水稻穗退化抑制因子RPD1调控顶端小穗细胞程序化死亡的分子机制研究

Panicle apical degeneration commonly results in short panicles and causes a dramatic reduction in grain number per panicle, which is a determinant trait for rice yield. Thus, panicle apical degeneration is a severe threat to rice production. However, our knowledge about the molecular mechanisms in regulation of panicle apical degeneration in rice is very limited. In order to characterize the transcriptional regulators during panicle development, we have selected the candidate transcription factors that are highly and specifically expressed during panicle development based on the analysis of expression profiles. One of candidate gene designated as RPD1, encoding a DNA-binding protein, has been focused on its role in panicle development because the rpd1 (repressor of panicle degeneration1) mutant showed a severe phenotype of panicle apical degeneration. Complementation experiments further confirm that panicle apical degeneration occurred in the rpd1 mutant is caused by the RPD1 mutation. Our cytological and biochemical analysis indicated that the tissue-specific programmed cell death was characterized in apical spikelets. In this study, we aim at dissecting the molecular mechanisms of RPD1 in regulation of panicle apical degeneration and the related programmed cell death processes. We will identify the downstream target genes of RPD1 and conduct the functional analysis of these target genes with respect to their roles in modulating programmed cell death occurred in apical spikelets. Our studies will reveal the molecular and biochemical patterns by which RPD1 regulates the activation of its downstream target genes. These results will provide insights into better understanding the genetic and molecular regulatory pathways about panicle apical degeneration in rice.

水稻穗顶部退化导致穗子短小以及重要的产量指标穗粒数显著降低，造成水稻产量的大幅下降。目前对于水稻顶端穗退化的形成的分子机制了解十分有限。通过水稻穗发育表达谱的系统分析，我们筛选了一批在水稻穗发育过程中特异表达的转录因子作为重点研究对象。其中，RPD1编码一个DNA结合蛋白，其突变体呈现明显的顶端穗退化的表型。遗传互补实验表明突变体的顶端穗退化表型是由于RPD1突变所导致的。细胞学和生化分析显示rpd1突变体顶端小穗呈现明显的细胞程序化死亡（PCD）特征。本项目的主要目标是解析以RPD1为关键调控节点的水稻顶端穗退化以及细胞程序化死亡调控机制，鉴定RPD1所调控的下游靶点基因，并系统分析下游基因参与PCD发生和进程调节的细胞生物学过程及分子机制。研究将阐释水稻顶端穗退化抑制因子RPD1调控其下游基因的分子生化特性和调控模式，建立较为明确的水稻穗顶端退化的遗传和分子调控路径。

水稻穗顶部退化导致穗子短小以及重要的产量指标穗粒数显著降低，造成水稻产量的大幅下降。本项目的研究揭示RPD1/SPL6为调控水稻穗发育和穗“秃顶”表型产生的关键因子。细胞学和生化分析显示rpd1-1（spl6-1）突变体顶端小穗呈现明显的细胞程序化死亡（PCD）特征。通过pRPD1::GUS转基因株系表达样式分析以及原位杂交验证，结果显示 RPD1在穗发育过程中呈现顶端高水平表达样式，其中在花药中呈现高水平表达。分离鉴定了RPD1的等位突变体rpd1-2，进行了相应的遗传和穗发育形态的分析，该等位突变体也呈现穗发育以及穗顶端“秃顶”的表型；结合已完成的遗传互补验证实验，进一步在遗传上明确了RPD1功能缺失突变是造成rpd1突变体顶端小穗退化的原因。我们通过进行野生型和rpd1-1突变体之间穗发育RNA-seq分析，确定了差异表达基因RRG1/IRE1 (Inositol-requiring enzyme 1)为我们的重点关注对象，该基因在细胞内质网胁迫（ER-stress）信号转导过程中具有重要的功能。我们对于IRE1下游且涉及ER-stress信号通路的关键转录因子bZIP50的表达水平也进行了分析，结果表明bZIP50在rpd1-1突变体穗组织中也呈现组成型高表达的样式，且在正常生长条件下有剪切的转录本存在。上述结果表明，IRE1在突变体的组成型高水平表达，导致下游关键信号原件蛋白bZIP50的非胁迫条件下的启动，从而使突变体穗组织细胞在正常生长条件下进入过激的ER-stress状态。证实RPD1蛋白和其下游调控基因IRE1启动子区域表达调控原件均具有较强的结合特性。明确RPD1对于IRE1表达具有转录抑制功能，形式转录抑制子的功能。研究结果揭示水稻SBP-box家族转录因子RPD1/SPL6抑制细胞内质网胁迫感应因子IRE1的转录，控制胁迫信号的输出强度；RPD1/SPL6在穗发育过程中呈现顶端高水平表达样式，其功能缺失导致IRE1过度表达，造成细胞内质网胁迫信号输出失控和下游基因过激表达，导致顶端小穗细胞衰老退化和穗“秃顶”性状产生。因此RPD1/SPL6作为细胞内质网胁迫信号途径的上游调控关键因子，调节胁迫信号输出的时空强度和平衡，进而决定水稻穗发育过程中的细胞存活命运。