Proteomic Response Of Nitrogen Metabolism In Flue-cured Tobacco Under Different Nitrogen Application Levels

Nitrogen is one of the important nutrients for the growth and development of flue-cured tobacco.Many nitrogen-containing compounds in tobacco leaves are related to the level of nitrogen supply,and the effects of nitrogen-containing compounds on tobacco leaf quality are heterogeneous.In order to regulate nitrogen-containing compounds in tobacco leaves,proteomics technology was used to analyze the protein expression differences of tobacco leaves under different nitrogen application levels,and to explore the differences in the metabolic pathways of main nitrogen-containing compounds under different nitrogen stresses,in order to deduce the key pathways for regulating nitrogen-containing compounds.The study came to the following conclusions:1.Using TMT quantitative proteomics analysis technology,7629 proteins were quantified in tobacco leaves at the cluster stage,and 6128 were quantified 10 days after topping;compared with 7.0kg/667m~2(normal nitrogen application rate), 5.5kg/667m~2(Low nitrogen)and 8.5kg/667m~2(high nitrogen)treatments had 157and 219 differential proteins at the cluster stage,respectively,and 646 and 35 at 10 d after topping.The low nitrogen treatment had more differential proteins at the mature stage,while the high nitrogen treatment had more at the cluster stage.3.The main nitrogen-containing compounds in flue-cured tobacco include nitrate nitrogen,ammonium nitrogen,protein,amino acids,nitrogen-containing pigments, heterocyclic compounds and nicotine.NRT1/PTR-6.4 at the cluster stage played a leading role on the nitrate nitrogen content of flue-cured tobacco;the increase of ammonium nitrogen content increased with the increase of nitrogen application level,and the ammonium nitrogen content at this time depended on the source of ammonium nitrogen.No nitrate-nitrogen and ammonium-nitrogen-related transporters were detected 10 days after topping.At this time,the source and consumption of ammonium-nitrogen were in a state of dynamic equilibrium.Low nitrogen promotes protein degradation,while high nitrogen inhibits the metabolism of aspartic acid, glutamic acid,phenylalanine and tyrosine and the mutual conversion of glutamic and oxalic amino acids in flue-cured tobacco at the mass tree stage,and promotes glutamic acid Amino acids are converted into each other and the expression of coenzymes is promoted at the same time;high nitrogen inhibits the metabolism of phenylalanine,tyrosine,glutamic acid,aspartic acid and the conversion of glutamine to glutamic acid 10 days after topping.Both high nitrogen and low nitrogen at the cluster stage inhibited the synthesis of chlorophyll;high nitrogen and normal nitrogen application levels promoted the degradation of chlorophyll.High nitrogen after topping promotes chlorophyll synthesis and degradation.Low nitrogen promotes pyrimidine synthesis and inhibits pyrimidine degradation at the cluster stage,while high nitrogen inhibits pyrimidine degradation;high nitrogen promotes pyrimidine synthesis and degradation 10 days after topping.At the clustering stage,increasing the nitrogen application rate was beneficial to the synthesis of nicotine in flue-cured tobacco,but when the nitrogen application rate exceeded the normal nitrogen application rate,the nicotine synthesis-related proteins were inhibited;10 days after topping,N-carbamoyl group in high nitrogen Putrescine amidase promotes nicotine synthesis.Both low nitrogen and high nitrogen promote the degradation of nicotine.4.Low nitrogen mainly affects carbohydrates in tobacco leaves,followed by nitrogenous compounds.High nitrogen mainly affects nitrogenous compounds and has little effect on carbohydrates.Under low nitrogen treatment,nitrogen content and protein were significantly reduced,nicotine did not change significantly,and water-soluble total sugar and reducing sugar were significantly increased;under high nitrogen treatment,nicotine and protein were significantly increased,total nitrogen,water-soluble total sugar,There was no significant change in reducing sugar,and chlorine content decreased.The nitrogen application rate had no effect on the potassium content of tobacco leaves.5.Nitrogen is preferentially allocated to structural proteins in flue-cured tobacco to meet the needs of growth and development.Under low nitrogen conditions,carbon and nitrogen metabolism decreases,and tobacco yield and quality decrease.By promoting the activities of nitrate reductase and nitrite reductase,nitrogen absorption is increased.It is an effective method to improve nitrogen metabolism.In addition,it can improve carbon metabolism by promoting the synthesis of delta-aminolevulinic acid to protoporphyrin IX,and the improvement of related enzyme activities in the synthesis pathway of protoporphyrin IX to chlorophyll.Under high nitrogen conditions,excessive accumulation of nitrate nitrogen leads to increased nicotine and protein.When nitrate absorption cannot be reduced,nicotine content can be reduced by inhibiting amine oxidase activity,or by increasing niacin-nucleotide Pyrophosphorylase activity promotes the degradation of nicotine and plays a role in regulating nicotine.After topping,it can promote protein degradation,and promote the expression of nitrate reductase and nitrite reductase,so that nitrogen is stored in the form of ammonium nitrogen and amino acid,which can achieve the purpose of reducing protein.