Genome‐wide analysis of coding and non‐coding RNA reveals a conserved miR164‐NAC regulatory pathway for fruit ripening

Abstract

Summary: Kiwifruit (Actinidia spp.) is a climacteric fruit with high sensitivity to ethylene, influenced by multiple ethylene‐responsive structural genes and transcription factors. However, the roles of other post‐transcriptional regulators (e.g. miRNAs) necessary for ripening remain elusive. High‐throughput sequencing sRNAome, degradome and transcriptome methods were used to identify further contributors to ripening control in the kiwifruit (A. deliciosa cv ‘Hayward’). Two NAM/ATAF/CUC domain transcription factors (AdNAC6 and AdNAC7), both predicted targets for miR164, showed significant upregulation by exogenous ethylene. Gene expression analysis and luciferase reporter assays indicated that Ade‐miR164 and one of its precursor miRNAs (Ade‐MIR164b) were repressed by ethylene treatment and negatively correlated with AdNAC6/7 expression. Subsequent analysis indicated that both AdNAC6 and AdNAC7 proteins are transcriptional activators and physically bind the promoters of AdACS1 (1‐aminocyclopropane‐1‐carboxylate synthase), AdACO1 (1‐aminocyclopropane‐1‐carboxylic acid oxidase), AdMAN1 (endo‐β‐mannanase) and AaTPS1 (terpene synthase). Moreover, subcellular analysis indicated that the location of the AdNAC6/7 proteins was influenced by Ade‐miR164. Multiple omics‐based approaches revealed a novel regulatory link for fruit ripening that involved ethylene‐miR164‐NAC. The regulatory pathway for miR164‐NAC is present in various fruit (e.g. Rosaceae fruit, citrus, grape), with implications for fruit ripening regulation.