生长素响应因子参与的植物花干细胞维持的分子机理研究

Floral stem cells are responsible for producing a defined number of floral organs, and then will be terminated programmatically at particular developmental stage ---be referred to as floral meristem determinacy. In Arabidopsis, WUSCHEL (WUS), a key stem cells maintenance gene, is expressed in the Organize Center to keep the stem cells property of the overlying cells. AGAMOUS, encoding a MADS domain transcriptional factor particular expressed in floral organ, is essential for the termination of floral stem cells fate by regulating WUS expression. How AG turns off WUS expression and then terminates floral stemcells is not very clear. Based on an AG weak mutant ag-10 EMS mutagenesis, we screened a mutant with enhanced floral meristem indeterminacy of ag-10. Using map based cloning we found a mutation occurring in one of the Auxin Response Factors (ARFs) and transgenic plant harboring genomic region of this gene show rescued phenotype which mean the mutation was responsible for the mutant defect. In this work, by in situ hybridation assay,gene transformation and ChIP techniques combined with biochemical and genetic analysis, we will figure out the molecular mechanism of the ARF gene controlling floral stem cells determinacy through a combinational approach of cellular and molecular biology, genetics and epigenetics.Morover, this will be the first study in which the plant hormones Auxin and cytokine as well as small RNA involved floral meristem determinacy is focused on. Our finding will unravel the regulation network controling the floral determinacy, which is also very significant for the crop high-yielding.

花干细胞在产生特定的花器官后，干细胞活性被程序性终止。该过程是通过AGAMOUS在特定时期对WUSCHEL（维持干细胞活性的关键基因）的终止表达实现的，具有精细时序调控特征，其作用的分子机制亟待澄清。申请者前期利用一个AGAMOUS弱的突变体ag-10的EMS诱变突变体库，筛选出一个参与AG调节WUS表达，控制花干细胞活性的突变体，经图位克隆证实是生长素反应因子ARF3基因发生突变。本项目拟利用原位杂交、ChIP等技术分析ARF3在调节花干细胞分化中的靶基因，并借助arf3突变体与花发育相关突变体的遗传杂交寻找ARF3作用的分子途径；同时将首次关注花干细胞活性终止过程中植物激素的作用，通过生化、细胞和分子生物学等手段全面考察ARF3在介导AG、生长素、细胞分裂素及小RNA等调节WUS终止表达的分子机理。研究结果为揭示花干细胞的调控网络的作用机制奠定基础，同时也对提高作物的高产具重要意义。

植物的各个器官来源于植物分生组织中的干细胞，于茎尖和根尖干细胞终生有活性不同的是，花干细胞在产生出特定的花器官后需要精确的分化终止。植物激素特别是生长素及细胞分裂素对于分生组织的维持及分化有重要作用，但是在花干细胞的活性调控的机理上未有相关报道。本研究课题立足于遗传筛选得到的生长素反应因子ARF3突变体进行研究。ARF3突变可以增强ag-10(AGAMOUS基因的弱突变体)花干细胞分化的缺陷表型；遗传学分析表明ARF3通过调节WUSCHEL基因来起作用；分子生物学实验证明ARF3介导了AGAMOUS和APETALA2在花干细胞分化调控中的作用。该研究成果发表于Plant J 并被F1000推荐。同时本课题组TOP1基因参与的植物分生组织分化调控机理研究中得到本基因的部分支持，该研究结果发表于Plant Cell杂志。