H3K27me3 and H3K4me3 profiles in chr4/pkr1 and WT [RNA-seq]

Several pathways conferring environmental flowering responses in Arabidopsis converge on developmental processes that act in the shoot apical meristem to mediate the floral transition. Many characterized mutations impair environmental flowering responses, however downstream developmental processes have been more refractory to mutagenesis. We constructed a quintuple mutant in which several environmental pathways are impaired and showed that its flowering responses to changes in photoperiod and ambient temperature are almost abolished. Analysis of the quintuple mutant by RNA-seq showed that expression of gibberellin biosynthetic genes and transcription factors that contribute to the age pathway correlate with flowering. Mutagenesis of the quintuple mutant recovered two late-flowering mutants, quintuple ems 1 (qem1) and qem2. The causal genes were identified by isogenic mapping and transgenic complementation. The qem1 mutation was an allele of ga20ox2, confirming the importance of gibberellin for flowering in the absence of environmental responses. By contrast, qem2 is in CHROMATIN REMODELING 4 (CHR4), which was not previously genetically implicated in floral induction. Using co-immunoprecipitation, RNA-seq and ChIP-seq, we show that CHR4 interacts with transcription factors involved in floral meristem identity and affects expression of key floral regulators. We conclude that CHR4 plays important roles in the inflorescence meristem to promote floral identity. Overall design: Examination of differentially expressed genes in betweeen WT and svp flc ft tsf soc1, svp flc ft tsf soc1 and qem2 and col and chr4-2 apices in a short day time course.

拟南芥中调控环境响应型开花的多条信号通路，最终汇聚于在茎尖分生组织（shoot apical meristem）中执行功能的发育程序，以介导成花转变。诸多已被表征的突变可削弱环境开花响应，但下游的发育程序却更难通过诱变手段获得功能缺失突变体。我们构建了一个多环境开花通路受损的五重突变体，并发现其对光周期与环境温度变化的开花响应几乎完全丧失。通过RNA测序（RNA-seq）对该五重突变体进行转录组分析，结果显示赤霉素（gibberellin）生物合成基因以及参与年龄通路（age pathway）的转录因子的表达水平与开花进程显著相关。对该五重突变体开展EMS诱变筛选，获得了两个晚开花突变体：五重EMS诱变突变体1号（quintuple ems 1, qem1）与qem2。通过同基因作图（isogenic mapping）与转基因互补实验（transgenic complementation），我们成功鉴定到了这两个突变体的致病基因。qem1突变是ga20ox2的等位基因突变，这证实了在环境响应缺失的遗传背景下，赤霉素对开花调控的关键作用。与之相反，qem2的突变位点位于染色质重塑因子4（CHROMATIN REMODELING 4, CHR4）的编码区中，而该基因此前并未被报道与成花诱导存在遗传关联。通过免疫共沉淀（co-immunoprecipitation）、RNA测序（RNA-seq）以及染色质免疫共沉淀测序（ChIP-seq）实验，我们证实CHR4可与参与花分生组织身份决定的转录因子发生相互作用，并调控关键成花调控因子的表达水平。综上，我们认为CHR4在花序分生组织中发挥重要调控功能，以促进花器官身份的建立。实验整体设计：在短日照时间序列样本中，分别检测野生型（WT）、svp flc ft tsf soc1五重突变体、qem2突变体以及哥伦比亚生态型（col）、chr4-2突变体的茎尖组织内的差异表达基因。