Physiologicals And Differential Proteomic Analysis Of Flue-cured Tobacco Seeding Under Different Nitrogen Level

Tobacco is highly sensitive to fertilizer. Fertilization is one of the important link of tobacco cultivation. Different flue-cured tobacco cultivar show different nitrogen nutritional requirements. Three flue-cured tobacco varieties, K326,CB-1,Red Flower Big Jinyuan (hereinafter referred to as Red Big), were used in this study. The effects of three nitrogen level [1.0 mmol/L (low), 4 mmol/L (normal) and 8.0 mmol/L (high) NH₄NO₃] on physiological characteristics of seedlings were investigated. Also, the differentially expressed proteins of tobacco K326 leaves under different nitrogen level were studied using two-dimensional electrophoresis to study. The major results are as follow:1. Chlorophyll (Chl) content and photosynthetic rate(Pn) in flue-cured tobacco leaves increased with increasing nitrogen supply. As nitrogen supply increased from 0.5 mmol/L to 2.0 mmol/L, the activities of carbon metabolism enzymes and the accumulation of photosynthetic product in leaves increased, then decreased under 8.0 mmol/L. By comparision, we found that photosynthesis was more sensitive to high nitrogen in K326 and CB-1 than in Big Red. The activities of carbon metabolism enzymes are highest in normal nitrogen level. No significant difference was found in sucrose synthase (SS),sucrose phosphate synthase (SPS) among different varieties. The activity of acid invertase (AI) was significantly higher in K326 leaves than in CB-1 and Red Big leaves. This is advantage for the formation of"luzhou-flovor"smoke. On the low and normal nitrogen levels, CB-1 had the highest accumulation of photosynthetic product, followed by Red Big and K326. On the high nitrogen level, Red Big ad the highest accumulation of photosynthetic product. Based on these results, it can be concluded that the ability of the carbon metabolism varied with tobacco varieties.2. Nitrogen supply increased the contents of total nitrogen, K⁺, Mg²⁺, amino acids, NO₃^-N and the activities of glutamine synthetase (GS) in leaves, but did not significantly affect the contents of total phosphorus and nicotine. Nitrate reductase (NR) activity was the highest in normal nitrogen leaves, both low and high nitrogen decreased its activity. Total contents of N, P and K+ were higher in K326 leaves than in Red Big and CB-1 ones.In high nitrogen level,GS activity and NR activity are high. compare with K326 and Red Big ,CB-1 has lowest N ,P K⁺ and Mg²⁺ the activity of NR is low also.3. Nitrogen supply increased the activity of catalase (CAT), decreased the contents of malondialdehyde (MDA) and H₂O₂ in leaves, thus decreasing the membrane-lipid peroxidation. Both low and high nitrogen increased leaf activity of superoxide dismutase (SOD) This might help to delay aging and increase the quality of tobacco. The activity of SOD was the highest in K326 leaves, followed in CB-1 and Red Big ones. Leaf content of MDA was lower in K326 leaves than in Red Big and CB-1 ones.. The activity of CAT was lower in Red Big leaves than in K326 and CB-1 ones, while the content of H₂O₂ was higher in the former. Based on these results, it can be concluded that the mechanism in response to nitrogen is different among three varieties.4. In tobacco K326 leaves, 18 proteins were differentially expressed under different nitrogen levels. These proteins are related to photosynthesis and carbon assimilation, stress, material and energy metabolism and function proteins. The expression of photosynthesis related proteins were up-regulated with increasing nitrogen supply, meaning that nitrogen play a significant role in promoting photosynthesis. The expression of stress-related proteins was up-regulated in both low nitrogen and high nitrogen. The expression of energy related proteins was up-regulated under high nitrogen, meaning that nitrogen metabolism in seedlings is activeIn conclusion, the response of flue-cured to nitrogen was induced by external signal. In tobacco cultivation, it could change nitrogen nutrient supply to regulate the carbon and nitrogen metabolisn in order to improve yields and quality of tobacco leaves. It could be concluded that normal nitrogen is conducive to growth and the production of high quality tobacco. K326 has the highest phosphorus content, lowest sucrose and soluble sugar contents, in high notrogen level,it has highest NO₃^-N content, this may concern about strong resistance to high notrogen and the formation of"Luzhou-flavor"tobacco style ; CB-1 The highest Chl content in leaves, on low and normal nitrogen levels,has higher sucrose content than K326 and Red Big, this may concern about to format the"Qing scent"tobacco style. CAT activity and H₂O₂ content is lowest in Red Big under each nitrogen levels, Pn and k⁺ content is lowest in high nitrogen level, this may relate to weaker relevant of Red Big.