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

在长日照植物拟南芥中，由几条途径转导的环境和内源信号在所谓的“花途径整合子”的转录调控下整合。FI基因座T是花发育途径的重要整合子之一，阐明其作用的分子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，其功能是FT促进开花所必需的。另一个是与FT同源性最近的拟南芥中的双姐妹（Twin Sister OF FT，TSF）基因。FD基因编码bZIP转录因子，优先在茎尖中表达。我们发现，花分生组织身份基因APETALA 1（AP 1）在花命运特化的最早阶段起作用，是FD的调节靶点，FD需要FT融合基因。 关于我们 nucleotide激活AP 1转录。FT和FD之间的相互依赖性是由蛋白质-蛋白质相互作用介导的。我们通过酵母双杂交试验、体外下拉试验和植物细胞双分子荧光互补试验证明了这一点。FT的表达在任一脉管系统，这是网站的表达，或拍摄顶端可以拯救FT突变体。相反，FD在茎尖中表达，而不是在脉管系统中表达是有效的拯救fd突变体。这些结果以及FT和FD通过蛋白质相互作用的相互依赖性强烈表明FT的作用位点是茎尖，在那里它可能调节FD的活性。这使我们提出FT可能代表了在开花中长期寻找的长距离信号，称为成花素（Abe等人，我们研究了TSF在调节开花中的作用。以前的工作表明，TSF在促进开花的潜在作用。我们发现，TSF和FT共享相似的调节模式：CONSTANS的直接正调节，昼夜振荡与晚高峰，光周期的反应，负调节FLOWERLOCUS C，和春化反应。TSF和FT也通过在短期内的早期BLTING和phyB共享调节。TSF在长日照和短日照条件下均增强FT表型。这些都有力地表明，TSF是一个花的途径集成冗余与FT。TSF和FT在韧皮部组织中共享优先表达，这导致我们提出韧皮部代表途径整合的位点之一（Yamaguchi et al.，我们目前的假设是：FT和TSF整合了来自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）以某种方式运输到茎尖，在那里它们调节bZIP转录因子FD的活性，FD反过来激活AP！转录并启动花的形态发生。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