亚细胞氧化还原及GSH稳态调控苹果实生树童性的研究

miR156 plays a pivotal role in regulation of vegetative phase change in angiosperms. The expression of miR156 is under control of a developmental signal from leaf primordia. So far, however, this signal and the regulating pathway are unidentified. We found that, in comparison to juvenile phase, reactive oxygen species (ROS) accumulated in chloroplast of leaves from adult phase, accompanied by significant decreases in GSH level, GSH/GSSG ratio, as well miR156 and pre-miR156 expressions at the leaf tissue level. No apparent change in ROS and GSH homeostasis was detected in pre-miR156 transgenic tobacco leaves. In this study, therefore, by using in vitro plantlets and field leaves of juvenile and adult phase of apple hybrids, chemical inducers or inhibitors of subcellular ROS or GSH will be applied in order to investigate the pathway of choloplastic ROS over production and to assay if and how chloroplast ROS and/or plastid-nucleus retrograde signals affect GSH homeostasis and juvenility. Then, the influences of cellular or subcellular GSH homeostasis on MIR156A5 and MIR156A12 expressions will be determined. Finally, the regulations of MIR156A5, MIR156A12 or their upstream genes will be predicted and examined via multiple-omics and bioinformatics analysis. The results will be important for understanding the mechanism of ontogenesis in higher plants and for further use in fruit breeding and propagation practices.

高等植物阶段转变受miR156调控，而miR156水平受叶原基的发育信号调控，但该信号是什么以及它如何调控miR156表达仍不清楚。我们已明确苹果实生树成龄期叶片叶绿体活性氧（ROS）积累，叶组织GSH水平及GSH/GSSG比值降低，miR156及其前体pre-miR156表达量显著降低；转pre-miR156烟草叶片ROS和GSH稳态未见明显规律性变化。因此，本项目以苹果实生树童期和成龄期组培苗和田间叶片为试材，首先通过外施ROS或GSH调控剂及烟草转基因等方法，研究成龄期叶绿体ROS积累的途径，分析叶绿体ROS及质体-细胞核逆行信号对GSH稳态及童性的影响；然后，研究细胞或亚细胞GSH稳态的变化对MIR156A5和MIR156A12表达的影响；最后，多组学联合分析预测并验证MIR156A5、MIR156A12或上游基因的调控途径。对认识阶段转变的机制，指导果树育种和良种繁育有意义。

项目组前期发现苹果实生树成龄期叶片叶绿体中H2O2水平显著高于童期。本项目结果表明，苹果实生树成龄期叶绿体中H2O2积累既不是因为叶绿体中光和电子传递链电子渗漏增加，也不是由于NTRC/2-Cys Prx系统清除ROS能力减弱所致，而与成龄期GSH含量和GSH/GSSG比值降低密切相关。.有报道的质体-核逆行信号（PNRS）中，1O2、GUN1、PAP、MEcPP、WHIRLY、heme等相关PNRS均与MdMIR156s或miR156无相关性。Mg-Proto IX相关PNRS与miR156表达模式呈现显著负相关，但Mg-Proto IX PNRS处于miR156下游，受miR156和GSH负调控。.苹果实生树miR156表达水平、GSH、GSH/GSSG比值都随节位的升高而下降，miR156的表达量与 GSH 的含量及 GSH/GSSG的比值显著性相关，GSH位于miR156的上游。DEM处理抑制细胞质中的GSH向细胞和运输，引起MdMIR156s和miR156水平降低。成龄期叶绿体和细胞核GSH含量降低源于MdGGT1表达量以及质外体可溶性 GGT 酶活的降低，因为GGT酶活降低导致质外体Cys再利用能力减弱，对叶绿体中GSH合成有影响。.童期与成龄期实生树MdMIR156s基因DNA甲基化类型、甲基化度和甲基化位点未见显著差异。成龄期MdMIR156上游转录因子MdWRKY40和MdCCA1基因表达量显著下调。MdWRKY40 可与 MdMIR156a12 启动子互作，正调控其转录；MdCCA1能够与 MdMIR156a5 启动子互作，正调控其转录。MdWRKY40表达量受GSH水平正调控。.上述结果对认识多年生木本植物个体发育，果树育种和良种繁育具有重要的理论依据。