Genetic characterization of phytochrome nuclear bodies in plant light signaling

描述(申请人提供)：核体(NBS)是存在于动物和植物细胞中的不同的亚核结构域。尽管许多NBS，如Cajal小体、PML(早幼粒细胞白血病)NBS和IGC(间染色质颗粒团)在哺乳动物系统中已被广泛研究，这些核小体的形态和组成的变化与人类疾病有关，但NBS的确切功能和调控仍不清楚。一个可能的原因是缺乏研究NB功能的遗传模型。我们建议利用光敏色素NBS在植物光信号中作为遗传模型系统来研究NB功能的一般原理。光敏色素是一种红色和远红光感受器，通过转录调控来调节植物的生长发育。在黑暗中，光敏色素定位于细胞质中。在光激活后，它们重新定位到细胞核并形成光敏色素NBS。我们证明了含有NBS的光稳定光敏色素B(PHYB)的形成受到光的直接调控，并且与光敏反应密切相关。基于这些观察，我们假设光敏色素NBS直接参与光敏色素信号事件。为了验证这一假设，我们建议：(1)通过基于共聚焦的遗传筛选来识别和表征PHYB-GFP NB形成所需的新成分。我们已经确定了29个这样的突变体。一个名为HMR的基因座已经被克隆，另外两个基因座已被粗略定位。值得注意的是，我们的初步研究表明，从这个筛选中鉴定出的第一个基因(HMR)对于PHYB-GFP NB的形成和早期的光敏色素信号事件都是必需的，包括光敏色素PhyA的光依赖蛋白分解。有趣的是，HMR在结构上类似于酵母蛋白RAD23的哺乳动物同源蛋白，RAD23是一种参与蛋白质降解的多泛素结合蛋白。这些结果首次提供了将光敏色素核体与蛋白质降解联系起来的遗传学证据，并表明这一遗传筛选将可能识别将光敏色素NB功能与早期光敏色素信号事件联系起来的新成分；(2)研究光敏色素NBS在早期光敏色素信号事件中的功能。我们提出了一些实验来直接测试光敏色素NBS是否是PhyA降解和/或转录调控的位点；(3)确定HMR在PhyA降解中的功能。我们将测试HMR是否通过将PhyA运送到蛋白酶体进行降解来发挥RAD23的作用。总的来说，拟议的实验应该会对更好地理解植物中的光信号做出重大贡献。更重要的是，他们还将开始解开NBS在细胞信号中的一般原理。 与公共卫生相关：核体是存在于动物和植物系统中的不同的亚核区域。许多哺乳动物核体的形态和组成的变化与人类疾病有关。我们将利用植物光敏色素核体系统作为遗传模型，在细胞信号转导的背景下研究核体的功能和调控。我们的研究将大大有助于我们理解人类疾病的细胞和分子基础。

DESCRIPTION (provided by applicant): Nuclear bodies (NBs) are distinct subnuclear domains present in both animal and plant cells. Although numerous NBs, such as Cajal bodies, PML (promyelocytic leukemia) NBs , and IGC (interchromatin granule cluster) have been extensively studied in mammalian systems, and changes in morphology and constitution of these nuclear bodies are associated with human diseases, the precise function and regulation of NBs are still poorly understood. One possible reason for this is the lack of genetic models to study NB functions. We propose to use phytochrome NBs in plant light signaling as a genetic model system to investigate general principles behind NB functions. Phytochromes are red and far-red photoreceptors regulating plant development and growth through transcription regulation. Phytochromes localize in the cytoplasm in the dark. Upon light activation, they relocate to the nucleus and form phytochrome NBs. We demonstrated that the formation of a photo-stable phytochrome B (PHYB) containing NBs is directly regulated by light and is tightly correlated to phytochrome responses. Based on these observations, we hypothesize that phytochrome NBs are directly involved in phytochrome signaling events. To test this hypothesis, we propose to: (1) identify and characterize new components required for PHYB-GFP NB formation by a confocal-based genetic screen. We have already identified twenty-nine such mutants. One locus, HMR, has been cloned, and two others rough-mapped. Strikingly, our preliminary studies show that the first gene (HMR) identified from this screen is required for both PHYB-GFP NB formation and early phytochrome signaling events including the light-dependent proteolysis of PHYA, a photo-labile phytochrome. Interestingly, HMR is structurally similar to the mammalian ortholog of a yeast protein RAD23, which is a multiubiquitin binding protein involved in protein degradation. These results provide the first genetic evidence linking phytochrome nuclear bodies with protein degradation, and demonstrate that this genetic screen will likely identify novel components linking phytochrome NB function and early phytochrome signaling events; (2) investigate the function of phytochrome NBs in early phytochrome signaling events. We propose a number of experiments to directly test whether phytochrome NBs are sites for PHYA degradation and/or transcription regulation; (3) define the function of HMR in PHYA degradation. We will test whether HMR acts as RAD23 by delivering PHYA to the proteasome for degradation. Collectively, the proposed experiments should contribute significantly to a better understanding of light signaling in plants. More importantly, they will also start to unravel general principles of NBs in cell signaling. PUBLIC HEALTH RELEVANCE: Nuclear bodies are distinct subnuclear domains present in both animal and plant systems. Changes in morphology and constitution of numerous mammalian nuclear bodies are associated with human diseases. We will take advantage of the plant phytochrome nuclear body system and use it as a genetic model to investigate the function and regulation of nuclear bodies in the context of cell signaling. Our studies should contribute significantly to our understanding of the cellular and molecular basis for human diseases.