Supplementary file 1_Fertilizer placement modulates the effects of soil metabolite formulations on nutrient status, phenylpropanoid–flavonoid metabolism, and sensory quality in flue-cured tobacco.docx

Relative to CK2, A3 (well-cellar application with soil metabolite formulation 1) and A4 (well-cellar application with soil metabolite formulation 2) increased PPO activity by 29.49% and 28.16%, respectively, and PAL activity by 81.08% and 73.91%, respectively, whereas SPS activity showed only a non-significant upward trend. A3, which combined well-cellar application with soil metabolite formulation 1, showed the best overall sensory profile, with improved aroma quality, reduced irritancy and off-odors, and greater smoke softness.

Transcriptomic and metabolomic analyses showed that treatment-responsive genes and metabolites were mainly associated with plant hormone signaling, photosynthesis, sugar and amino acid metabolism, and phenylpropanoid and flavonoid biosynthesis. Integrated omics analysis revealed treatment-dependent changes in phenylpropanoid–flavonoid metabolism. Weighted gene co-expression network analysis (WGCNA) identified a darkgreen module containing 4,363 genes that was significantly correlated with several flavonoid-related metabolites, including quercetin, kaempferol, myricetin, and 5,7-dihydroxyflavanone (r = 0.56–0.69), and eight candidate hub genes were identified.</p>Conclusion<p>Well-cellar application combined with soil metabolite formulation 1 may improve nitrogen and potassium status in mature leaves and coordinate phenylpropanoid–flavonoid metabolism and quality-precursor accumulation, thereby enhancing the overall sensory quality of cured leaves.

These findings highlight fertilizer placement as an important determinant of soil metabolite formulation performance and provide a theoretical basis for precision rhizosphere fertilization and the rational use of soil metabolite formulations in flue-cured tobacco production.</p>