Promoter cis ‐element variations in the CgWRKY1 ‐ CgZAT12 ‐ CgGAD4 module regulate citric acid degradation in Citrus grandis

Citric acid plays a key role in determining the quality and flavor of citrus fruits. However, the transcriptional regulatory mechanisms controlling citric acid levels are not well-understood. In this study, we identified a natural bud mutation of the 'Wu Suan (WS)' pomelo (Citrus grandis) variety, which has significantly reduced citric acid content. By comparing the gene expression profiles of 'WS' with the moderate-acid variety 'Gao Ban (GB)', we found that the gene CgGAD4, associated with citric acid degradation, was highly expressed in 'WS'. Overexpression of CgGAD4 decreased citric acid levels, while suppressing its expression increased acidity. CgZAT12 was identified as a transcription factor (TF) that activates CgGAD4. A single nucleotide change in the promoter region of CgGAD4 (from AGTGT to AATGT) in 'WS' enhanced the binding of CgZAT12, leading to higher gene transcriptional activity. Further analysis revealed a variation in the W-box element of the CgZAT12 promoter (from GGTCAA in 'GB' to GATCAA in 'WS'). This change prevents the upstream TF CgWRKY1 from suppressing CgZAT12 in 'WS', unlike in 'GB'. These findings reveal the key genetic variations in the WRKY1-ZAT12-GAD4 regulatory module involved in citric acid degradation and provide new insights into improving citrus fruit quality.