ABSTRACT
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.
ABSTRACT
Jujube (Ziziphus jujuba Mill.), the most economically important fruit tree in Rhamnaceae, was domesticated from sour jujube (Z. jujuba Mill. var. spinosa (Bunge) Hu ex H.F.Chow.). During domestication, fruit sweetness increased and acidity decreased. Reduction in organic acid content is crucial for the increase in sweetness of jujube fruit. In this study, the determination of malate content among 46 sour jujube and 35 cultivated jujube accessions revealed that malate content varied widely in sour jujube (0.90-13.31 mg g-1) but to a lesser extent in cultivated jujube (0.33-2.81 mg g-1). Transcriptome sequencing analysis showed that the expression level of Aluminum-Dependent Malate Transporter 4 (ZjALMT4) was substantially higher in sour jujube than in jujube. Correlation analysis of mRNA abundance and fruit malate content and transient gene overexpression showed that ZjALMT4 participates in malate accumulation. Further sequencing analyses revealed that three genotypes of the W-box in the promoter of ZjALMT4 in sour jujube associated with malate content were detected, and the genotype associated with low malate content was fixed in jujube. Yeast one-hybrid screening showed that ZjWRKY7 binds to the W-box region of the high-acidity genotype in sour jujube, whereas the binding ability was weakened in jujube. Transient dual-luciferase and overexpression analyses showed that ZjWRKY7 directly binds to the promoter of ZjALMT4, activating its transcription, and thereby promoting malate accumulation. These findings provide insights into the mechanism by which ZjALMT4 modulates malate accumulation in sour jujube and jujube. The results are of theoretical and practical importance for the exploitation and domestication of germplasm resources.
