PuMYB21/PuMYB54 coordinate to activate PuPLD beta 1 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 PuPLD beta 1 (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 PuPLD beta 1 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 PuPLD beta 1. In summary, we demonstrate that PuMYB21 and PuMYB54 may have roles in membrane lipid metabolism by directly binding to the downstream structural gene PuPLD beta 1 during the low temperature-induced peel browning of Nanguo pears.