Light Signaling Connectivity in Arabidopsis

描述（由申请人提供）：本研究将主要在布宜诺斯艾利斯大学Jorge J. Casal博士的实验室进行，作为NIH资助RO 1GM 52413（光感受器信号的初始事件）的扩展。控制转录组的不同信号的协调对于多细胞生物的正常发育是必不可少的。长期的目标是以拟南芥为模型系统，研究光信号连接的机制。该系统将发育的复杂性和复杂的遗传学与可逆激活不同受体的能力相结合。通过使用Affytron全基因组芯片，我们将在详细的动力学实验中鉴定其丰度由光感受器光敏色素B（PHYB）和隐花色素1（cryptochrome 1，PHY 1）协同控制的转录物。将使用数据的无监督和监督多变量分析的组合来研究转录组的主要模式。该信息将提供网络架构的动态图像。将根据早期性和协同作用的重要性对基因进行优先排序，并利用已鉴定出破坏基因的公开可用的大量T-DNA突变体，通过使用反向遗传学研究其功能。作为对该分析的补充，我们将使用一种新的遗传筛选来鉴定PHYB和PGE 1之间协同相互作用所必需的信号元件。将使用通过T-DNA随机诱变的种子和通过化学试剂诱变的群体进行筛选。突变基因将通过对T-DNA插入片段周围区域进行测序或通过染色体步移来克隆。由正向或反向遗传学产生的突变体的特征在于它们在暴露于组合不同波长的光时整合信号的能力。不同的光感受器调节重叠但多样的发育反应的机制一直是一个长期存在的问题。光信令可以使用共享组件。或者，信号通路可能会集中在一组相似的靶基因上。这里提出的实验设计作为一个综合的方法来开始回答这个重要的问题。

DESCRIPTION (provided by applicant): This research will be done primarily at the laboratory of Dr. Jorge J. Casal, University of Buenos Aires as an extension of NIH grant RO 1GM52413, Initial Events in Photoreceptor Signaling. The coordination of different signals controlling the transcriptome is essential for proper development of multicellular organisms. The long-term aim is to investigate the mechanisms of signaling connectivity by using light signaling in Arabidopsis thaliana as a model system. The system combines developmental complexity and sophisticated genetics with the ability to reversibly activate different receptors. By using Affymetrix full-genome chips we will identify transcripts whose abundance is controlled synergistically by the photoreceptors, phytochrome B (PHYB) and cryptochrome 1 (CRY1), in detailed kinetic experiments. A combination of unsupervised and supervised multivariate analysis of the data will be used to investigate the main patterns of the transcriptome. This information will provide a dynamic picture of the architecture of the network. The genes will be prioritized according to earliness and significance of the synergism and their function will be investigated by using reverse genetics, taking advantage of the publicly available large population of T-DNA mutants where disrupted genes have been identified. Complementary to this analysis, we will identify signaling elements necessary for the synergistic interaction between PHYB and CRY1 using a novel genetic screen. Seeds randomly mutagenized by T-DNA and populations mutagenized by chemical agents will be used for the screens. The mutated genes will be cloned either by sequencing the areas surrounding the T-DNA insert or by chromosome walking. Mutants resulting from forward or reverse genetics will be characterized by their ability to integrate signals when exposed to light combining different wavelengths. The mechanism by which different photoreceptors regulate an overlapping, yet diverse, set of developmental responses has been a long-standing question. Light signaling may use shared components. Alternatively, the signaling pathways may converge on a similar set of target genes. The experiments proposed here are designed as an integrated approach to begin answering this important question.