The H3K27me3 demethylase REF6 promotes leaf senescence through directly activating major senescence regulatory and functional genes in Arabidopsis

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. Author summary Leaves of higher plants start yellowing and subsequently die (senescence) at particular developmental stages as a result of both internal and external regulations. Leaf senescence is evolved to facilitate nutrient remobilization to young/important organs to meet their rapid development, and a large number of genes (Senescence associated genes, SAGs) are activated to regulate/facilitate the process. It has been intriguing how these genes are kept transcriptionally inactive to ensure an effective photosynthesis before the initiation of leaf senescence. Here, we reveal an epigenetic mechanism responsible for the prohibition of their premature transcription. We found that an H3K27me3 demethylase, RELATIVE OF EARLY FLOWERING 6 (REF6), directly promotes the expression of its ten target senescence regulatory and functional genes (EIN2, ORE1, NAP, AtNAC3, NTL9, NYE1/2, LOX1, PAD4, and PPDK), which are involved in major phytohormones' signaling, biosynthesis, and chlorophyll degradation. Crucially, REF6 is substantially involved in promoting the H3K27me3 demethylation of both their promoter and/or coding regions during the aging process of leaves. We therefore provide conclusive evidence that H3K27me3 methylation is an epigenetic mechanism hindering the premature transcriptional activation of key SAGs, which helps to explain the aging effect of senescence initiation.