmonate (JA) response in Arabidopsis. Transcript level analysis of OsTIFY genes in tissues and organs revealed different tempo-spatial expression patterns. The expression of all the OsTIFY genes was investigated in leaf tissues under drought, salt and cold stresses, also treated with JA or abscisic acid (ABA). Among 18 OsTIFY genes checked, all were induced by drought and salt; 8 OsTIFY genes belonged to JAZ family were induced by cold and JA treatment; and only 4 OsTIFY genes induced by ABA. To further identify the putative JAZ members in rice, we used the protein synthesis inhibitor cyclohexamide (CHX) to treat the seedling rice before treated with JA. The real-time PCR analysis indicated that the expressions of 9 OsTIFY genes that were induced by JA were not suppressed by CHX. In yeast-two-hybrid (Y2H) assay, eight members of the OsJAZ subfamily interacted with OsCOI1a and OsCOIlc, but no interaction was found for OsCOIlb in the presence of 50μM coranatine.Two members of the OsTIFY family (OsTIFY11a and OsTIFY11b) were selected for further functional study in this research.OsTIFY11a encodes a rice JAZ protein with a defective Jas motif. Rice plants over-expressing OsTIFY11a showed decreased sensitivity to JA, increased salt tolerance, and slightly decreased K+ content in root.OsTIFY11a-suppressed plants showed sensitivity to JA, decreased salt tolerance, and obviously decreased K+ content in root. Expression analysis of salt stress-related genes in the OsTIFY11a over-expression and suppression transgenic lines identified several genes regulated by OsTIFY11a. These genes include OsNHX1, OsAKT1, and OsSKC1. OsSKC1 is a HKT-type transporter. The expression of OsSKC1 was suppressed by JA. OsTIFY11a could not interact with the putative OsCOI1s in the presence or absence of coronatine in Y2H assay. These results suggest that OsTIFY11a may act as a suppressor in the initial response of jasmonate-regulated gene expression toward salt tolerance.OsTIFY11b encoded a JAZ protein with a conserved motif in the N-terminal of the proteins and integrated Jas motif. Overexpression and T-DNA insertion mutation of the OsTIFY11b did not resulted in phenotypic changes under drought, salt, and cold stresses. These transgenic plants were more sensitive to cold stress than wild-type. We studied the roles of the three conserved domains of OsTIFY11b in protein interactions. The TIFY motif and Jas domain were critical in the interaction between OsTIFY11b and OsCO11s. The Jas domain was critical in the interaction between OsTIFY11b and OsMYC2. We designed construct by targeted deletion of the N-terminal conserved domain of OsTIFY11b (OsTIFY11b⊿A). The OsTIFY11b⊿A protein interacted with OsICEl in both Y2H and BiFC assays. We analyzed the expression levels of the cold-responsive genes in the OsTIFY11b⊿A-overexpression. transgenic lines and wild-type rice. After cold treatment, the expression level of OsDREB1a was higher in the OsTIFY11b⊿A transgenic lines than that in wild-type, while the expression of MybS3 was lower in the transgenic lines than that in wild-type. These results suggest that OsTIFY11b was involved in regulation of genes response to cold stress in rice.