PuMYB21/PuMYB54 coordinate to activate PuPLDβ1 transcription during peel browning of cold-stored “Nanguo” pears

Refrigeration is commonly used to extend the storage life of "Nanguo" pears, but fruit in long-term refrigeration is prone to peel browning, which is related to membrane lipid degradation. To determine the mechanism of membrane lipid degradation, we identified two R2R3-MYB transcription factors (TFs), PuMYB21 and PuMYB54, from "Nanguo" pears, which were notably expressed in response to cold stress and during the peel-browning process. The results from yeast one-hybrid, electrophoretic mobility shift, and transient expression assays indicated that both PuMYB21 and PuMYB54 directly bind to the promoter of PuPLDβ1 (a key enzyme catalyzing the hydrolysis of membrane phospholipids) and activate its expression, which probably enhances the degradation of membrane phospholipids and eventually results in peel browning. Moreover, the overexpression of PuMYB21 and PuMYB54 can greatly activate the transcription of endogenous PuPLDβ1 in both "Nanguo" pear fruits and calli, and their silencing can inhibit its transcription. Furthermore, yeast two-hybrid, bimolecular fluorescence complementation, and pull-down assays verified that PuMYB21 interacts with PuMYB54 to enhance the expression of PuPLDβ1. In summary, we demonstrate that PuMYB21 and PuMYB54 may have roles in membrane lipid metabolism by directly binding to the downstream structural gene PuPLDβ1 during the low temperature-induced peel browning of "Nanguo" pears.