Widely targeted metabolomics reveals maturity-associated metabolic differences and their associations with style characteristics in flue-cured tobacco from distinct ecological regions of Guizhou.

To clarify maturity-associated metabolic differences in flue-cured tobacco grown in distinct ecological regions of Guizhou and their relationships with post-curing style characteristics, middle leaves of Yunyan 87 were collected from Dafang County (DF) and Weining County (WN), representing honey-sweet and fresh-sweet regions, respectively, at 16 (T1), 18 (T2), 20 (T3), 22 (T4), and 24 (T5) days after topping. Leaf phenotypes, color parameters, SPAD values, key enzyme activities, and sensory style scores were assessed across the five stages, whereas widely targeted metabolomics was performed on cured leaves from T1 and T5, resulting in four metabolomic groups: DF_T1, DF_T5, WN_T1, and WN_T5. Local environmental and soil variables and RT-qPCR validation of selected pathway genes were incorporated to strengthen ecological and transcriptional interpretation. With delayed harvest, leaves from both regions changed from green to yellow-green and pale yellow, the midrib gradually lost greenness and turned whitish, and trichome shedding increased; these phenotypes appeared earlier in DF. SPAD values generally declined in both regions and remained higher in WN than in DF. POD, LOX, PPO, and SPS activities increased with maturity, with generally higher activities in DF. Sensory evaluation indicated that the target style characteristics of cured leaves from both regions were enhanced with increasing maturity. Widely targeted metabolomics of cured leaves identified 72 differential metabolites in DF between T5 and T1, including 44 upregulated and 28 downregulated metabolites, with high proportions of flavonoids, alkaloids, terpenoids, and other phenolic-related metabolites. In WN cured leaves, 51 differential metabolites were identified, including 19 upregulated and 32 downregulated metabolites, mainly associated with sugar metabolism, glutathione metabolism, flavonoid biosynthesis, and ABC transporters. Environmental correlation analysis suggested that radiation-, temperature-, precipitation-, and soil-related variables were associated with region-specific flavonoid and antioxidant-related metabolic patterns. RT-qPCR of selected flavonoid biosynthesis and sugar metabolism/transport genes in fresh leaves supported pathway-level transcriptional divergence, although partial transcription-metabolite decoupling was also observed. Collectively, these results reveal regional differences in physiological and metabolic remodeling at key maturity stages and provide preliminary metabolomics-based evidence for optimizing harvest timing and style-oriented quality regulation of flue-cured tobacco in Guizhou.