已表明miR840a结合在双反向靶基因WHIRLY3和PPRs的3’UTR上，过表达和敲除miR840a促进和延缓拟南芥叶片衰老和改变衰老相关基因的表达，双突变whirly3 pprs具有和过表达miR840a相同的早衰表型。基于此，本项目利用拟南芥miR840a/WHIRLY3/PPRs的单、双和三敲除和过表达纯合突变体，综合CHIP-seq、bulk cDNA sequencing、RIP-seq和MALDI-MS等技术，探索miR840a如何剪切、抑制或修饰WHIRLY3和PPRs的转录、翻译，WHIRLY3和PPRs及其互作如何对靶基因的转录本进行剪辑，miR840a和WHY3-PPRs RNA剪辑的靶基因如何调控植物衰老的发生；阐明miR840a对靶基因WHIRY3和PPRs的作用机制，揭示miR840a与WHIRLY3-PPRs共同介导的靶基因的RNA剪辑调控植物衰老的分子机理

Plant senescence is controlled by genes and affected by internal and external environmental factors or signals including delivered from organelle, and it plays an important role in the whole life cycle of plants. Understanding its regulatory mechanism of plant senescence initiation and processes has important biological significance and theoretical and application value on increasing crop biomass, improving crop agronomic traits. Our previous results showed that miR840a is a natural antisense miRNA (nat-miRNA), which was expressed from the opposite strand of its predicted target gene, WHIRLY3 (WHY3), and within the annotated 3´ untranslated region (3´UTR) of a PPR (PPRs) mRNA. MiR840a is developmentally expressed at the onset of plant senescence, its overexpression or knockdown results in a opposite leaf senescent phenotype as compared to the WT plants. Using oemiR840a and kdmiR840a transgenic plants, the results of quantitative RT-PCR and immunodetection reveal that miR840a repress PPRs transcription by targeting and cleaving mRNA or inhibit WHY3 protein accumulation by targeting 3’UTR and interference its translation, but does not synergistically change WHY3 transcription, indicating that miR840a independently affect PPRs at transcriptional level and WHY3 at translational level. However, single mutation neither of pprs nor why3 could result in leaf senescent phenotype; and the double mutations of pprs why3 partly achieved senescence phenotype. The expression of senescence related genes that are differentially expressed in oemiR840a transcriptome show mostly consistent expression in the pprs why3 double mutants. It demonstrates that miR840a affected both targeted genes WHY3 and PPRs independently, which both synergistically co-regulated leaf senescence events. Based on existing knowledge, WHY3 functions as a cofactor of WHY1 for double strand break (DSBs) related DNA damage repair in plastids, for RNA processing, and for DNA binding protein of kenesin gene in the nucleus; The typical PPR protein have high consensus function domain, is targeted to mitochondria or chloroplasts in plants, binds one or several organelle transcripts, and influences their expression by altering RNA sequence, turnover, processing, or translation. The PPRs is an E subclass of PPR protein, is targeted to chloroplast. Most PPR proteins have functions in one of the four processes, RNA editing, splicing, maturation, and translation activation. All the editing functions reported so far are associated with either PPR-DYW or PPR-E type proteins. Based on this and our previous research, this project is intended to use the identified miR840a, why3, pprs homozygous single mutant, double mutant and triple mutants in Arabidopsis, by comprehensively using CHIP-seq, bulk cDNA sequencing, RNA-CoIP-seq, MALDI-MS, CoIP, BIFC, Y2H etc techniques and bioinformatics methods, it is explored that : (1) how miR840a targets, cleavages, inhibits or modifies target genes both WHY3 and PPRs transcription, translation, or expression; (2) how WHY3，PPRs and both effect on downstream targeted RNA processing including RNA recognized site, RNA editing, splicing, maturation, and translation activation, (3) how the plastid target genes of both miR840a and WHIRLYY3-PPRs mediated RNA processing affect the incidence of plant aging. It will illustrate the function and genetic interaction of miR840a with two anti-orientation target genes WHY3 and PPRs, reveal the regulatory mechanism of miR840a and WHY3-PPRs mediated RNA splicing and editing on plant senescence, and demonstrate the action of plastid gene RNA editing on the nuclear events of plant aging.