芽从头发生的表观遗传调控机制

The ability of plant de novo shoot organogenesis reflects the plasticity of plant cells and their pluripotency. In tissue culture, adventitious shoots could be regenerated from a pluripotent cell mass, called callus, under the control of phytohormones. Here we focus on the research of the epigenetic regulation in de novo shoot organogenesis from callus. To start this research, we screened for mutants of epigenetic factors using the de novo shoot organogenesis system and identified several mutants that were defective in shooting. Mutations in the ISWI and PKL chromatin remodeling factors and the H2A ubiquitin E3 ligase AtRING1a and AtRING1b showed a very typical shooting defect. Our primary data showed that these epigenetic factors may function in either establishment of pluripotency in callus or promotion of shoot apical genes during de novo shoot organogenesis. Based on these preliminary research data, our research will continue to focus on 1) studying the downstream targets of epigenetic factors in shooting using the molecular genetic methods; 2) characterization of their interacting proteins and their functional specificity using biochemical approaches; and 3) analyzing their roles in changing the chromatin status at their target loci. We anticipate that these studies will provide an overall view on how epigenetic mechanism is involved in de novo shoot organogenesis and will also add evidence on how plant cells are highly plastic and pluripotent.

植物细胞的多能性和高度可塑性在芽从头发生过程中充分体现。在组织培养中，具有多能性的愈伤组织能够在激素刺激下实现不定芽的从头发生，继而发育成为完整植株。本项申请集中在芽从头发生的表观遗传调控机制。我们利用芽从头发生体系对模式植物拟南芥的表观因子突变体进行了表型筛选，发现了多个芽从头发生缺陷的表观遗传突变体，包括ATP依赖型染色质重塑因子ISWI和PKL以及组蛋白H2A泛素化E3酶AtRING1a和AtRING1b。初步研究基础还显示，这些表观遗传突变体可能作用在两条遗传通路上：1）愈伤组织多能性的获得；2）芽特征基因的激活。基于这些前期研究基础，我们将从分子遗传角度解析这些表观因子的下游基因；利用生化方法解析这些表观因子的互作蛋白和特异性来源；并分析表观因子对下游基因座位上染色质结构改变的功能。我们期望全面和系统的解析芽从头发生的表观遗传调控机制，为植物细胞可塑性和多能性提供理论依据。

项目执行过程中主要回答计划书提出的科学问题，即表观因子和其调控的转录因子参与芽从头再生的机制是通过维持愈伤组织的多能性还是促进芽尖干细胞的出现。通过研究，我们的答案是：ISWI染色质重塑因子CHR11/17，PcG复合体成员CLF/SWN/EMF2、H3K27me3去甲基化酶REF6、ELF6等表观因子及表观因子调控的LBD16转录因子，WOX11转录因子，WOX5/7转录因子，参与维持愈伤组织的多能性；而ISWI染色质重塑蛋白复合体成员CHR11/17-RLT1/2，PcG复合体成员CLF/SWN、AtRING1a/b等参与促进芽尖干细胞出现。因此，ISWI和PcG同时参与了愈伤组织多能性维持和芽尖干细胞出现。同时我们还发现组蛋白H3K36me3甲基转移酶SDG8参与伤口响应。 总体来看，该项目的研究成果揭示了在植物器官再生过程中一套由转录因子、激素通路、表观因子组成的复杂网络，一起调控了从伤口到细胞命运转变的过程，最终实现植物再生。项目执行期内，共发表了论文15篇（14篇为SCI论文），其中有11篇为第一资助，4篇为第二资助。第一资助的代表性研究成果有Nature Plants (2019), Nature Plants (2021), Journal of Genetics and Genomics (2019), Regeneration (2017), Chromosoma (2017) 等。参与了4次科普活动，培养了3位硕士研究生获得学位，8位博士研究生获得学位，2位博士后出站。项目主持人获得多项人才奖励。