Two bHLH transcription factors regulate PbGME1 expression to modulate ascorbate biosynthesis and salt tolerance in pear.

Salt stress severely limits plant growth and productivity. GDP-mannose-3',5'-epimerase (GME), a key enzyme in ascorbic acid (AsA) biosynthesis, has been implicated in plant stress responses, but its role in pear salt tolerance remains unclear. Here, we identified PbGME1 as a positive regulator of salt tolerance in pear. The basic helix-loop-helix transcription factor PbbHLH67 directly binds to the PbGME1 promoter and activates its transcription, as shown by yeast one-hybrid, electrophoretic mobility shift, and dual-luciferase assays. Overexpression of PbbHLH67 enhanced salt tolerance in pear seedlings, pear calli, and Arabidopsis, whereas silencing of PbbHLH67 increased salt sensitivity in Pyrus betulifolia, accompanied by corresponding changes in AsA accumulation. We further identified PbbHLH53 as a PbbHLH67-interacting protein, which also binds the PbGME1 promoter and activates its transcription. Similar to PbbHLH67, overexpression of PbbHLH53 enhanced salt tolerance, whereas its silencing increased salt sensitivity. Notably, heterodimerization of PbbHLH67 and PbbHLH53 further strengthened PbGME1 transcriptional activation. Moreover, the AsA biosynthesis inhibitor lycorine largely abolished the enhanced salt tolerance conferred by PbbHLH67 or PbbHLH53 overexpression. Together, these findings demonstrate that the PbbHLH67-PbbHLH53 module promotes pear salt tolerance by activating PbGME1 expression and increasing AsA accumulation.