Cloning And Functional Characterization Of Heat Responsive Transcription Factor TabZIP60 And TabZIP28 In Wheat (Triticum Aestivum L.)

Heat stress has become to a major limiting factor for wheat production, which dramatically reduces its yield and quality, but the molecular mechanisms underlying wheat tolerance to extreme temperature are largely unknown. In this study, we identified two heat responsive bZIP transcription factors TabZIP60 and TabZIP28 based on the previous transcriptome analysis, and analyzed their biological functions in response to heat stress, the main results are as follows:1. We cloned wheat bZIP transcription factor TabZIP60, which expression level is induced by heat stress and DTT treatment. TabZIP60 had conserved transmembrane domain for endoplasmic reticulum membrane location. Sequence alignment revealed that TabZIP60 have high homology to the corresponding genes of Arabidopsis, rice, Barley and Brachypodium. Centriolfold software predicted that the mRNA of TabZIP60 can form two kissing hairpin loops, a secondary structure of splicing by TaIRE1, which is furtuer confirmed by using specific primers.2. To dissect the biological function of TabZIP60, we generated transgenic plants overexpressing unspliced form(TabZIP60) and spliced form(TabZIP60D), respectively, and the former transgenic plants exhibited no significant variation in response to heat stress and endoplasmic reticulum stress (ER stress), while the later transgenic plants showed enhanced tolerance to heat and ER stress. For example, the survival rate (48.16% on average) is signficnatly higher in transgenic lines compared to the wild type. In addition, the longest root length of the wild-type and the TabZIP60D-OE lines had no significant difference at normal condition, however, the longest root length of the transgenic line OE1 (24.80 mm) were significantly longer than the wild-type (16.79 mm) under DTT treatment. Similarly, the longest root length of the transgenic line OE1 (41.29 mm) showed significantly difernernce compared to the wild-type (27.31 mm) under TM treatment. We next performed transcriptome analysis of the wild-type plant and 35S::TabZIP60D-MYC transgenic plants subjected to heat stress at 1 h, and found that 25 genes were significantly differentially expressed (Fold change≧2, FDR<0.05) between each other at normal conditions, and 19 genes are ER stress-related. After heat stress, there are 17 genes significantly differentially expressed, among which,7 genes are involved in ER stress. Chromatin immunoprecipitation (ChIP) varified that TabZIP60D was capble of binding to 9 genes (BiP3, PDI9, PDI6, PDI10, bZIP60, BI-1, OS9 and AT1G70490) possessing one ERSE-I element,2 genes (AT1G29310 and SAR1B) with ERSE-Ⅰ and UPRE-Ⅲ,3 genes (AT5G42050, TIN1 and PDI11) with UPRE-Ⅰ,2 genes (AT4G16260, AT2G01345) with UPRE-Ⅲ and one gene (AT3G51980) with ERSE-Ⅰ and XBP1-BS.To further investigate the potential role of TabZIP60 in the abiotic stress, TabZIP60 RNA interference (RNAi) lines in KN199 background were generated, which showed impaired tolerance to ER stress and heat stress.3. AtIRE1 is responsible for the splicing of AtbZIP60 in Arabidopsis. Wheat TaIRE1 was cloned and mapped to chromosome 6AL,6BL and 6DL, respectively. We produced wheat transgenic lines with silencing of TaIREl in KN199 background, which also showed decreased tolerance to heat and ER stress.4. We cloned a heat induced transcription factor TabZIP28 from wheat, which contains the open reading frame (ORF) of 1713 bp, encoding 570 amino acid residues. Phylogenetic analysis revealed that TabZIP28 clustered in the same clade as AtbZIP17, AtbZIP28 and AtbZIP49. Sequence analysis indicated that TabZIP28 possessed a conserved bZIP domain, a transmembrane domain (TMD) and a canonical SIP cleavage site. The promoter region of TabZIP28 contained several stress related cis-elements. The expression patterns of TabZIP28 were analyzed by Real-time PCR (qRT-PCR) under stress conditions. The results showed that:TabZIP28 was indued by heat and peaked at 1 h; the transcript level of TabZIP28 was increased to the highest level by PEG treatment for 6 h and reduced thereafter; TabZIP28 was found to be slightly upregulated by treatment of H2O2 for 12 h and TabZIP28 cannot be induced by DTT treatment. We overexpressed TabZIP28 in Arabidopsis and found the thermotolerance of the transgenic lines was significantly increased indicated by the survival rate and germination rate under heat stress.