Phytochrome A: Structure/Function and Signaling Pathways

光敏色素（phytochromes, phyA - phyE）是一类独特的感觉光感受器，可调节发育中重要基因的表达，以响应来自环境的信息光信号。这项研究计划的长期目标是确定这些感觉分子感知、解释和转导光信号到光响应核基因的分子和细胞机制。最近来自本实验室和其他实验室的证据导致了关于这种信号传递过程中潜在的细胞内通路和机制的概念的范式转变。数据表明，其中一种途径涉及光激活的物理分子从细胞质到细胞核的易位，随后与启动子结合的基本螺旋-环-螺旋（bHLH）类转录因子（PIF3）特异性相互作用，以及随后的靶基因转录激活。此外，基于寡核苷酸微阵列的表达谱分析表明，这些靶基因可能包括一组转录调节基因，这些基因在植物调节的转录网络的各个分支中协调表达。尽管取得了这一进展，但仍缺乏关于物理分子直接参与转录调节及其可能机制的明确证据，并且组成主要物理调节转录网络的成分和电路仍有待充分定义。我们建议使用phyA来解决这些缺陷，phyA是家族中最具特征和实验最易于处理的成员。该提案的具体目标是：(a)识别和表征phyA信号转导中涉及的分子成分，特别关注phyA途径特有的分子成分；(b)考虑到phyA与bHLH因子PIF3的相互作用，探索phyA转录调控的分子基础；(c)探讨phyA信号向PIF3传递的生化机制；(d)绘制介导phyA调控的幼苗脱殖的主要转录网络。实验方法将包括：(a)克隆和分子表征在特定植物信号中间体的遗传筛选中鉴定的成分；(b)利用一种新的酵母双杂交筛选技术克隆和鉴定了其他植物相互作用蛋白；(c)用phya或pif3融合蛋白通过融合的dna结合域人工靶向报告基因启动子转化的植物细胞的转录激活试验；(d)用重组野生型和信号受损突变型phyA蛋白进行酶促试验，以确定phyA制剂中生化检测到的PIF3蛋白激酶活性是否与体内phyA信号活性相关；(e)野生型和phyA信号缺陷拟南芥突变体的综合寡核苷酸微阵列表达谱。了解真核细胞感知和转导细胞外信息信号的分子和细胞机制的全谱仍然是生物医学研究的中心目标。本文提出的实验系统和策略有可能为实现这一目标做出重大贡献。

The phytochromes (phyA to phyE) are a family of unique sensory photoreceptors that regulate expression of developmentally important genes in response to informational light signals from the environment. The long-term goal of this research program is to define the molecular and cellular mechanisms by which these sensory molecules perceive, interpret and transduce light signals to photoresponsive nuclear genes. Recent evidence from this and other laboratories has resulted in a paradigm shift in concepts regarding the potential intracellular pathways and mechanisms utilized in this signaling process. The data suggest that one pathway involves light- activated translocation of phy molecules from the cytoplasm to the nucleus, followed by specific interaction with a promoter- bound basic helix-loop-helix (bHLH)-class transcription factor (PIF3), and consequent transcriptional activation of target genes. In addition, oligonucleotide microarray-based expression profiling suggests that these target genes may include a master set of transcriptional-regulator genes that orchestrate expression in various branches of a phyA-regulated transcriptional network. Despite this progress, definitive evidence of the postulated direct involvement of phy molecules in transcriptional regulation and the possible mechanisms involved are lacking, and the components and circuitry comprising primary phy-regulated transcriptional networks remain to be fully defined. We propose to address these deficiencies using phyA, the best characterized and experimentally most tractable member of the family. The specific objectives of this proposal are: (a) to identify and characterize molecular components involved in phyA signal transduction, with particular focus on those specific to the phyA pathway; (b) to explore the molecular basis of transcriptional regulation by phyA, given its established interaction with the bHLH factor PIF3; (c) to explore the biochemical mechanism of phyA signal transfer to PIF3; and (d) to map the primary transcriptional network that mediates phyA- regulated seedling deetiolation. The experimental approaches will include: (a) cloning and molecular characterization of components identified in genetic screens for phyA-specific signaling intermediates; (b) molecular cloning and characterization of additional phyA-interacting proteins using a novel yeast two-hybrid screen; (c) transcriptional activation assays in plant cells transformed with phyA-or PIF3-fusion proteins artificially targeted to reporter-gene promoters via fused DNA-binding domains; (d) enzymatic assays with recombinant wild-type and signaling-compromised mutant phyA proteins to determine whether protein kinase activity toward PIF3 detected biochemically in phyA preparations is correlated with phyA signaling activity in vivo; and (e) comprehensive oligonucleotide microarray-based expression profiling of wild-type and phyA- signaling-defective Arabidopsis mutants. Understanding the full spectrum of molecular and cellular mechanisms by which eukaryotic cells perceive and transduce extracellular informational signals remains a central goal of biomedical research. The experimental system and strategies proposed here have the potential to contribute significantly to this goal.