Fertilizer placement modulates the effects of soil metabolite formulations on nutrient status, phenylpropanoid–flavonoid metabolism, and sensory quality in flue-cured tobacco

Abstract

Background The quality of flue-cured tobacco depends on nutrient acquisition, carbon–nitrogen allocation, and quality-related secondary metabolism, particularly phenylpropanoid and flavonoid pathways. However, whether fertilizer placement and exogenous soil metabolite formulations can jointly improve leaf nutritional physiology and cured-leaf quality remains unclear. Methods A field experiment was conducted with the flue-cured tobacco cultivar Yunyan 87 (Nicotiana tabacum L.). Six treatments combined two fertilizer placement methods, strip application and well-cellar application, with two soil metabolite formulations at the same total fertilizer application rate. Nitrogen, phosphorus, and potassium contents and the activities of polyphenol oxidase (PPO), phenylalanine ammonia-lyase (PAL), and sucrose-phosphate synthase (SPS) were determined in mature middle leaves. Sensory evaluation of cured leaves was integrated with transcriptomic, metabolomic, multi-omics, and weighted gene co-expression network analyses. Results Compared with conventional strip application (CK1), well-cellar application (CK2) increased leaf nitrogen, phosphorus, and potassium contents by 4.45%, 7.57%, and 6.89%, respectively, with significant increases in nitrogen and potassium. 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. Conclusion 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.