PbWRKY26 positively regulates malate accumulation in pear fruit by activating PbMDH3

Malate is the main organic acid that affects fruit acidity and flavor in pear (Pyrus spp.). However, the regulatory mechanism of malic acid accumulation in pear remains unclear. We identified PbWRKY26 as a candidate gene using mRNA-seq, and quantification analysis verified the expression level. The expression of PbWRKY26 was positively correlated with the malic acid content in two P. pyrifolia cultivars ('Cuiguan', 'Hongsucui') and two P. ussuriensis cultivars ('Qiuxiang', 'Hanhong'), with respective correlation coefficients of 0.748*, 0.871**, 0.889**, and 0.910** (*, P < 0.05; **, P < 0.01). The expression of PbWRKY26 enhanced the malate content in overexpression transgenic pear fruit and callus. In contrast, silencing PbWRKY26 decreased the pear fruit malic acid content. Analysis of the neighbor-joining phylogenetic tree indicated that PbWRKY26 was a PH3 homolog. The WRKY26 (PH3) has been identified to regulate a proton pump gene, PH5, in a lot of plant species, but the LUC and Y1H assays showed that PbWRKY26 could not bind to PbPH5 promoter in our study. Interestingly, a malate dehydrogenase gene, PbMDH3, was identified to be regulated by PbWRKY26. This study might be valuable to understand the metabolic regulatory network associated with malate accumulation.