Regulation Of Different Growth Temperatures And Nitrogen Levels On Flavonoids Content And Their Metabolic Pathways In Tobacco Leaves

Tobacco(Nicotiana tobacum L.) is an important cash crop in China, temperature and nitrogen levels are important environmental factors which play important roles on the growth and development of tobacco plants, leaf quality and secondary metabolites. Therefore, comprehensive and in-depth studying on effects of temperature and nitrogen levels on tobacco quality has important significance, the research showed that flavone compounds rutin is an important index affecting the quality of tobacco,This paper, taking the tobacco plants(Yunyan 87) as materals, studied the effects of different growth temperature(temperatures averaged 18.5, 23.5 and 28.5 ℃) and different nitrogen levels(60, 90 and 120 Kg/hm2) on the metabolism of flavonoids in tobacco leaves, in order to provide theoretical basis for the effect of temperature and nitrogen levels on flavonoids metabolism of tobacco leaves.The major results were shown as follows, 1. Regulation of different growth temperatures on flavonoids content and their metabolic pathways in tobacco leavesIn this study, the tobacco cultivar "Yunyan 87" was used as experimental material growed in artificial climate chamber with three different growth temperatures respectively(temperatures averaged 18.5, 23.5 and 28.5 ℃). Through using liquid chromatography coupled with mass spectrometry, spectrophotometry and fluorescence quantitative PCR methods to study the effects of different growth temperature on the contents of flavones rutin and kaempferol-3-O-rutin, the related enzyme activities and gene expression during the growth and development of tobacco leaves. The results showed that, with the growth and development of tobacco leaves, the expression of the genes related to flavonoids metabolism firstly decreased and then increased, and it regulated the related enzyme activities and the content of rutin firstly decreased and then increased. And make the content of flavonoids rutin firstly decreased and then increased, while kaempferol 3-O-rutin content was gradually decreased. In addition, compared with the tobacco leaves grew under higher temperature(temperatures averaged 23.5 and 28.5 ℃), the tobacco leaves grew under lower temperature(temperature averaged 18.5 ℃) enhanced the gene expression of PAL, C4 H, 4CL, CHS, F3 H, F3’H and FLS, improved the key enzyme activities of PAL, C4 H, 4CL and CHI, and finally promoted accumulation of the flavones rutin and kaempferol 3-O-rutin(P<0.05). It indicated that lower growth temperature(temperature averaged 18.5 ℃) is in favour of the accumulation of flavonoids in tobacco leaves. 2. Regulation of different nitrogen levels on flavonoids content and their metabolic pathways in tobacco leavesIn this study, the tobacco cultivar "Yunyan 87" was used as experimental material, it set with three different nitrogen levels respectively(60, 90, 120 Kg/hm2), Through using ultra performance liquid chromatography, spectrophotometry and fluorescence quantitative PCR methods to study the effects of different nitrogen levels on the contents of flavone compounds rutin, kaempferol-3-O-rutin and total flavonoids, the related enzymes activities and the expression of related genes in tobacco leaves during the growth and development of tobacco leaves. The results show that, with the growth and development of tobacco leaves, the gene expression of flavonoid metabolism related gene PAL, C4 H, 4CL, CHS, CHI, F3 H, F3’H, FLS, A3 RT was gradually decreased in general, and it regulated the content of Kaempferol-3-O- rutin and total flavonoids showed a trend of gradually reduce,while the content of rutin was first increased slightly and then decreased. Under different nitrogen levels, at the early stage of the process(50 and 83 d), the tobacco leaves grown under lower nitrogen level 60 Kg/hm2 enhanced gene expression of the flavonoids metabolism related genes PAL, C4 H, 4CL, CHS, CHI, F3 H, F3’H, FLS, A3 RT and ANS, raised the key enzyme activities of PAL and CHI related to flavonoids metabolism, and finally promoted accumulation of the flavonoids rutin, kaempferol 3-O-rutin and total flavonoids(P<0.05) in tobacco leaves; At the late period of treatment(106 d), the tobacco leaves grown under higher nitrogen level 120 Kg/hm2 enhanced gene expression of the flavonoids metabolism related genes PAL, C4 H, 4CL, CHS, CHI, F3 H, F3’H, A3 RT and ANS, improved the key enzyme activities of PAL and CHI related to flavonoids metabolism, and finally promoted accumulation of the flavonoids rutin, kaempferol 3-O-rutin and total flavonoids(P<0.05) in tobacco leaves. Furthermore, With the growth and development, the key enzyme activity of flavonoids metabolic PAL and CHI were gradually increased, there was a very significant positive correlation between the gene expression of anthocyanin synthase ANS(P<0.01). It is speculated that PAL, CHI enzyme activity regulates the synthesis of anthocyanin compounds, with the growth and development, the anthocyanin compounds may be gradually accumulation.