叶绿体蛋白PagHCF106参与调控杨树气孔运动和抗旱性的机理研究

Stomatal movement play important regulatory roles in plant growth and water metabolism. However, the mechanism of stomatal movement in woody plants remains largely unknown. Preliminary results suggest that poplar chloroplast protein HCF106 (PagHCF106) might participate in the regulation of stomatal movement via regulating (affecting) the levels of reactive oxygen species (ROS) and starch metabolism in chloroplast of guard cells. Here, we will develop the poplar plants with different PagHCF106 expression levels through poplar DNA-free genome editing system and transgene technology. Functions of PagHCF106 in drought tolerance, stomatal movement, the ROS and starch metabolism in chloroplast of guard cells will be revealed through analyzing the physiological response characteristics of each poplar plants upon exposure to water stress. We will further elucidate the signal transduction pathway and molecular mechanism for PagHCF106 participates in stomatal movement by ChIP-seq, co-IP-MS and RNA-seq. We expect to provide theory and technique basis for further understanding the regulatory mechanism of stomatal movement in woody plants and developing drought tolerance and water-saving poplar plants.

气孔运动对林木生长和水分代谢调控起重要的作用，但针对林木植物气孔运动机制的研究较少。项目申请人前期研究发现，杨树叶绿体蛋白HCF106(PagHCF106)可能通过调节(影响)保卫细胞叶绿体活性氧和淀粉代谢参与调控气孔运动，进而影响植株的抗旱性。本项目拟通过杨树DNA-free基因组编辑技术和转基因技术，培育不同PagHCF106基因表达水平的杨树株系；通过分析各株系对水分胁迫的生理响应特征，揭示PagHCF106在抗旱性、气孔运动及保卫细胞叶绿体活性氧和淀粉代谢中的功能；通过染色质免疫共沉淀结合高通量测序、免疫共沉淀联合质谱和转录组测序，明确PagHCF106在气孔运动中的信号转导途径和分子机制。本项目的研究结果为丰富木本植物气孔运动调节机制和培育抗旱节水型杨树奠定理论与技术基础。

通过调节气孔发育和气孔运动是培育抗旱节水植物潜在的有效途径。本项目中，我们对杨树(Populus alba × Populus glandulosa)的PagHCF106基因进行了克隆和功能分析。研究发现，PagHCF106的表达主要集中在成熟叶片，呈现组织特异性表达特征；PagHCF106蛋白主要定位于叶绿体中。通过在拟南芥hcf106突变株系中过量表达PagHCF106发现，相较于突变体，回补株系的气孔开度和抗旱能力基本回复到野生型水平。通过GUS染色试验对PagHCF106启动子的活性核心区域进行鉴定发现，-400以内区域为PagHCH106基因表达所必须区域。随后对该区域进行基于CRISPR/Cas9基因组编辑发现，杨树突变株系的气孔开度和水分散失降低，而在保卫细胞中累积了高水平的过氧化氢信号分子。综上所述，PagHCF106可能通过累积活性氧信号分子来调节气孔开度，进而负调控抗旱能力；通过修饰HCF106启动子可能用于提升木本植物的抗旱性。