GSH/GSSG介导蛋白质翻译后修饰调控苹果实生树MdMIR156a5/a12转录的研究

MicroRNA156 (miR156) directly controls the vegetative phase change and floral transition in higher plants. Mature miR156 is under transcriptional regulation. We found that of the seven dominantly expressed miR156 precursor genes in apple genome, MdMIR156a5 and MdMIR156a12 control the vegetative phase change of apple seedlings in response to GSH level and GSH/GSSG ratio. In this study, transcription factors (TF) binding to MdMIR156a5/a12 promoters will be screened by co-expression analysis using our previous transcriptomic data and yeast single hybrid, after validation via VIGS, EMSA and transgenesis, the dimerization and S-glutathionylation of the TFs will be analysed. The changes in deposition of H2A.Z、H3K4me3、H3K27ac and H3K27me3 marks on MdMIR156a5/a12 chromatin during ontogenesis of apple seedlings will be detected using ChiP-qPCR strategy. Then, to test whether modifications on TFs, histones subunits or both of them regulate MdMIR156a5/a12 expression, the responses of the post-translational modifications of the TFs and histone subunits to GSH and GSH/GSSG are verified using exogenous BSO/OTC treated in vitro shoots or MdGGT1-ox/RNAi GL-3 transformants. Finally, to test whether the transcription regulation of MdMIR156a5/a12 by GSH is controlled by TFs glutathionylation mediated chromatin remodeling, the dimerizing or the glutathionylated cysteine residues are mutated and the mutant full length cDNA of TFs will be transiently transformed to tobacco and stably transformed to GL-3. The oncoming data would promote novel understanding on the molecular mechanism of plant ontogenesis.

miR156调控高等植物阶段转变和成花转变。我们发现调控苹果实生树阶段转变的miR156前体基因MdMIR156a5和MdMIR156a12的表达随谷胱甘肽（GSH）含量和GSH/GSSG比值的变化而变化。本研究首先通过分析已有转录组数据，并采用酵母单杂筛选与MdMIR156a5/a12启动子结合的重要转录因子，经EMSA、VIGS和转基因苹果验证后，检测苹果实生树不同节位MdMIR156a5/a12转录因子二聚体化和谷胱甘肽化的变化，以及MdMIR156a5/a12组蛋白H2A.Z、H3K4me3、H3K27ac、H3K27me3富集的变化；以外施和转基因GL-3组培苗验证转录因子和组蛋白修饰对GSH水平和GSH/GSSG的响应。最后，将关键Cys残基点突变后转化苹果，分析转录因子翻译后修饰是否通过组蛋白修饰调控MdMIR156a5/a12转录。对认识植物阶段转变的分子机制有意义。

miR156控制高等植物阶段转变，但miR156前体基因转录调控的分子机制尚未破解。本项目组前期发现苹果阶段转变过程中GSH/GSSG稳态参与调控miR156主要前体基因MdMIR156a5和MdMIR156a12转录。本项目首先证实MdWRKY40响应GSH稳态，调控MdMIR156a12转录；CCA1家族成员MdRVE1和MdRVE7结合MdPIF4靶向MdMIR156a5启动子并响应GSH/GSSG稳态正调控其转录。其次，排除了GSH通过蛋白质谷胱甘肽化修饰调控MdMIR156a5和MdMIR156a12转录。再次，排除MdWRKY40、MdRVEs和MdPIF4通过二硫键直接形成二聚体参与miR156前体基因转录调控，但MdRVEs和MdPIF4上游转录因子MdBEH4及互作蛋白MdTRXh5.1和MdTRXh5.2在成龄期的二硫键水平明显高于童期，说明MdBEH4很可能通过MdTRXh5响应GSH/GSSG稳态变化，并通过靶向MdRVEs和MdPIF4参与MdMIR156a5转录调控。此外，苹果实生树成龄期MdRVE1启动子H3K27me3水平明显高于童期，说明MdRVEs基因的组蛋白修饰参与调控MdMIR156a5的转录调控。上述研究结果为进一步揭示高等植物阶段转变过程中miR156转录调控的分子机制指明了研究方向。