Analysis of molecular mechanism of action of flowering-time genes in Arabidopsis thaliana

在长日照植物拟南芥中，多种途径转导的环境和内源信号被整合在所谓的“花通路整合子”的转录调控上。开花位点T（flower LOCUS T， FI）是花途径整合子之一，阐明FT基因产物的分子作用机制是理解开花调控的重要一步。考虑到这一点，我们通过分析两个基因来研究FT作用的分子机制。一个是开花时间基因FD，它的功能是FT促进开花所必需的。另一个是与拟南芥中FT最接近的同源基因TSF （TWIN SISTER OF FT）。我们通过基于图谱的方法鉴定了FD基因。FD基因编码一个优先在茎尖表达的bZIP转录因子。我们发现，在花的命运决定中起最早作用的花分生组织身份基因aptala1 （AP1）是FD的调控靶点，FD需要FT的更多功能来激活AP1的转录。FT和FD之间的相互依赖是通过蛋白相互作用介导的。我们通过酵母双杂交实验、体外拉下实验和植物细胞双分子荧光互补实验证明了这一点。无论是在表达部位的脉管系统还是茎尖表达FT，都可以挽救FT突变体。相反，FD在茎尖表达，而在脉管系统中不表达，对FD突变体的拯救效果更好。这些结果以及FT和FD通过蛋白相互作用的相互依赖强烈提示FT的作用位点在茎尖，并可能调节FD的活性。这使我们提出，FT可能代表了开花过程中长期寻找的长距离信号，称为“致花因子”（Abe et al., Science, in press）。我们研究了TSF在调控开花中的作用。先前的研究表明，TSF在促进开花方面具有潜在作用。研究表明，TSF和FT具有相似的调控模式：由CONSTANS直接正调控，有一个晚峰的昼夜振荡，对光周期的响应，由开花位点C负调控，以及春化响应。TSF和FT也由EARLY BLTING IN SHORT DAYS和phyB共同调控。TSF在长日照和短日照条件下均增强了ft表型。这些有力地表明，TSF是一个与FT一起冗余作用的花通路整合子。TSF和FT在韧皮部组织中有优先表达，这使我们提出韧皮部代表了通路整合的一个位点（Yamaguchi等人，Plant & Cell Physiology, in press）。我们目前的假设是：FT和TSF整合韧皮部组织中各种途径的信号，它们的基因产物（可能是蛋白质）以某种方式被运送到茎尖，在那里它们调节bZIP转录因子FD的活性，进而激活AP！转录和启动花的形态发生。验证这一点是理解开花的重要一步。少

In the long-day plant, Arabidopsis, environmental and endogenous signals transduced by several pathways are integrated at the transcriptional regulation of so-called "floral pathway integrators". FLOWERING LOCUS T(FI) is one of the floral pathway integrators, and elucidation of molecular mechanism of action of FT gene product is an important step toward understanding of regulation of flowering. With this in mind, we investigated molecular mechanism of the FT action through analysis of two genes. One is a flowering-time gene FD, whose function is required for FT to promote flowering. The other is TWIN SISTER OF FT (TSF) which is a closest homelog of FT in Arabidopsis.We identified the FD gene by a map-based approach. The FD gene encodes a bZIP transcription factor preferentially expressed in the shoot apex. We showed that a floral meristem identity gene APETALA1 (AP1) which acts at the earliest step in floral fate specification is a regulatory target of FD and that FD requires the FT fu … More nction to activate AP1 transcription. Mutual dependence between FT and FD is mediated by protein-protein interaction. which we demonstrated by yeast two-hybrid assay, in vitro pull-down assay, and bimolecular fluorescent complementation assay in plant cells. Expression of FT in either vasculature, which is the site of expression, or shoot apex could rescue ft mutants. By contrast, FD expressed in the shoot apex, but not in vasculature was effective to rescue fd mutants. These results and mutual dependence of FT and FD via protein interaction strongly suggest that the site of action of FT is shoot apex where it may modulate the activity of FD. This led us to propose that FT may represent a long-sought long distance signal in flowering, called florigen (Abe et al., Science, in press).We investigated the role of TSF in regulation flowering. Previous work has suggested a potential role of TSF in promotion of flowering. We showed that TSF and FT share similar modes of regulation : direct positive regulation by CONSTANS, diurnal oscillation with an evening peak, response to photoperiod, negative regulation by FLOWERING LOCUS C, and vernalization response. TSF and FT also share regulation by EARLY BLTING IN SHORT DAYS and phyB. tsf enhanced ft phenotype in both long-day and short-day conditions. These strongly suggest that TSF is a floral pathway integrator acting redundantly with FT. TSF and FT share preferential expression in phloem tissues, which led us to propose that the phloem represents on of the sites of pathway integration (Yamaguchi et al., Plant & Cell Physiology, in press).A current hypothesis from our work is : FT and TSF integrate signals from various pathways in-phloem tissues and their gene products (possibly proteins) are somehow transported to the shoot apex where they modulate the activity of a bZIP transcription factor FD which in turn activate AP! transcription and initiate floral morphogenesis. To test this is an important step forward in understanding flowering. Less