Comprehensive study on softness mechanisms and the metabolic pattern of key cell wall components in tobacco leaves during the curing stage

Abstract

Background: Curing is an essential process for transforming tobacco leaves into an economic product. Optimizing curing parameters to regulate the orderly senescence and apoptosis of tobacco cells serves as a direct approach to enhancing the softness of leaves and a critical guarantee of quality. Therefore, understanding the intrinsic mechanisms underlying leaf softening during curing is vital for curing conditions and improving tobacco usability.

Results: To elucidate the degradation patterns of key cell wall components and dynamic changes in related enzyme activities during the softening of tobacco leaves during curing stage, we systematically analyzed the softening progression, cell wall structure, and dynamic transformation of key metabolites in the flue-cured variety Yunyan 87. The results demonstrated that leaf softness peaked during the yellowing stage (minimal softness value: 11.83mN). The cell wall structure progressively disintegrated throughout curing. Pectin methylesterase (PME), polygalacturonase (PG), and pectin lyase (PL) exhibited higher activities in fresh leaves and the yellowing stage, with corresponding values of 8.33 and 20.56 U/g for PME, 1.20 and 1.33 U/g for PG, and 2.39 and 4.93 U/g for PL. The cell wall matrix content decreased significantly during curing, reaching 412.84 mg/g in the drying stage.LC-MS/MS analysis identified 1,220 metabolites with significant alterations during curing, 54 of which were closely associated with cell wall metabolism. We further delineated metabolic pathways for cellulose, pectin, and lignin.

Conclusions: This study comprehensively investigated the metabolic basis of tobacco leaf softening during curing, identifying the fresh leaves and yellowing stage as critical regulatory nodes. These findings provide valuable references for optimizing tobacco curing parameters.

Keywords: Cell wall components; Curing stage; Flue-cured tobacco; Metabolic pattern; Softness mechanisms.

Fig. 1

Multiscale evolution of tobacco leaf properties during curing. a Curing process and morphological features: Fresh leaves (FL), Yellowing stage (YS), Fixing stage (FS), Drying stage (DS). The blue line indicates the dry-bulb temperature in the curing chamber, and the red line represents the wet-bulb temperature. Red arrows point to representative leaf morphology at corresponding stages. b Bar graph shows softness values (left Y-axis; higher values indicate poorer flexibility). Line graph displays relative moisture content (right Y-axis, %). Different lowercase letters denote significant differences in softness and moisture content across stages (Duncan’s test, p < 0.05). c Ultrastructural Observations: Transmission electron microscopy (TEM) images (Scale bar = 5 μm, consistent across all panels). From left to right: FL, YS, FS, and DS

Fig. 2

Dynamic changes in cell wall components and key enzymatic activities of tobacco leaves during curing stage. Enzyme activities of PME a, PL b, PG c, endo-β−1,4-glucanase d, exo-β−1,4-glucanase e and β-gal f; The contents of cellulose g, hemicellulose h, lignin i, protopectin j, soluble pectin k, and cell wall matrix content l; The horizontal axes of all subplots consistently represent the curing stages from left to right as: Fresh leaf (FL), yellowing stage (YS), fixing stage (FS), and drying stage (DS)

Fig. 3

Integrated metabolomic profiling of tobacco leaves during curing stage. a Principal component analysis (PCA) plot of metabolomic data showing separation of sample groups across curing stages. b Bar graph displaying the count of metabolites upregulated and downregulated in the yellowing stage (YS), fixing stage (FS), and drying stage (DS) relative to the fresh leaves(FL). c Venn diagram illustrating the overlap of differentially abundant metabolite profiles identified in the YS, FS, and DS stages compared to FL. d Heatmap showing relative abundance of clustered metabolites associated with cell wall components, selected from the global metabolome via pathway screening (Table 1), color scale represents z-scores of log₂-transformed relative abundance with red indicating above-average abundance, blue indicating below-average abundance, and saturation corresponding to the absolute value of the z-score.

Fig. 4

Metabolic pathways of cellulose and pectin in tobacco leaves during the curing stages. The bars arranged left to right showing relative abundances of key metabolites in the fresh leaf (FL, green), yellowing stage (YS, yellow), fixing stage (FS, blue), and drying stage (DS, red). *, **, ***, and **** indicate significant differences at p < 0.05, p < 0.01, p < 0.001 and p < 0.0001, respectively (Student’s t test)

Fig. 5

Metabolic pathways of lignin in tobacco leaves during the curing stages. The bars arranged left to right showing relative abundances of key metabolites in the fresh leaf (FL, green), yellowing stage (YS, yellow), fixing stage (FS, blue), and drying stage (DS, red). *, **, ***, and **** indicate significant differences at p < 0.05, p < 0.01, p < 0.001 and p < 0.0001, respectively (Student’s t test)