Molecular Mechanism of Master Growth Repressor DELLA Signaling in Arabidopsis

植物生长激素赤霉素(GA)在种子萌发、叶片展开、茎伸长和花果发育中起着关键作用。在20世纪60-70年代的绿色革命期间，改变谷类作物(如小麦和水稻的矮秆品种)的GA产量或反应，极大地提高了粮食产量。这些例子说明了这种激素在调节植物发育和农业中的重要性。GA促进的植物发育是由Della蛋白调控的，Della蛋白是一种主要的生长抑制因子，它整合了多种细胞途径来响应环境信号(如光条件、病原体、低温和干旱胁迫)。虽然Della在调节多种植物生长过程中发挥着核心作用，但人们对Della如何控制多条途径以及Della活动如何动态调节知之甚少。本项目将集中于这两个重要方面，并阐明其中涉及的分子机制。这项研究将促进更有效的策略来操纵植物的生长和胁迫反应，以改善农业，因为Dellas在植物中高度保守。该研究项目还将对培养青年科学家产生广泛影响，包括1名博士后、1名研究生和4名本科生。最近的研究表明，Dellas通过直接与关键转录因子(TF)进行蛋白质-蛋白质相互作用，在多种信号通路中发挥中枢调节作用。DELLA不包含DNA结合域，并可能通过隔离其相互作用的TF来抑制靶基因的表达。Dellas还可以通过与特定的转录因子结合来激活转录。然而，Dellas如何调节靶染色质和基因表达的分子机制仍不清楚。首席研究员实验室的初步结果表明，RGA(拟南芥)特定亚区的两个错义突变不会影响与TFS的相互作用。相反，它们在结合组蛋白和表观遗传调节方面受到了损害。这些结果提出了一个令人兴奋的可能性，即组蛋白结合可能会增强Della与目标染色质的结合，并且Della可能会招募表观遗传调节因子来诱导染色质结构的变化。这个工作模型将在特定的目标1中进行测试。通过酵母双杂交和下拉试验分析额外的错义RGA等位基因对与TF、组蛋白和表观遗传调节因子结合的影响，以及通过芯片-qPCR分析与目标染色质的关联，来确定不同亚域在RGA中的作用。Della的活性需要根据内部和外部的信号进行严格的调节，众所周知，GA会诱导Della的降解。最近的研究也表明，della的活性受到翻译后修饰的动态调节。初步数据表明，在GA缺乏的条件下，Della的磷酸化会发生改变，尽管这种修饰的作用尚不清楚。具体目标2将通过MS分析鉴定RGA中的亚磷酸盐，并通过遗传和生化分析表征潜在的蛋白激酶对这些修饰的功能。这项研究项目将阐明DELLAS如何调节目标染色质和基因表达的分子机制，并阐明磷酸化在微调DELLA活动中的作用。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

The plant growth hormone gibberellin (GA) plays a key role in seed germination, leaf expansion, stem elongation and flower and fruit development. Altered GA production or responses in cereal crops, e.g., the dwarf cultivars of wheat and rice, greatly increased the grain yields during the "Green Revolution" in the 1960s-1970s. These examples illustrate the importance of this hormone in regulating plant development and in agriculture. GA-promoted plant development is modulated by DELLA proteins, which are master growth repressors that integrate multiple cellular pathways in response to environmental cues (such as light conditions, pathogens, cold and drought stresses). Although DELLA plays such a central role in regulating diverse plant growth processes, very little is known about how DELLA controls multiple pathways and how DELLA activity is dynamically regulated. This project will focus on these two important aspects and elucidate the molecular mechanisms involved. This research will facilitate more effective strategies to manipulate plant growth and stress responses for agricultural improvements since DELLAs are highly conserved in plants. This research project will also have a broad impact in training young scientists, including a postdoctoral fellow, a graduate student and four undergraduate students. Recent studies indicate that DELLAs function as central regulators of multiple signaling pathways by direct protein-protein interactions with key transcription factors (TFs). DELLAs do not contain a DNA-binding domain, and are likely to repress expression of target genes by sequestering their interacting TFs. DELLAs can also activate transcription by binding to specific TFs. However, the molecular mechanism of how DELLAs regulate target chromatin and gene expression remains unclear. Preliminary results from the principal investigator's laboratory showed that two missense mutations in a specific subdomain of RGA (an Arabidopsis thaliana DELLA) did not affect interaction with TFs. Instead, they were impaired in binding histone and an epigenetic regulator. These results raise an exciting possibility that histone binding may enhance DELLA association with the target chromatin, and DELLA may recruit epigenetic regulators to induce chromatin structural changes. This working model will be tested in Specific Aim 1. The roles of different subdomains in RGA will be defined by analyzing the effects of additional missense rga alleles on binding to TFs, histone, and epigenetic regulators using yeast two-hybrid and pulldown assays, and association with target chromatin by ChIP-qPCR. DELLA activity needs to be strictly modulated in response to internal and external cues, and it is known that GA induces DELLA degradation. Recent studies also indicated that DELLA activity is dynamically modulated by post-translational modifications. Preliminary data indicate that DELLA phosphorylation is altered under GA-deficient conditions, although the role of this modification remains unclear. Specific Aim 2 will identify phosphosites in RGA by MS analysis, and characterize functions of potential protein kinases for these modifications by genetic and biochemical analyses. This research project will shed new light on the molecular mechanism of how DELLAs regulate target chromatin and gene expression, and elucidate the role of phosphorylation in fine-tuning DELLA activity.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.