CONSTANS alters the circadian clock in Arabidopsis thaliana. CONSTANS alters the circadian clock in Arabidopsis thaliana

Plants are sessile organisms that have acquired highly plastic developmental strategies to adapt to the environment. Among these processes, the floral transition is essential to ensure reproductive success and is finely regulated by several internal and external genetic networks. The photoperiodic pathway, which controls the plant response to day length, is one of the most important pathways controlling flowering. In Arabidopsis photoperiodic flowering, CONSTANS (CO) is the central gene activating the expression of the florigen FLOWERING LOCUS T (FT) in the leaves at the end of a long day. CO expression is strongly regulated by the circadian clock. However, to date, no evidence has been reported regarding a feedback loop from the photoperiod pathway back to the circadian clock. Using transcriptional networks, we have identified relevant network motifs regulating the interplay between the circadian clock and the photoperiod pathway. Gene expression, chromatin immunoprecipitation experiments and phenotypic analysis allowed us to elucidate the role of CO over the circadian clock. Plants with altered CO expression showed a different internal clock period, measured by daily rhythmic movements in the leaves. We show that CO is able to activate key genes related to the circadian clock, such as CCA1, LHY, PRR5 and GI, at the end of a long day by binding to specific sites on their promoters. Moreover, a significant number of PRR5 repressed target genes are upregulated by CO, and this could explain the phase transition promoted by CO. The CO-PRR5 complex interacts with the bZIP transcription factor HY5 and helps to localize the complex in the promoters of clock genes. Our results indicate that there may be a feedback loop in which CO communicates back to the circadian clock, feeding seasonal information to the circadian system. Overall design: Chromatin immunoprecipitation (ChIP-seq) for CONSTANS in Arabidopsis. Seedlings were grown in LD and harvested at 7DAG. CO ovexpressing lines were used to get high protein amount. CO mutants (co-10) were employed as control. The immunoprecipitation was carried out using specific anti-CO antibodies

植物是固着生长的生物，演化出高度可塑性的发育策略以适应外界环境。其中，成花转变是保障生殖成功的核心环节，受到多条内源与外源遗传网络的精细调控。调控植物对日照长度响应的光周期途径，是控制开花的关键途径之一。在拟南芥（Arabidopsis）的光周期开花调控过程中，CONSTANS（CO）是在长日照结束时，于叶片中激活成花素FLOWERING LOCUS T（FT）表达的核心基因。CONSTANS的表达受到生物钟（circadian clock）的强烈调控，但截至目前，尚无关于光周期途径向生物钟反馈环路的研究报道。
本研究通过转录调控网络分析，鉴定出调控生物钟与光周期途径互作的关键网络基序。通过基因表达检测、染色质免疫沉淀实验与表型分析，我们阐明了CONSTANS对生物钟的调控作用：CONSTANS表达量改变的植株，其内源生物钟周期发生变化，该周期可通过叶片的每日节律性运动进行测定。研究证实，CONSTANS可在长日照结束时，通过结合CCA1、LHY、PRR5与GI等生物钟关键基因启动子的特定区域，激活这些基因的表达。此外，PRR5的大量抑制靶基因可被CONSTANS上调，这或可解释CONSTANS介导的节律相位转变。CONSTANS-PRR5复合物可与bZIP转录因子HY5相互作用，并协助该复合物定位至生物钟基因的启动子区域。本研究结果表明，存在一条由CONSTANS向生物钟反馈的环路，该环路可将季节信息传递至生物钟系统。
整体实验设计：对拟南芥中的CONSTANS进行染色质免疫沉淀测序（ChIP-seq）。实验材料为长日照（LD）条件下生长7天的幼苗（7DAG）；为获得足量靶蛋白，采用CONSTANS过表达株系进行实验，同时以CONSTANS突变体（co-10）作为对照；免疫沉淀实验使用特异性抗CONSTANS抗体完成。