State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai, China.
School of Life Sciences, Qilu Normal University, Jinan, China.
PLoS Genet. 2019 Apr 10;15(4):e1008068. doi: 10.1371/journal.pgen.1008068. eCollection 2019 Apr.
The roles of histone demethylation in the regulation of plant flowering, disease resistance, rhythmical response, and seed germination have been elucidated recently; however, how histone demethylation affects leaf senescence remains largely unclear. In this study, we exploited yeast one-hybrid (Y1H) to screen for the upstream regulators of NONYELLOWING1 (NYE1), and identified RELATIVE OF EARLY FLOWERING6 (REF6), a histone H3 lysine 27 tri-methylation (H3K27me3) demethylase, as a putative binding protein of NYE1 promoter. By in vivo and in vitro analyses, we demonstrated that REF6 directly binds to the motif CTCGYTY in NYE1/2 promoters through its zinc finger domain and positively regulates their expression. Loss-of-function of REF6 delayed chlorophyll (Chl) degradation, whereas overexpression of REF6 accelerated Chl degradation. Subsequently, we revealed that REF6 positively regulates the general senescence process by directly up-regulating ETHYLENE INSENSITIVE 2 (EIN2), ORESARA1 (ORE1), NAC-LIKE, ACTIVATED BY AP3/PI (NAP), PYRUVATE ORTHOPHOSPHATE DIKINASE (PPDK), PHYTOALEXIN DEFICIENT 4 (PAD4), LIPOXYGENASE 1 (LOX1), NAC DOMAIN CONTAINING PROTEIN 3 (AtNAC3), and NAC TRANSCRIPTION FACTOR-LIKE 9 (NTL9), the key regulatory and functional genes predominantly involved in the regulation of developmental leaf senescence. Importantly, loss-of-function of REF6 increased H3K27me3 levels at all the target Senescence associated genes (SAGs). We therefore conclusively demonstrate that H3K27me3 methylation represents an epigenetic mechanism prohibiting the premature transcriptional activation of key developmentally up-regulated senescence regulatory as well as functional genes in Arabidopsis.
最近，组蛋白去甲基化在调控植物开花、抗病性、节律响应和种子萌发方面的作用已经阐明；然而，组蛋白去甲基化如何影响叶片衰老在很大程度上仍不清楚。在这项研究中，我们利用酵母单杂交（Y1H）筛选 NONYELLOWING1（NYE1）的上游调控因子，并鉴定出 RELATIVE OF EARLY FLOWERING6（REF6），一种组蛋白 H3 赖氨酸 27 三甲基化（H3K27me3）去甲基酶，是 NYE1 启动子的一个假定结合蛋白。通过体内和体外分析，我们证明 REF6 通过其锌指结构域直接结合到 NYE1/2 启动子中的 motif CTCGYTY，并正向调节它们的表达。REF6 的功能丧失会延迟叶绿素（Chl）降解，而过表达 REF6 则加速 Chl 降解。随后，我们揭示了 REF6 通过直接上调 ETHYLENE INSENSITIVE 2（EIN2）、ORESARA1（ORE1）、NAC-LIKE，ACTIVATED BY AP3/PI（NAP）、PYRUVATE ORTHOPHOSPHATE DIKINASE（PPDK）、PHYTOALEXIN DEFICIENT 4（PAD4）、LIPOXYGENASE 1（LOX1）、NAC DOMAIN CONTAINING PROTEIN 3（AtNAC3）和 NAC TRANSCRIPTION FACTOR-LIKE 9（NTL9），这些是主要参与调控发育性叶片衰老的关键调控和功能基因，正向调控一般性衰老过程。重要的是，REF6 的功能丧失会增加所有目标 Senescence associated genes（SAGs）的 H3K27me3 水平。因此，我们得出结论，H3K27me3 甲基化代表了一种表观遗传机制，阻止了关键发育性上调的衰老调控和功能基因在拟南芥中的过早转录激活。
