Gene expression profile during wound-induced callus formation in Arabidopsis thaliana

Wounding is a primary trigger of organ regeneration but how wound stress reactivates cell proliferation and promotes cellular reprogramming remains elusive. In this study we combined the transcriptome analysis with quantitative hormonal analysis to investigate how wounding induces callus formation in Arabidopsis thaliana. Our time-course RNA-seq analysis revealed that wounding induces dynamic transcriptional changes that can be categorized into five clusters with distinct temporal patterns. Gene ontology analyses uncovered that wounding modifies the expression of hormone biosynthesis and response genes, and quantitative analysis of endogenous plant hormones revealed accumulation of cytokinin prior to callus formation. Mutants defective in cytokinin synthesis and signalling display reduced efficiency in callus formation, indicating that de novo synthesis of cytokinin has major contribution in wound-induced callus formation. We further demonstrate that type-A ARABIDOPSIS RESPONSE REGULATOR (ARR)-mediated cytokinin signalling regulates the expression of CYCLIN D3;1 (CYCD3;1) and mutations in CYCD3;1 and its homologs CYCD3;2-3 cause defects in callus formation. Our transcriptome data, in addition, showed that wounding activates multiple developmental regulators, and we found novel roles of ETHYLENE RESPONSE FACTOR 115 (ERF115) and PLETHORA3 (PLT3), PLT5, PLT7 in wound-induced callus formation. Together, this study provides novel mechanistic insights into how wounding reactivates cell proliferation during callus formation. Overall design: Examination of transcriptome at 0, 1, 3, 6, 12,24 h after wounding.

创伤是器官再生的核心触发因子，但创伤应激如何重新激活细胞增殖并促进细胞重编程，其具体机制仍有待阐明。本研究将转录组分析与定量激素分析相结合，旨在探究创伤如何诱导拟南芥（Arabidopsis thaliana）愈伤组织形成。我们的时间序列RNA-seq分析显示，创伤可诱导动态转录变化，这些变化可被划分为五个具有独特时间表达模式的转录簇。基因本体（Gene Ontology, GO）分析显示，创伤会调控激素合成与响应相关基因的表达；对内源植物激素的定量分析则发现，在愈伤组织形成前，细胞分裂素（cytokinin）会出现积累。细胞分裂素合成与信号转导缺陷的突变体，其愈伤组织形成效率显著降低，表明细胞分裂素的从头合成在创伤诱导的愈伤组织形成中发挥主要作用。本研究进一步证实，A型拟南芥反应调节因子（ARR）介导的细胞分裂素信号转导，可调控细胞周期蛋白D3;1（CYCD3;1）的表达；CYCD3;1及其同源基因CYCD3;2-3发生突变后，会导致愈伤组织形成缺陷。此外，我们的转录组数据还显示，创伤会激活多种发育调控因子；本研究还发现了乙烯响应因子115（ERF115）以及PLETHORA3（PLT3）、PLT5、PLT7在创伤诱导的愈伤组织形成中的全新功能。综上，本研究为创伤诱导愈伤组织形成过程中细胞增殖的重新激活机制，提供了全新的见解。实验设计：于创伤处理后0、1、3、6、12、24小时六个时间点进行转录组检测。