Transcription factors PpWRKY50 and PpWRKY75 play positive roles in malate accumulation in pear

Malate is a predominant organic acid in many fruit and received considerable attention thanks to its role in establishing the flavor, perception of sweetness, and nutritional quality of fruit. The transcription regulation of malate-metabolizing enzymes and vacuolar transporters has been studied extensively. However, the knowledge regarding transcription regulation of malate accumulation by WRKY transcription factors remains poorly understood in pear (Pyrus spp.). Our previous study demonstrated that PpWRKY44 function as a positive regulator of malate accumulation. In this study investigated the functional role of two WRKY transcription factors, PpWRKY50 and PpWRKY75, in controlling malate accumulation. The results indicated that the transient overexpression of PpWRKY50 and PpWRKY75 resulted in significantly higher malate content than the control. Reverse transcription quantitative PCR analysis showed that the expression levels of genes related to malate synthesis and transport, including the cytosolic malate dehydrogenase (PpcyMDH) and the vacuolar proton pump genes (PpVHA-A, PpVHP1 and PpVHP2), were more highly expressed in the PpWRKY50-overexpressing calli, while the PpWRKY75-overexpressing calli significantly increased the expression levels of PpcyMDH, PpVHA-A, PpVHP1, PpVHP2, aluminum-activated malate transporter 9 (PpALMT9), and tonoplast dicarboxylate transporter 1 (PptDT), compared to the control. Furthermore, transiently overexpressed PpWRKY50 and PpWRKY75 in tobacco leaves significantly enhanced the accumulation of malate. These findings suggest that PpWRKY50 and PpWRKY75 play positive roles in controlling malate accumulation in pear and provide new insights into the molecular function of WRKY transcription factors in malate accumulation.