MicroRNA840 (MIR840) accelerates leaf senescence by targeting the overlapping 3'UTRs of PPR and WHIRLY3 in Arabidopsis thaliana

MicroRNAs negatively regulate gene expression by promoting target mRNA cleavage and/or impairing its translation, thereby playing a crucial role in plant development and environmental stress responses. In Arabidopsis, the MIR840 gene is located within the overlapping 3'UTR of the PPR and WHIRLY3 (WHY3) genes, both being predicted targets of miR840* and miR840, the short maturation products of MIR840. Gain- and loss-of-function of MIR840 in Arabidopsis resulted in opposite senescence phenotypes. The highest expression levels of the MIR840 precursor transcript pre-miR840 were observed at senescence initiation, and pre-miR840 expression is significantly correlated with a reduction in PPR, but not WHY3, transcript levels. Although a reduction of transcript level of PPR, but not WHY3 transcript levels were not significantly affected by MIR840 overexpression, its protein levels were strongly reduced. Mutating the cleavage sites or replacing the target sequences abolishes the miR840*/miR840-mediated degradation of PPR transcripts and accumulation of WHY3 protein. In support for this, concurrent knockdown of both PPR and WHY3 in wild-type plants resulted in a senescence phenotype resembling that of the MIR840-overexpressing plant. This indicates that both PRR and WHY3 are targets in the MIR840-mediated senescence pathway. Moreover, single knockout mutants of PPR and WHY3 show a convergent upregulated subset of senescence-associated genes, which are also found among those induced by MIR840 overexpression. Our data provide evidence for a regulatory role of MIR840 in plant senescence.