MicroRNA840 accelerates leaf senescence by targeting the overlapping 3’UTRs of PPR and WHIRLY3 in Arabidopsis thaliana

Abstract MicroRNAs (miRNAs) negatively regulate gene expression by cleaving the target mRNA and/or impairing its translation, thereby playing a crucial role in plant development and environmental stress responses. In Arabidopsis, MIR840 is located within the overlapping 3’UTR of PPR and WHIRLY3 (WHY3), both being predicted targets of miR840. Gain- and loss-of-function of miR840 in Arabidopsis resulted in opposite senescent phenotypes. Highest expression of pri-miR840 is observed at senescence initiation, and is negatively correlated with a significant reduction of PPR transcripts but not of WHY3. Although WHY3 transcript levels were not significantly affected by miR840 overexpression, its protein synthesis was strongly reduced. Mutating the cleavage sites or replacing the target sequences abolishes the miR840-mediated degradation of PPR transcripts and inhibition of WHY3 translation. In support for this, concurrent knock-down of both PPR and WHY3 in the WT resulted in the senescent phenotype resembling that of the miR840-overexpressing mutant. This indicates that both PRR and WHY3 are targets in the miR840-regulated senescent pathway. Moreover, single knockout mutant of PPR or WHY3 shows a convergent up-regulated subset of senescence-associated genes, which are also found among those induced by miR840 overexpression. Our data provide evidences for a regulatory role of miR840 in plant senescence. Highlight MicroRNA840 (miR840) has a unique miRNA-target configuration regulating PPR and WHIRLY3 genes in Arabidopsis. MiR840 is highly expressed at the onset of plant senescent stage. Both PPR and WHIRLY3 transcripts are specifically targeted in vivo within their 3’UTR region by mature miR840 or its star strand in vivo. Interestingly, PPR expression is mainly repressed on mRNA transcript level by cleavage, while WHIRLY3 is predominantly translationally inhibited. We conclude that miR840 enhances plant senescence via post transcriptional gene silencing of PPR and WHIRLY3 , which appear to be novel negative joint regulators of plant senescence. Footnote: The author(s) responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the intructions for Authors is: Ying Miao ( ymiao@fafu.edu.cn )