The rapid softening of sweet cherry fruits during ripening results in the deterioration of fruit quality. However, few genes related to sweet cherry fruit ripening and softening have been identified, and the molecular regulatory mechanisms underlying this process are poorly understood. Here, we identified and functionally characterized PavNAC56, a NAC transcription factor that positively regulates sweet cherry fruit ripening and softening. Gene expression analyses showed that PavNAC56 was specifically and abundantly expressed in the fruit, and its transcript levels increased in response to abscisic acid (ABA). A subcellular localization analysis revealed that PavNAC56 is a nucleus‐localized protein. Virus‐induced gene silencing of PavNAC56 inhibited fruit ripening, enhanced fruit firmness, decreased the contents of ABA, anthocyanins, and soluble solids, and down‐regulated several fruit ripening‐related genes. Yeast one‐hybrid and dual‐luciferase assays showed that PavNAC56 directly binds to the promoters of several genes related to cell wall metabolism (PavPG2, PavEXPA4, PavPL18, and PavCEL8) and activates their expression. Overall, our findings show that PavNAC56 plays an indispensable role in controlling the ripening and softening of sweet cherry fruit and provides new insights into the regulatory mechanisms by which NAC transcription factors affect nonclimacteric fruit ripening and softening.

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Sweet cherry, an economically important horticultural crop, has strong antioxidant activity. The fruits contain compounds potentially beneficial to human health—particularly anthocyanins, which are synthesized in cytosol and predominantly accumulated in vacuoles. Although anthocyanin levels differ among dark-red, blush, and yellow sweet cherry cultivars, the regulatory mechanism of anthocyanin transport and accumulation is not well understood in this species. In this study, we identified 53 glutathione S-transferase genes (PavGSTs) from sweet cherry and found that PavGST1 expression was well correlated with anthocyanin accumulation in cultivars with different fruit skin colors. TRV-mediated virus-induced silencing of PavGST1 decreased anthocyanin accumulation in sweet cherry fruits and downregulated the expressions of anthocyanin biosynthetic and regulatory genes. In addition, transient overexpression of PavGST1 promoted anthocyanin accumulation. Furthermore, yeast one-hybrid and dual-luciferase assays revealed that PavMYB10.1 and PavMYB75 directly bind to different MYB binding sites of the PavGST1 promoter (MBS-1 and MBS-3) to activate PavGST1 transcription. According to our results, PavGST1 plays a central role in sweet cherry fruit anthocyanin accumulation. Our findings provide novel insights into the coordinative regulatory mechanisms of PavGST1 and PavMYBs in anthocyanin accumulation in sweet cherry.