Proteomics analysis of nitrogen stress on the influence of carbon and nitrogen metabolism of flue-cured tobacco.

Nitrogen stress is the main abiotic stress factor affecting the carbon and nitrogen balance of flue-cured tobacco. Using tandem mass tag (TMT) proteomics, we identified 157 differentially expressed proteins (DEPs) under low nitrogen (LN, 60 kg/hm2; 65 with increased abundance, 92 with decreased abundance) and 219 DEPs under high nitrogen (HN, 150 kg/hm2; 123 with increased abundance, 96 with decreased abundance). In nitrogen metabolism, LN downregulated proteins involved in nitrogen absorption and transport (e.g., nitrite reductase, glutamine synthetase), weakening pathway activity; however, it upregulated tyrosine aminotransferase to promote tyrosine catabolism and generate nitrogen-containing intermediates, compensating for nitrogen deficiency. HN, by contrast, upregulated proteins in porphyrin/chlorophyll metabolism (e.g., protoporphyrinogen oxidase), amino acid metabolism, and alkaloid biosynthesis, enhancing nitrogen-related processes. In carbon metabolism, LN inhibited glycolysis, the tricarboxylic acid (TCA) cycle, and the pentose phosphate pathway via downregulating key enzymes. HN promoted starch, sucrose, fructose, and mannose synthesis (e.g., sucrose synthase upregulation) and the pentose phosphate pathway, but downregulated TCA cycle-related proteins, leading to carbon-nitrogen metabolic imbalance.Notably, both LN and HN enhanced the biosynthesis of isoquinoline, tropane, piperidine, and pyridine alkaloids, but via distinct enzymes: LN relied on tyrosine aminotransferase, while HN depended on amine oxidase. This study reveals the “stress-specific regulation” mechanism of carbon-nitrogen metabolism in flue-cured tobacco under nitrogen stress, providing a theoretical reference for precise nitrogen fertilizer management and carbon-nitrogen balance optimization in tobacco production.