Exogenous calcium modulates oxalic acid metabolism via repression of LcAAE3 and mediates calcium accumulation in litchi pericarp and fruit pedicel.

Calcium plays a crucial role in fruit development and quality formation, yet the regulatory mechanisms underlying calcium distribution and oxalic acid metabolism in litchi remain poorly understood. In this study, the effects of exogenous calcium on calcium accumulation, fruit quality, and oxalic acid metabolism in litchi were investigated through stem infusion experiments using calcium chloride (CaCl2) and ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) treatments. The results demonstrated that exogenous CaCl2 application significantly enhanced calcium accumulation in pedicel tissues, particularly in the pith, phloem, and xylem, whereas EGTA treatment chelated endogenous calcium ions (Ca2+) and inhibited this accumulation. Notably, despite increased calcium levels in the pericarp, exogenous CaCl2 and EGTA applications did not affect fruit quality parameters, including appearance, weight, seed weight, pericarp weight, color, or total soluble solids content. Furthermore, CaCl2 treatment promoted the formation of insoluble calcium oxalate crystals in both fruit pedicels and pericarp without significantly altering soluble oxalic acid content, suggesting a homeostatic maintenance of oxalic acid levels. Through bioinformatic analysis and enzymatic assays, acyl-activating enzyme 3 (LcAAE3), an oxalyl-CoA synthetase that catalyzes the initial step of oxalic acid degradation, was identified and characterized. LcAAE3 exhibited specific catalytic activity toward oxalic acid and LcAAE3 gene showed markedly higher expression levels than its paralog LcAAE3-1 across litchi tissues. Exogenous CaCl2 suppressed LcAAE3 expression, while EGTA treatment induced its expression. Virus-induced gene silencing of LcAAE3 in fruit pedicels resulted in significant accumulation of both oxalic acid and calcium oxalate in pedicels and pericarp. In contrast, overexpression of LcAAE3 in tobacco significantly inhibited the accumulation of calcium oxalate and oxalic acid, confirming its role in promoting oxalic acid degradation. These findings reveal that exogenous calcium enhances tissue-specific calcium accumulation and calcium oxalate formation without compromising fruit quality, and establish LcAAE3 as a key regulator of oxalate homeostasis in litchi. This study provides insights into calcium-mediated oxalic acid metabolism and offers a theoretical basis for improving litchi fruit quality through calcium management.