Analysis of the causes of ash hanging in flue-cured tobacco upper leaves in sandy mud field in Guangdong tobacco-growing areas based on metabolomics and proteomics

【Objective】 Based on the combined analysis of metabolomics and proteomics，the differences in material metabolism and protein expression of flue-cured tobacco upper leaves in sandy mud field and pruple soil of Guangdong tobacco-growing areas were analyzed to provide reference for reducing ash deposition of tobacco leaves in sandy mud field. 【Method】 Purple soil flue-cured tobacco leaves that were less prone to ash hanging after curing（control）and sandy mud field flue-cured tobacco leaves that were prone to ash hanging after curing were used as the research objects. The fresh tobacco leaves of the upper leaves of the 2 soil types at 4 stages of before maturity，just mature，mature and overmature were selected to determine the activity of polyphenol oxidase（PPO）. Subsequently，a combined metabolomic and proteomic analysis was conducted on mature tobacco leaves to identify differential metabolites and differentially expressed proteins present in mature upper leaves from sandy mud fields. Additionally，this study aimed to analyze and screen for key pathways influencing browning in these upper tobacco leaves grown in sandy mud field. 【Result】 During the maturation of the upper leaves，the PPO activity of the purple soil tobacco leaves was extremely significantly lower than that of the sandy mud field tobacco leaves（P<0.01），and the difference between the two was the largest during the maturation period. A total of 129 differential metabolites and 1314 differentially expressed proteins were screened from the mature tobacco samples of the 2 soil types，and the differential metabolites and differentially expressed proteins were mainly up-regulated. The clustering of differential metabolites and KEGG metabolic pathway analysis were mainly enriched in tyrosine metabolism，phenylalanine-tyrosine-tryptophan biosynthesis，phenylpropanoid biosynthesis. GO functional annotation and KEGG signal pathway enrichment analysis of differentially expressed proteins were mainly enriched in phenylpropanoid biosynthesis，tyrosine metabolism，isoquinoline alkaloids biosynthesis and plant hormone signal transduction. During the biosynthesis of polyphenols，the differentially expressed proteins of phenylalanine ammonia lyase，4-coumaric acid coenzyme A ligase and hydroxycinnamoyl coenzyme A shikimate/quinic acid hydroxycinnamoyl transferase were down-regulated in sandy mud field tobacco leaves. The differentially expressed proteins of phytoene desaturase，zeaxanthin epoxidase，9-cis-epoxycarotenoid dioxygenase and xanthooxin dehydrogenase were down-regulated in sandy mud field tobacco leaves. Differential metabolites phaseic acid and dihydrophaseic acid were down-regulated. 【Conclusion】 Compared with the flue-cured tobacco upper leaves in purple soil，the flue-cured tobacco upper leaves in sandy mud field are more susceptible to environmental stress during the growth process，resulting in a decrease in the quality of tobacco leaves，an increase in abscisic acid synthesis，activation of hormone signals，enhancement of phenolic metabolism，and an increase in PPO activity. During the curing process，it is easy to lead to more severe enzymatic browning，resulting in easy ash hanging of cured tobacco leaves.