Application and Effect of Micropeptide miPEP164c on Flavonoid Pathways and Phenolic Profiles in Grapevine “Vinhão” Cultivar

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Climate change increasingly challenges viticulture, demanding innovative and sustainable strategies to preserve grapevine productivity and grape quality. MicroRNA-encoded peptides (miPEPs) have emerged as natural regulators of gene expression, providing a novel mechanism for fine-tuning plant metabolism. Here, we evaluated whether exogenous application of miPEP164c, previously shown to repress VviMYBPA1 in vitro, can modulate flavonoid pathways in field-grown grapevines (Vitis vinifera L. cv. Vinhão). Grape clusters were sprayed with 1 µM miPEP164c before and during véraison, and molecular, biochemical, and metabolomic analyses were performed at harvest. miPEP164c treatment significantly upregulated pre-miR164c transcripts, leading to post-transcriptional silencing of VviMYBPA1 and strong downregulation of the proanthocyanidin-related genes VviLAR1, VviLAR2, and VviANR. Correspondingly, LAR and ANR activities were reduced by up to 75%, and total proanthocyanidin content decreased by nearly 30%. Metabolomic profiling showed reduced flavan-3-ols and moderate shifts in phenolic acids and stilbenoids, while anthocyanins increased slightly. Overall, miPEP164c reprogrammed flavonoid metabolism under vineyard conditions, selectively lowering tannin biosynthesis without affecting other key phenolics. These findings establish miPEPs as promising biostimulants for precise modulation of grape berry composition, offering new tools for urgently needed sustainable and precision viticulture and improved wine quality under climate change and the increasing environmental challenges it poses.