Characterization of a pericarp browning related LACCASE 14-4 from longan fruit with a focus on (epi)catechin oxidative polymerization

Abstract

Polyphenol oxidase (PPO) has long been considered as the key enzyme responsible for the pericarp browning of longan fruit after harvest. However, due to the overlapping substrate ranges of PPOs and laccases, the contribution of laccases in longan pericarp browning is unclear. In this study, we found that the contents of flavan-3-ols and procyanidin A2/B2 in longan pericarp decreased during browning after harvest. It might be caused by high activity of laccases rather than PPOs. A 100-kDa laccase was purified from the pericarp and its protein sequence was identical to the deduced sequence of LAC14-4. The laccase showed more favored to (−)-epicatechin (EC) than other flavan-3-ols and procyanidin A2/B2 identified in the pericarp. Using EC and (+)-catechin (CT) as substrates, brown products were generated by LAC14-4 and exogenously expressed LAC14-4-3 × HA in vitro; isomers of procyanidin polymers were detected in the products. Transient expression of LAC14-4-GFP in tobacco leaves showed that LAC14-4 was located in the vacuoles and endoplasmic reticulum. In situ hybridization showed that LAC14-4 mainly expressed in the outer part of mesocarp closed to the brachysclereid layer. The transcript, protein and activity levels of LAC14-4 in pericarp were upregulated during browning. In summary, longan pericarp contains high levels of LAC14-4 that catalyzes the procyanidin polymerization leading to longan pericarp browning.