Functional and regulatory mechanisms of the NtPPO5 gene in the browning reaction of tobacco

Tobacco (Nicotiana tabacum L.) is a vital economic crop, yet it frequently suffers excessive enzymatic browning during the curing process, which negatively impacts its leaf color and commercial value. In this study, we constructed NtPPO5 (Nitab4.5_0003171g0010.1) gene overexpression and knock-out vectors and generated transgenic tobacco plants to investigate the role of NtPPO5 in curing-induced browning. Investigation of agronomic traits demonstrated that NtPPO5 plays a positive regulatory role in plant height and leaf area. The curing experiment revealed that overexpression of NtPPO5 exacerbated enzymatic browning, whereas knockout of NtPPO5 effectively controlled the browning reaction. To elucidate the underlying mechanisms, we conducted enzyme activity assays, physiological and biochemical measurements, and non-targeted metabolomics analyses. The results demonstrated that knockout of NtPPO5 significantly reduced polyphenol oxidase (PPO) activity while inducing compensatory upregulation of superoxide dismutase (SOD) activity. Additionally, peroxidase (POD) activity exhibited distinct peak stages compared to wild-type cultivar Cuibi-1 (CB-1). Metabolomics analysis revealed significant changes in the levels of key phenolic substrates involved in enzymatic browning during the curing process, particularly shikimic acid and phenylpropanoic acid compounds, which were predominantly enriched in the Flavonoid Biosynthesis Pathway (map00941). The knockout of NtPPO5 optimized Flavonoid Biosynthesis Pathway by regulating precursor availability and metabolic fluxes, enhancing antioxidant defenses, and reducing curing-induced browning. This study provides theoretical insights and practical evidence for mitigating browning in flue-cured tobacco and offers valuable guidance for optimizing the curing process.