Secondary metabolic pathways in grape berries are tightly regulated by an array of molecular mechanisms, including microRNA-mediated post-transcriptional regulation. As recently discovered, before being processed into mature miRNAs, the primary transcripts of miRNAs (pri-miRNAs) can encode for small miRNA-encoded peptides (micropeptides - miPEPs) that ultimately led to an accentuated downregulation of the respective miRNA-targeted genes. Although few studies about miPEPs are available, the discovery of miPEPs reveals a new layer of gene regulation at the post-transcriptional level and may present a key advantage in agronomy. Here, we identified a miPEP encoded in non-mature miR164c putatively targeting grapevine transcription factor VvMYBPA1 (miPEP164c/miPEP-MYBPA1), a positive regulator of key genes in the proanthocyanidin-biosynthetic pathway, one that competes directly for substrate with the anthocyanin-biosynthetic pathway. Thus, the objective of this work was to test the hypothesis that the exogenous application of miPEP164c (miPEP-MYBPA1) can modulate the secondary metabolism of grape berry cells by inhibiting PA biosynthetic pathway while simultaneously stimulating anthocyanin synthesis. The exogenous application of miPEP164c to suspension-cultured cells from grape berry (cv. Gamay) enhanced the transcription of its corresponding pri-miR164c, thus leading to a more pronounced post-transcriptional silencing of its target VvMYBPA1. This led to a significant inhibition of the proanthocyanidin pathway, mostly via inhibition of leucoanthocyanidin reductase and anthocyanidin reductase enzymatic activities and VvLAR1 downregulation. In parallel, the anthocyanin-biosynthetic route was stimulated. Anthocyanin content was 31 % higher in miPEP164c-treated cells, in agreement with the higher activity of VvUFGT and the corresponding VvUFGT1 transcripts.

中文翻译：

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外源应用未成熟的miRNA编码的miPEP164c抑制原花青素的合成并刺激花青素在葡萄浆果细胞中的积累

葡萄浆果中的次级代谢途径受到一系列分子机制的严格调控，包括microRNA介导的转录后调控。正如最近发现的那样，在被加工成成熟的miRNA之前，miRNA的初级转录本（pri-miRNA）可以编码小的miRNA编码的肽（micropeptide-miPEPs），最终导致各个miRNA靶向基因的加重下调。尽管很少有关于miPEPs的研究，但miPEPs的发现揭示了转录后水平上基因调控的新层面，并可能在农学上表现出关键优势。在这里，我们确定了在非成熟miR164c中编码的miPEP，该miR164c可能靶向葡萄转录因子VvMYBPA1（miPEP164c / miPEP-MYBPA1），这是原花色素生物合成途径中关键基因的正向调节剂，直接与花青素生物合成途径竞争底物的一种。因此，这项工作的目的是检验以下假设：miPEP164c（miPEP-MYBPA1）的外源应用可以通过抑制PA生物合成途径同时刺激花色苷的合成来调节葡萄浆果细胞的次级代谢。将miPEP164c外源应用于来自葡萄浆果（cv。Gamay）的悬浮培养细胞可增强其相应pri-miR164c的转录，从而导致其靶标VvMYBPA1的转录后沉默更加明显。这导致对原花色素途径的显着抑制，主要是通过抑制白花色素还原酶和花色素还原酶的酶活性以及VvLAR1下调。在平行下，花青素的生物合成途径受到刺激。在miPEP164c处理的细胞中，花色素苷含量高出31％，这与VvUFGT和相应的VvUFGT1转录物的更高活性相一致。