拟南芥转录因子LBD29调控愈伤形成的分子机制

During in vitro organogenesis, the somatic cells from plant tissues have the capacity to reverse their state of differentiation and form an entire plant through the processes of dedifferentiation and redifferentiation. Our previous studies demonstrate that Arabidopsis LATERAL ORGAN BOUNDARIES DOMAIN (LBD)/ASYMMETRIC LEAVES2-LIKE (ASL) transcription factors are key regulators of the somatic cell fate switch in the auxin-induced callus initiation process. However, little is known of the molecular mechanism underlying LBDs-directed somatic cell fate switch in callus initiation in vitro. To elucidate molecular basis of LBDs, we first identified 242 up-regulated genes and 108 down-regulated genes in the inducible LBD29 transgenic seedlings incubated on MS medium with inducer within 8 h by RNA sequencing (RNA-Seq). Genome-wide binding site analysis through chromatin immunoprecipitation sequencing (ChIP-Seq) revealed more 300 genes associated with LBD29 binding sites in the overexpressing LBD29 transgenic seedlings. CACGTG and TGGGC[C/T] motifs were highly enriched in LBD29 binding peaks. We found that CACGTG and TGGGC[C/T] binding motifs were significantly enriched in the promoter of 3 kinds of genes (including methyltransferase, transcription factor and FAD-binding genes) by combining RNA-Seq with ChIP-Seq data. Moreover, our preliminary results demonstrated that LBD29 could directly bind to the promoter of a few of methyltransferase and transcription factor genes through Yeast-one-hybrid and ChIP-qPCR. Ectopic expression of these methyltransferase and transcription factor genes induced spontaneous callus formation without exogenous phytohormones. On the basis, we will try to dissect the target genes of LBD29 in the process of LBD29-regulated somatic cell fate switch with Yeast-one-hybrid, EMSA and CHIP-qPCR and investigate the upstream and downstream relationship between LBD29 and candidate target genes using genetic analysis. Moreover, we will explore the molecular mechanisms of candidate target genes on switching the somatic cell fate according to their characteristics. The elucidation of target genes of LBD29 genome-wide will contribute to understanding the molecular mechanisms underlying the somatic cell fate switch and to facilitating plant cell totipotentcy to agricultural production during in vitro organogenesis.

在离体再生体系统中，植物体细胞能逆转已分化的细胞状态经过脱分化和再分化形成完整的植株。拟南芥LBDs转录因子是生长素诱导愈伤形成过程中转变体细胞命运的关键因子。为了解析LBDs转变体细胞命运的分子机制，我们采用RNA-Seq和ChIP-Seq技术测定了LBD29的早期诱导表达谱和结合基序，发现启动子区含有CACGTG 和 TGGGC[C/T] 基序的3类基因（包括一些甲基转移酶，转录因子和FAD-binding基因）可能是LBD29的靶基因。初步地实验证明LBD29能结合候选的甲基转移酶和转录因子基因，且它们的异位表达自发诱导愈伤。在此基础上，本项目拟通过生物化学，遗传学和分子生物学等技术，鉴定LBD29在愈伤起始过程中的靶基因，明确LBD29和靶基因的遗传关系并对靶基因的具体作用机制进行阐释，该研究结果有助于我们理解愈伤起始过程中体细胞命运转变的分子机制和更好地将细胞全能性用于生产实践。

植物细胞长期以来被认为具有全能性，即高度分化的组织和器官在生长素的作用下形成具有高分化能力的愈伤组织。生长素诱导的愈伤组织形成是经典的植物离体再生体系中的起始步骤，在这个过程中植物器官中的中柱鞘和类中柱鞘细胞通过根的发育通路发生重编程形成多能性的愈伤组织。申请人前期的研究发现四个拟南芥LATERAL ORGAN BOUNDARIES DOMAIN（LBD）转录因子是生长素诱导愈伤发生的关键因子。在本项目资助下，申请人按计划以解析LBD调控愈伤形成的分子机制这一重要科学问题为目标，开展了多个方面的工作。这些研究包括：首先，利用ChIP-Seq和RNA-Seq技术从全基因组水平上对LBD29的靶基因以及愈伤形成的早期分子事件进行了解析；其次，发现转录因子BASIC REGION/LEUCINE ZIPPER MOTIF 59 （bZIP59）与LBD形成复合体共同直接结合并激活FAD-BINDING BERBERINE（FAD-BD）来调控愈伤形成；再次，初步证明LBD29直接结合并调控的INDOLE GLUCOSINOLATE O-METHYLTRANSFERASE IGMTs）通过影响ROS稳态平衡参与植物愈伤组织的发生过程。相关研究工作发表在Nat. Plants、Plant Cell Physiol.、aBIOTECH和植物学报等期刊上。除已发表的研究外，其它研究也已经取得了阶段性进展，部分成果在审稿或准备投稿阶段。这些研究不仅为解析生长素诱导的植物愈伤组织形成和再生分子机理提供了新的思路，而且为鉴定植物离体再生过程中调控细胞命运转变的关键因子提供了有价值的资源。综上，本项目经过四年的实施，按计划取得了预期的进展和成果，并为研究组后续在该领域的深入研究奠定了重要基础。