Abiotic Stress Response Of Tea Plant BZIP Family Members And Functional Analysis Of Group C Genes CsbZIP6 And CsbZIP4

It is known that transcription factors play critical roles in regulation of stress signal transduction in the plant life cycle. Basic region leucine zipper(b ZIP) family genes have been extensively characterized in plant genomes, and numerous studies have indicated that b ZIP genes are involved in diverse biologic processes, such as development and stress response. In this study, on the basis of previous studies, we isolated b ZIP genes from tea plant by transcriptome sequencing analysis again. Expression patterns in different tissues and in response to abiotic stresses were analyzed. Besides, the stress function of Csb ZIP6 and Csb ZIP4 have been studied in transgenic Arabidopsis. The main results are as follows:1. Cloning and bioinformatic characteristics of bZIP genes from tea plant Seven novel Csb ZIP genes, Csb ZIP12-Csb ZIP18 were indetified from the tea plant. Bioinformatic analyses showed that 18 Csb ZIP proteins contain highly conserved b ZIP domain in the basic region with an invariant N-x7-R/K motif and leucine zipper motif. The 18 Csb ZIP proteins were clustered according to sequence similarities of their basic region and conserved motifs with 75 Atb ZIPs and MEME analysis, classified into ten groups, including groups A, B, C, D, E, F, H, I, S and group K.2. Expression patterns of Csb ZIP genes in different tissues and in response to abiotic stresses in tea plant 1) We observed that Csb ZIP genes were ubiquitously expressed in the tissues of roots, stems, leaves and flowers with variable transcript levels. Csb ZIP18 exhibited very low transcript abundance in all organs, Csb ZIP12, Csb ZIP14 and Csb ZIP18 had relatively high expression levels in roots, as compared to the other three tissues. In contrast, Csb ZIP16 was abundantly expressed in leaves, stems and flowers, but not in roots. 2) we found that conditions of short-term stress(0, 1, 3, 9, 24, 72 and 120-h) on the tea plant had a marked effect on the expression profile of Csb ZIPs. Expression analysis of Csb ZIP genes in response to four abiotic stresses showed that most Csb ZIP genes exhibited 1.5 folds up-and /or down-regulation in leaves and /or roots. Notably, Csb ZIP16, Csb ZIP17 and Csb ZIP18 showed significant regulation in response to these same stresses, suggesting a vital functional role in stress response.3. Csb ZIP6 is a negative regulator of freezing tolerance in Arabidopsis 1) Csb ZIP6 protein was located to the nucleus. 2 201 bp full-length promoter sequence of Csb ZIP6 were isolated and submitted into promoter database for element forecast analysis. The results showed that cold responsive elements ABRE, CBFHV and LTRE were discovered in the promoter region of Csb ZIP6. 2) ABA sensitivity: Compared to wild type, transgenic Arabidopsis plants overexpressing Csb ZIP6 showed decreased tolerance to ABA with lower germination rate and shorter root length. 3) Cold tolerance: Csb ZIP6-OE lines showed severe frostbite phenotype and lower survival rates than that of wild plants. The lectrolyte leakage and MDA accumulation in Csb ZIP6-OE plants were significantly higher than that of WT. Meanwhile, soluble sugar contents were decreased in Csb ZIP6-OE plants. These results indicated that the Csb ZIP6-OE lines were less tolerant to freezing stress. Gene ontology analysis showed that the differentially expressed genes were mapped to several important processes such as the response to cold, water stress and response to glucose signal transduction pathways. Microarray analysis showed that most cold responsive genes, such as COR413IM1, DREB1A/CBF3, KIN1 and LEA4-5, as well as sugar signaling related genes APL3, APL4 and SBE2.2 were down-regulated, and MYB15, which was the negative regulator of cold response, BT5 and DIN10 which confered freezing tolerance were up-regulated under freezing stress in Csb ZIP6-OE plants. These results suggested that Csb ZIP6 is a negative regulator of the cold stress response in Arabidopsis through repressing cold and osmosis responsive genes.4. CsbZIP4 is a positive regulator of salinity tolerance in Arabidopsis. Csb ZIP4 protein localized in nucleus. Over-expression of Csb ZIP4 in Arabidopsis resulted in increased tolerance to salinity and ABA at germination stage. Plants were subjectd to Na Cl treatment, and the result showed that Csb ZIP4-OE lines exhibited higher salt tolerance than that of WT. SPAD value of the leaves of Csb ZIP4-OE plants were significantly higher than that of WT under salinity stress. These results indicated that the Csb ZIP4-OE lines were more tolerant to salt stress. Meanwhile, the salt stress responsive gene SOS1 was strongly induced in Csb ZIP4-OE lines under salt stress, which indicated that Csb ZIP4 might function as a positive regulator of salt stress response by SOS1 pathway.