The PuNAC2–PutDT regulatory module drives late-stage vacuolar citrate accumulation in pear fruit

Abstract

In most fleshy fruits, including apple, cherry, plum, and grape, citric acid content declines during the late stages of fruit development, a process that largely determines fruit flavor. In contrast, pear (Pyrus spp.) exhibits a less common developmental pattern characterized by continued citric acid accumulation during late fruit development. Although similar patterns have been reported in a limited number of other fruit species and germplasms, the molecular mechanisms underlying this developmental trajectory remain largely unknown. Here, comparative analyses of organic acid dynamics among cultivated pear varieties revealed that citric acid predominantly accumulates during late fruit development. Transcriptome analysis combined with M-fuzz soft clustering identified the vacuolar dicarboxylate transporter PutDT and the NAC transcription factor PuNAC2 as candidate genes closely associated with this developmental pattern. Electrophysiological analyses demonstrated that PutDT functions as a pH-dependent vacuolar transporter for both citrate and malate, with a higher affinity for citrate under acidic conditions. Functional analyses further demonstrated that PutDT promotes citric acid accumulation in pear fruit, callus, and a heterologous tomato system. Furthermore, PuNAC2 directly activated PutDT transcription by binding to the SNBE motif in its promoter, and its positive effect on citric acid accumulation depended on PutDT activation. Collectively, these findings reveal a PuNAC2–PutDT regulatory module controlling vacuolar citric acid accumulation during pear fruit development and provide molecular insights into the regulation of fruit acidity, as well as potential molecular targets for acidity improvement.