Genome-wide characterization of polyphenol oxidase (PPO) gene family in tobacco and the molecular improvement of browning reaction for K326 tobacco leaves

Polyphenol oxidase (PPO) oxidizes polyphenols, causing browning in tobacco ( Nicotiana tabacum L.) leaves during roasting, which reduces their quality and commercial value. In this study, 13 NtPPO genes were identified through genome mining, and their bioinformatic analyses were performed, including gene structure, protein structure, and physicochemical properties. Tissue-specific expression analysis found that the NtPPO genes were substantially expressed in leaves, whereas NtPPO9 and NtPPO10 were also highly expressed in flowers. During leaf growth, NtPPO1, 3, 5, 6, and 7 expression levels changed dramatically, whereas NtPPO2, 9, 10, and 13 expression patterns remained steady. Notably, NtPPO1 displayed the highest expression levels during the maturity stage of tobacco leaves. Subcellular localization revealed that NtPPO1, 6, 9, and 12 were primarily distributed in chloroplasts. KO-87 was generated through CRISPR/Cas9 technology, including mutations in the NtPPO1, 2, 4, 6, 8, 9, 11, 12, and 13 genes. KO-87 exhibits similar growth to the wild-type, but with decreased PPO activity in leaves and browning levels after roasting. Metabolomics analysis indicated that polyphenol metabolites were significantly upregulated, while quinone metabolites were significantly downregulated in the leaves of mutant plants. This study systematically explored the functions of NtPPO genes, providing preliminary insights into their key roles in the browning process of tobacco leaves. These findings offer new research directions and potential solutions for addressing the browning problem during tobacco leaf roasting. • NtPPO multi-gene mutant KO-87 constructed via CRISPR/Cas9. • Mutation of NtPPOs reduces the degree of browning in tobacco leaves after roasting. • Tobacco mutants show elevated polyphenolics, reduced quinones.