Effects Of Different Growth Temperature And Nitrogen Fertilization On Amino Acid Content And It’s Metabolism In Tobacco Leaves

Amino acids are the basic units of proteins. Growth temperature and soil nutrients have significant impact on the tobacco plant growth and tobacco leaf flavor.Tobacco is important cash crop in China, it’s quality is influenced by many factors,among them, free amino acids content in tobacco leaves is closedly related with tobacco aroma and taste. It is found that free amino acids content in the leaves of tobacco is related with different growth temperature and nitrogen fertilizer level.However, the mechanism of this effects awaits to be further investigated in tobacco.In this thesis, the total free amino acids content were detected and proline and phenylalanine metabolisms were studied in tobacco(Yunyan87) leaves under different growth temperatures and nitrogen fertilization level. The results were showed as the follows:1. Effects of different growth temperature on free amino acid content as well as proline and phenylalanine metabolism in tobacco leavesTo investigate the effects of different growth temperature on free amino acids content, as well as proline and phenylalanine metabolism in tobacco leaves, tobacco seedlings were grown in greenhouse after sowing and floated nursery, then the tobacco plants were transfered to phytotron with three dynamic temperatures set as average 18.5℃, 23.5℃ and 28.5 ℃, and treated by these temperatures throughout the period after rosette stage. Total amino acid content is detected by spectrophotometry,free amino acids content is analysed under different growth temperature by gas chromatography and mass spectrometry. The results show that, with different growth temperature, the content of total amount of amino acids and single amino acids changes significantly. Among them, with different growth temperature, the content of total amount of amino acids, proline and phenylalanine at the average temperature of18.5℃ is the highest, the content at average temperature 23.5℃ followed, and the content at average temperature 28.5℃ is the lowest. In addition, this study investigated the proline and phenylalanine metabolism related enzyme activity and gene expression. Proline metabolism related enzymes P5 CS, OAT’s activity isreduced with tobacco plants’ growth. At average temperature 18.5℃, the activity is highest. Pro DH’s activity is declined first and ascended with tobacco plants’ growth.And the activity is highest at average temperature 28.5℃. The trend of P5 CS, OAT and Pro DH gene expression is similar with the related enzyme’s activity changes.2. Effects of different nitrogen fertilizer level on amino acid content, proline and phenylalanine metabolism in tobacco leavesIn order to understand the influence of different nitrogen fertilizer level on free amino acids of the maturation stage in the middle tobacco leaves, tobacco seedlings were grown in greenhouse after sowing and floated nursery, then the tobacco plants were transplanted to field with the nitrogen fertilizer level set as 60kg/hm2, 90kg/hm2 and 120kg/hm2. Total amino acid content was detected by spectrophotometry and free amino acid content at different growth temperature is analysed by two-step derivatization and gas chromatography mass spectrometry. The results show that,with the difference nitrogen level, content of the total amount of amino acids and free amino acids changes significantly. Among them, with the increasing of nitrogen,content of the total amino acid, proline and phenylalanine is rising. Content of leucine,isoleucine, alanine, and 4-aminobutyric acid is the highest in the nitrogen levels60kg/hm2, among them, content of leucine and isoleucine is the lowest in nitrogen levels is 90kg/hm2, content of alanine, and 4-aminobutyric acid is the lowest in nitrogen levels 120kg/hm2. Activity of proline metabolism related enzymes and phenylalanine metabolism related enzymes is enhanced with nitrogen application levels, and the same of related gene expression quantities. Proline metabolism related enzymes P5 CS, OAT and Pro DH ’s activity is increased with increased nitrogen level,the related genes of OAT and Pro DH expression quantity is similar with the trend of its related enzyme activity, and P5 CS gene expression in the 60kg/hm2 is the highest,and 90kg/hm2 is the lowest. Activity of phenylalanine metabolism related enzymes C4 H and PAL is increasing with the increase of nitrogen fertilizer rate; The gene expression is positively correlated with enzyme activity.