Leaf senescence is a finely tuned and genetically programmed degeneration process, which is critical to maximize plant fitness by remobilizing nutrients from senescing leaves to newly developing organs. Leaf senescence is a complex process that is driven by extensive reprogramming of global gene expression in a highly coordinated manner. Understanding how gene regulatory networks involved in controlling leaf senescence are organized and operated is essential to decipher the mechanisms of leaf senescence. It was previously reported that the trifurcate feed-forward pathway involving EIN2, ORE1, and miR164 in Arabidopsis regulates age-dependent leaf senescence and cell death. Here, new components of this pathway have been identified, which enhances knowledge of the gene regulatory networks governing leaf senescence. Comparative gene expression analysis revealed six senescence-associated NAC transcription factors (TFs) (ANAC019, AtNAP, ANAC047, ANAC055, ORS1, and ORE1) as candidate downstream components of ETHYLENE-INSENSITIVE2 (EIN2). EIN3, a downstream signalling molecule of EIN2, directly bound the ORE1 and AtNAP promoters and induced their transcription. This suggests that EIN3 positively regulates leaf senescence by activating ORE1 and AtNAP, previously reported as key regulators of leaf senescence. Genetic and gene expression analyses in the ore1 atnap double mutant revealed that ORE1 and AtNAP act in distinct and overlapping signalling pathways. Transient transactivation assays further demonstrated that ORE1 and AtNAP could activate common as well as differential NAC TF targets. Collectively, the data provide insight into an EIN2-mediated senescence signalling pathway that coordinates global gene expression during leaf senescence via a gene regulatory network involving EIN3 and senescence-associated NAC TFs.

Plant leaves, harvesting light energy and fixing CO₂, are a major source of foods on the earth. Leaves undergo developmental and physiological shifts during their lifespan, ending with senescence and death. We characterized the key regulatory features of the leaf transcriptome during aging by analyzing total- and small-RNA transcriptomes throughout the lifespan of Arabidopsis (Arabidopsis thaliana) leaves at multidimensions, including age, RNA-type, and organelle. Intriguingly, senescing leaves showed more coordinated temporal changes in transcriptomes than growing leaves, with sophisticated regulatory networks comprising transcription factors and diverse small regulatory RNAs. The chloroplast transcriptome, but not the mitochondrial transcriptome, showed major changes during leaf aging, with a strongly shared expression pattern of nuclear transcripts encoding chloroplast-targeted proteins. Thus, unlike animal aging, leaf senescence proceeds with tight temporal and distinct interorganellar coordination of various transcriptomes that would be critical for the highly regulated degeneration and nutrient recycling contributing to plant fitness and productivity.

Methylcytosine-binding proteins containing SRA (SET- and RING-Associated) domain are required for the establishment and/or maintenance of DNA methylation in both plants and animals. We previously proposed that Arabidopsis VIM/ORTH proteins with an SRA domain maintain DNA methylation and epigenetic gene silencing in heterochromatic regions. However, their endogenous targets of epigenetic gene silencing have not been analyzed globally and the mechanisms by which VIM proteins coordinate DNA methylation and epigenetic silencing are largely unknown. In this study, a genome-wide transcript profiling analysis revealed 544 derepressed genes in a vim1/2/3 triple mutant, including 133 known genes. VIM1 bound to promoter and transcribed regions of the up-regulated genes in vim1/2/3 and VIM deficiency caused severe DNA hypomethylation in all sequence contexts at direct VIM1 targets. We found a drastic loss of H3K9me2 at heterochromatic chromocenters in vim1/2/3 nuclei. Furthermore, aberrant changes in transcriptionally active and repressive histone modifications were observed at VIM1 targets in vim1/2/3. VIM1-binding capacity to target genes was significantly reduced in the met1 background, indicating that VIM1 primarily recognizes CG methylation deposited by MET1. Overall, our data indicate that VIM proteins regulate genome-wide epigenetic gene silencing through coordinated modulation of DNA methylation and histone modification status in collaboration with MET1.