SdCLE多肽及其受体SdR/MOL1调控拟南芥维管形成层维持与分化的功能与分子机理研究

The plant vascular meristem is a stem-cell tissue comprising cambial cells that give rise to xylem and phloem cells in opposite sides. How cambial stem cell fate is modulated is one of the most intriguing research questions in plant vascular development. Previously, it was shown that CLE41, a phloem-derived CLE peptide, is perceived by PXY, a leucine-rich repeat receptor-like kinase located in the plasma membrane of procambial cells. This signal suppresses xylem cell differentiation of procambial cells and promotes their proliferation. However, this system is unbalanced because of missing xylem-derived intercellular signals for phloem-fate commitment. In this study, we identified a novel CLE peptide, SdCLE that producing in xylem cells, regulates vascular stem cell fate through a receptor-like kinase SdR/MOL1. We firstly will assess the role of SdCLE by overexpressing SdCLE and investigating alteration of tissue-specific markers upon SdCLE applied externally. In addition, the expression pattern of SdCLE and SdR/MOL1, as well as the subcellular localization of SdR/MOL1, will be determined by reporter constructs. The direct interaction between SdCLE and its receptor SdR/MOL1 will be confirmed by photoaffinity labeling using photoactivatable SdCLE and a SdR/MOL1 variant (SdR/MOL1-△KD-Halotag) in which the cytoplasmic kinase domain was replaced by the HaloTag. In order to decipher the relationship between SdCLE-SdR/MOL1 and CLE41-PXY pathways, the genetic interaction among SdCLE, SdR/MOL1, CLE41, PXY and WOX4 will be examined. This proposal will advance our understanding of the function of SdCLE and SdR/MOL1 in regulation of vascular cambium and the extent to how SdCLE and SdR/MOL1 modulate cambial stem cell fate. The information gained will be of great use in manipulating plant signaling for yield in woody plants.

维管形成层具有多能干细胞的特征，是植物维管系统发育的起始中心，其分生组织内干细胞增殖与分化的分子调控机制是研究的核心问题。韧皮部特定表达的CLE41与其受体PXY调控形成层干细胞数目并抑制干细胞向木质部细胞分化。但此调控体系是不平衡的，因为缺少一个源自木质部的信号分子及其受体与CLE41-PXY共同调控维管发育。我们分离到一个在拟南芥木质部特异表达的SdCLE多肽，并鉴定其受体为SdR。拟过表达SdCLE并分析SdCLE对维管系统标记基因表达的影响，明确其在维管发育中的功能；检测SdCLE与SdR之间的相互作用；分析它们的表达和蛋白定位，以及与CLE41、PXY和WOX4的遗传关系，揭示SdCLE-SdR与CLE41-PXY介导的信号途径之间的关联与异同。最终阐明SdCLE和SdR调控形成层分生组织维持与分化的分子机理，实现植物尤其是木本植物维管系统发育的精确调控，达到改良林木品质的目标。

维管形成层具有多能干细胞的特征，是植物维管系统发育的起始中心，其分生组织内干细胞增殖与分化的分子调控机制是研究的核心问题。韧皮部特定表达的CLE41与其受体PXY调控形成层干细胞数目并抑制干细胞向木质部细胞分化。但此调控体系是不平衡的，因为缺少一个源自木质部的信号分子及其受体与CLE41-PXY共同调控维管发育。我们分离到一个在拟南芥木质部特异表达的SdCLE多肽，并鉴定其受体为SdR。拟过表达SdCLE并分析SdCLE对维管系统标记基因表达的影响，明确其在维管发育中的功能；检测SdCLE与SdR之间的相互作用；分析它们的表达和蛋白定位，以及与CLE41、PXY和WOX4的遗传关系，揭示SdCLE-SdR与CLE41-PXY介导的信号途径之间的关联与异同。超表达SdCLE基因和SdCLEG6T基因纯合体植株茎基部横切结果显示，维管组织韧皮部区域出现木质化细胞，表明SdCLE基因和SdCLEG6T基因参与细胞木质化过程。同时，超表达SdCLE基因促进形成层细胞分裂，超表达SdCLEG6T基因不影响形成层细胞分裂，但明显改变维管组织细胞排列。外施SdCLE多肽和SdCLEG6T多肽造成ATHB8pro:GUS表达提前，但不改变其表达部位和范围，表明SdCLE多肽和SdCLEG6T多肽具有相同的功能参与下胚轴形成层和初生木质部的发育。同时外施SdCLE多肽造成TMO5pro:GFP的GFP信号密度和标记细胞层数增加，表明SdCLE多肽参与下胚轴初生木质部的发育。SdCLE基因在幼苗时期主要在根中表达，后期在维管组织中表达。为此，本实验选择SdCLE基因集中在维管组织表达的植株，进行根横切实验以观察SdCLE基因在维管组织中的表达情况。树脂包埋超薄切片结果显示，SdCLE基因在整个中柱范围内均有表达。35S::SdCLE转基因植株中韧皮部区域均出现了木质化细胞，间苯三酚染色结果也同样证明了韧皮部区域出现了木质化细胞。相同情况也出现在mol1-1背景下稳定表达35S::SdCLE的转基因植株中，而mol1-1植株韧皮部位置并未出现木质化细胞，表明MOL1可能是SdCLE的受体。我们的研究明确了SdCLE和SdR/MOL1参与了调控形成层分生组织维持与分化的功能，丰富和完善形成层分生组织的分子调控机理，可以在农林作物尤其是木本植物的分子育种研究中借鉴我们的相关成果。