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. 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.

拟南芥中介导环境开花响应的多条信号通路，最终汇聚于作用于茎尖分生组织（shoot apical meristem）以调控成花转变（floral transition）的发育进程。已有诸多已鉴定的突变会削弱环境开花响应，但下游发育进程却更难通过诱变手段得以解析。我们构建了一处多环境通路受损的五重突变体（quintuple mutant），并发现其对光周期（photoperiod）与环境温度（ambient temperature）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分析后发现，赤霉素生物合成基因（gibberellin biosynthetic genes）与参与年龄途径（age pathway）的转录因子的表达水平与开花进程显著相关。对该五重突变体进行诱变后，我们筛选得到两株晚花突变体（late-flowering mutants）：五重EMS诱变突变体1（qem1）与qem2。通过同基因作图（isogenic mapping）与转基因互补（transgenic complementation）实验，我们鉴定出了这两株突变体的致病基因。qem1突变是GA20氧化酶2（GA20ox2）的等位基因突变，这一结果证实了在环境响应缺失的情况下，赤霉素对于开花进程的关键作用。与之相反，qem2的突变位点位于染色质重塑因子4（CHROMATIN REMODELING 4, CHR4）基因中，而该基因此前并未被遗传学研究证实参与开花诱导过程。通过免疫共沉淀（co-immunoprecipitation）、RNA测序（RNA-seq）与染色质免疫共沉淀测序（ChIP-seq）实验，我们发现CHR4可与参与花分生组织属性（floral meristem identity）调控的转录因子相互作用，并影响关键开花调控因子的表达。综上，我们认为CHR4在花序分生组织（inflorescence meristem）中发挥重要作用，以促进花属性（floral identity）的建立。我们在短日照时间序列实验中，对野生型（WT）与svp flc ft tsf soc1突变体、svp flc ft tsf soc1与qem2、Col株系与chr4-2突变体的茎尖组织开展了差异表达基因分析。