A natural mutation in the promoter of the aconitase gene ZjACO3 influences fruit citric acid content in jujube.

Jujube ( Ziziphus jujuba Mill.) is the most economically important fruit tree of the Rhamnaceae and was domesticated from wild or sour jujube ( Z. jujuba Mill. var. spinosa Hu). During the process of domestication, there was a substantial reduction in the content of organic acids, particularly malate and citrate, which greatly influence the taste and nutritional value of the fruit. We previously demonstrated that ZjALMT4 is crucial for malate accumulation. However, the mechanism of citrate degradation in jujube remains poorly understood. In the present study, aconitase ZjACO3 was shown to participate in citric acid degradation in the cytoplasm through the GABA pathway. Interestingly, we discovered an E-box mutation in the ZjACO3 promoter (-484A > G; CAAGTG in sour jujube mutated to CAGGTG in cultivated jujube) that was strongly correlated with fruit citrate content; 'A' represented a high-citrate genotype and 'G' represented a low-citrate genotype. We developed and validated an ACO-based Kompetitive allele-specific PCR (KASP) marker for determining citric acid content. Yeast one-hybrid screening, transient dual-luciferase assays, and overexpression analyses showed that the transcription factor ZjbHLH113 protein directly binds to CAGGTG in the promoter of ZjACO3 in cultivated jujube plants, transcriptionally activating ZjACO3 expression, and enhancing citric acid degradation. Conversely, binding ability of the ZjbHLH113 protein to CAAGTG was weakened in sour jujube, thereby promoting citrate accumulation in the fruit. These findings will assist in elucidating the mechanism by which ZjACO3 modulates citrate accumulation in sour jujube and its cultivars.