Polyphenol oxidase gene editing changed the flavonoid composition and browning process of litchi (Litchi chinensis Sonn.) callus

•The PPO gene-edited callus exhibited a slower browning process compared to WT callus.•The total flavonoid content significantly increased in the PPO gene-edited callus.•(−)-Epicatechin was the primary direct substrate for PPO-mediated enzymatic browning reaction in litchi callus. Postharvest pericarp browning, caused primarily by the enzymatic oxidation of phenols, reduces the shelf life and market value of litchi fruit and is considered a major limitation for the development of the litchi industry. Previous studies have shown that polyphenol oxidase (PPO) is a key enzyme and that flavonoids are important substrates for enzymatic browning; however, direct evidence is still lacking. This study investigated the differences in the browning process among the wild type (WT) and four PPO gene-edited litchi calli to verify the function of PPO in the browning of litchi tissues. Compared to the WT callus, the proliferation rate, relative expression of litchi PPO gene (LcPPO), PPO activity and color changes significantly decreased or slowed down in all gene-edited calli, indicating that the latter exhibited a slower browning process. Using a liquid chromatography tandem mass spectrometry approach (LC-MS/MS), 83 metabolites of flavonoids were identified, of which 58 were differentially accumulated metabolites (DAMs). Venn analysis revealed 12 common DAMs across different genotypic contrasts that were mostly enriched in the flavonoid biosynthesis pathway. It was presumed that the decrease of LcPPO expression in gene-edited calli led to the reduced PPO activity, then reduced the (−)-epicatechin oxidation. The accumulation of (−)-epicatechin caused the common upregulation of procyanidin B2 and upstream substances such as dihydrokaempferol, taxifolin, naringenin chalcone, 7,4′-dihydroxyflavone, and rutin in their biosynthesis pathways. The results provide novel evidence that (−)-epicatechin acts as the primary direct substrate in the enzymatic browning reaction mediated by PPO.