CLE26多肽及其受体CLV2和CLE26R1调控拟南芥维管韧皮部分化的分子机理研究

Plant vascular tissues, xylem and phloem, differentiate in intricate patterns as an integral transport system for water, nutrients and signaling molecules throughout the plant body. Despite the importance of plant vascular tissues, details about how their development are modulated remain elusive. Particularly, the molecular mechanisms responsible for the formation and differentiation of phloem are largely unknown. Previously, it was shown that CLE45, a phloem-expressed CLE peptide, is perceived by a RLK (Receptor-Like Kinase) BAM3 to inhibit Arabidopsis phloem differentiation. However, it is suggested that additional CLE-receptor regulatory signaling modules are existing parallelly in modulating phloem development due to the facts that bam3 is partially insensitive to the CLE45 peptide treatment and no visible phenotype is observed in the bam3 mutant. In this study, we identified a CLE peptide, CLE26 that is specifically producing in root phloem cells, repressed the phloem development at a physiologically relevant concentration. Unlike CLE45, the bam3 mutant is sensitive to CLE26, excluding the possibility that BAM3 is the receptor for CLE26. Investigation with a collection of rlk and rlp (receptor-like protein) mutant transformed with APLpro:GUS/APLpro:GFP revealed that the APL signals in the clv2 mutant and one rlk mutant cle26r1 (cle26 receptor 1) are insensitive to the treatment of CLE26 peptides, thereby suggesting that CLE26R1 and CLV2 are involved in the perception of CLE26 signals in specifying root phloem differentiation. In order to decipher by what mechanisms the signaling module CLE26-CLV2/CLE26R1 control phloem patterning, the genetics of CLE26, CLV2 and CLE26R1 in the phloem formation will be firstly studied by generating different combinations of genetic mutants. Similarly, the genetic interaction among CLV2, CLE26R1, and BAM3 will be examined. Furthermore, the expression of CLE26, CLV2 and CLE26R1 will be carefully explored using custom histological techniques and confocal microscopy. Given the fact that CLV2 lacks a cytoplasmic domain, its association with (a) co-receptor(s) to initiate intracellular signaling is anticipated. Starting from this point, the formation of potential receptor complex(es) among CLV2, CLE26R1 and BAM3 will be unraveled through proper techniques. The direct interaction between the CLE26 ligand and its receptors will be confirmed by photoaffinity labeling using photoactivatable CLE26 with tagged CLV2 and CLE26R1. Finally, how CLV2- and CLE26R1-dependent CLE26-mediating signaling pathway is integrated into the established regulatory signaling module CLE45-BAM3 for phloem development will be investigated. The fulfillment of this proposal will advance our understanding of phloem formation and differentiation, strengthening the existing regulatory network in modulating phloem development. Additionally, the knowledge obtained from this study will be of great use in manipulating plant vascular development for better yield.

植物维管系统是植物运输水分、矿物质和有机养料的输导系统，形态结构复杂，难以观测，因此对其发育调控的研究相对滞后，特别是鉴定参与韧皮部细胞分化关键基因的工作较少。研究表明CLE45及其受体BAM3调控了拟南芥根韧皮部细胞的分化，然而对此信号途径的研究暗示还存在其他未知的信号分子及其受体参与维管韧皮部细胞分化的调控。本研究分离到一个在拟南芥根韧皮部特异表达的小肽CLE26，其显著抑制韧皮部细胞分化，初步鉴定CLV2和CLE26R1为其受体。本项目将围绕CLE26-CLV2/CLE26R1在韧皮部发育中信号的识别与转导展开，构建不同遗传材料，借助表型观察、基因表达和蛋白定位与互做检测、遗传互作分析以及配体与受体互作验证等实验，探究CLE26-CLV2/CLE26R1信号通路调控韧皮部细胞分化的机制及其与CLE45-BAM3信号通路之间的关联和异同，为实现植物维管系统发育的精确调控提供新思路。

植物维管系统是植物运输水分、矿物质和有机养料的输导系统，形态结构复杂，难以观测，因此对其发育调控的研究相对滞后，特别是鉴定参与韧皮部细胞分化关键基因的工作较少。研究表明CLE45及其受体BAM3调控了拟南芥根韧皮部细胞的分化，然而对此信号途径的研究暗示还存在其他未知的信号分子及其受体参与维管韧皮部细胞分化的调控。本研究分离到一个在拟南芥根韧皮部特异表达的小肽CLE26，其显著抑制韧皮部细胞分化，初步鉴定CLV2和CLE26R1为其受体。以此为基础，本课题进一步研究表明CLE26以及其同源基因CLE25和CLE45均在植物根维管韧皮部表达，其中CLE26和CLE45表达呈现特异性。我们借助CRISPR/Cas9基因编辑技术分别获得了CLE25/26/45的单突、双突和三突等基因功能敲除突变体。对于这些不同基因敲除突变体的表型分析发现，在这些突变体中韧皮部细胞分化提早，同时细胞分裂增强造成在韧皮部出现细胞聚集呈现多于野生型的韧皮部细胞数目，这和外施这三个CLE多肽抑制韧皮部细胞分化的功能正相反，表明这三个CLE基因影响韧皮部细胞分化和分裂。同样地，我们也发现cle26r1突变体中呈现较早的韧皮部细胞分化和较多的韧皮部细胞数目，表明CLE25/26/45和CLE26R1在一个相同的信号途径中发挥作用。进一步分析发现，CLE26介导的韧皮部细胞分化功能需要CLE26R1，明确了CLE26R1的确参与了CLE26信号的识别。基因表达分析发现CLE26R1在植物根维管中强烈表达。通过免疫沉淀发现CLV2和CLE26R1可以相互作用，说明CLV2和CLE26R1可能形成受体复合体共同接收CLE26介导的韧皮部细胞分化信号。尽管如此，我们的研究还需要进一步证实CLE25/26/45多肽与CLE26R1的生化互做以及解析CLE25/26/45-CLE26R1信号模块的下游因子等来分析这一信号通路。

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