Phytochrome A: Structure/Function and Signaling Pathways

光敏色素(PhyA To PhyE)是一个独特的感光感受器家族，它调节发育重要基因的表达，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官分子感知、解释光信号并将其转导到光响应核基因的分子和细胞机制。最近来自这个实验室和其他实验室的证据已经导致了关于潜在的细胞内途径和机制在这个信号过程中使用的概念的范式转变。数据表明，其中一个途径涉及光激活的PHY分子从细胞质到细胞核的移位，随后与启动子结合的碱性螺旋-环-螺旋(BHLH)类转录因子(PIF3)特异性相互作用，从而导致靶基因的转录激活。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主要的转录调节基因，这些基因在PhyA调控的转录网络的不同分支中协调表达。尽管取得了这一进展，但还缺乏确凿的证据表明PHY分子直接参与转录调控以及可能涉及的机制，并且组成初级PHY调控转录网络的组件和电路仍未完全确定。我们建议使用Phya来解决这些缺陷，Phya是该家族中最具特点和实验上最易驯服的成员。这一建议的具体目标是：(A)鉴定和表征PhyA信号转导中涉及的分子成分，特别是那些PhyA途径特有的分子成分；(B)探索PhyA与bHLH因子PIF3相互作用的转录调控的分子基础；(C)探索PhyA信号传递到PIF3的生化机制；以及(D)绘制介导PhyA调控幼苗去黄化的初级转录网络。这些实验方法将包括：(A)克隆和分子鉴定PHYA特异信号中间产物的遗传筛选组分；(B)利用新的酵母双杂交筛选法克隆和鉴定更多与PHYA相互作用的蛋白；(C)通过融合DNA结合域人工靶向报告基因启动子的PHYA或PIF3融合蛋白转化植物细胞的转录激活分析；(D)重组野生型和信号受损突变体PHYA蛋白的酶分析，以确定在PHHA制剂中生化检测到的针对PIF3的蛋白激酶活性是否与体内PHHA信号活性相关；和(E)基于寡核苷酸微阵列的野生型和PhyA信号缺陷突变体的表达谱分析。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。这里提出的实验系统和战略有可能对这一目标作出重大贡献。

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.