线粒体定位的杨树PPR519蛋白调控次生木质部发育的分子机制研究

Secondary xylem in trees is the biological basis for wood formation. Mitochondrial signals are important for developmental plasticity and environmental responses in plants. However, the mechanisms for mitochondrial regulation on secondary xylem development remain unclear. We have identified a poplar PPR protein designated PPR519, over-expression of which severely affected secondary xylem development, and this phenotype could be rescued by exogenous auxin application. Therefore, this study will focus on functional characterization of PPR519 as below. 1) The expression patterns of PPR519, phenotypes of PPR519 over-expressing and CRISPR-generated knockout poplar lines, and expression levels of downstream genes will be analyzed to reveal the PPR519-dependent regulation on secondary xylem development. 2) The expression levels and RNA splicing of mitochondrion encoding genes, the parameters for indication of mitochondrial functions, and the phenotypes of secondary xylem resulting from exogenous chemical inhibitors for mitochondrial respiratory chain will be checked to demonstrate PPR519-dependent regulation on mitochondrial function. 3) The effects on auxin signaling by mitochondrial dysfunction resulting from PPR519 over-expression and exogenous inhibitors will be tested. Moreover, phenotypic modification of mitochondrion-regulated secondary xylem development will be detected under exogenous auxin application and endogenous auxin-blocked condition to explore the requirement of auxin for mitochondrial regulation on secondary xylem development. In summary, this study will demonstrate mitochondrial regulation on wood formation and the required downstream signaling pathway, and provide key information for elucidation of mitochondrion-dependent cell proliferation.

树木的次生木质部是木材生产的生物学基础。线粒体稳态在调控植物发育和环境应答方面具有重要作用，而线粒体功能信号对次生木质部发育的调控作用和机制尚不清楚。我们筛选到一个线粒体定位的杨树PPR蛋白（PPR519），其超表达显著影响次生木质部发育，而该表型能被外施生长素恢复。本项目拟：1）分析表达模式、超表达和敲除株系表型及下游基因表达，确定PPR519对次生木质部的调控作用；2）检测线粒体编码基因表达和转录本内含子剪接、线粒体功能指标以及外施呼吸链抑制剂对次生木质部发育的影响，阐明PPR519调节线粒体功能的分子机理；3）分析PPR519和外源抑制剂干扰线粒体功能对生长素信号的影响，检测外施生长素及阻断生长素信号情况下线粒体调控次生木质部的表型变化，探究生长素介导线粒体调控次生木质部发育的作用。将揭示线粒体对木材发育的调控作用及所依赖的下游信号途径，促进对线粒体调控细胞增殖分化的机理解析。

树木的次生木质部是木材生产的生物学基础，其形成依赖于维管形成层细胞的增殖和分化活性。木材形成受到诸多内源因子和外部环境因素的调控和影响。细胞能量状态信号如何影响木材发育的分子机制目前尚不清楚。作为细胞能量代谢的重要细胞器，线粒体功能是否影响木材发育及其分子机制也不清楚。本项目在前期筛选到一个可能影响杨树木材发育的PPR类蛋白RFL30/PPR519，结合外源施加抑制剂干扰线粒体稳态，探究了线粒体功能变化对次生木质部发育的影响，揭示了线粒体功能对木材发育的调控作用，并通过分析线粒体与生长素通路的调控关系，阐明生长素是参与介导线粒体对木材发育调控的重要信号途径。本项目取得的重要结果和关键数据包括：（1）开展启动子报告株系分析，发现RFL30是一个主要在杨树维管形成层部位表达的PPR基因；（2）通过构建过表达和表达抑制株系并开展木材发育表型分析，证实RFL30蛋白是杨树形成层活性和木材发育中的负向调控因子，并能显著抑制多个与形成层和木材发育相关基因的表达；（3）开展线粒体编码基因表达水平和线粒体功能指标检测，表明RFL30蛋白能够影响大多数线粒体编码基因的表达，其过表达干扰了线粒体功能稳态；（4）外源施加呼吸链抑制剂破坏RFL30表达抑制株系的线粒体功能，发现RFL30调控木材发育依赖于线粒体功能；（5）比较转录组分析表明，多个生长素通路的基因在RFL30过表达株系中表达水平显著下调；（6）开展生长素含量检测、外施生长素处理和极性转运抑制剂处理实验，证实生长素信号参与介导RFL30蛋白及线粒体功能稳态对杨树木材发育的调控。以上结果表明，RFL30蛋白通过影响线粒体功能稳态并级联下游的生长素从而调控杨树木材发育。本项目揭示了线粒体功能级联生长素通路对木材发育的调控作用，为全面理解线粒体功能调控植物生长发育的作用提供了新认识。