Relationship Between GS Isoenzyme Expression Characteristics And Nitrogen Transport In Tobacco At Maturity

The aggregate forms and relative molecular weights of two glutamine synthetase isoenzymes in flue-cured tobacco leaf were studied by using Native-PAGE and Blue-native PAGE techniques combined with reactive staining.The gene abundance of GS isoenzymes in mesophyll and main veins of tobacco leaves were measured by using the Real-time PCR,the protein subunit contents of GS isoenzymes in mesophyll and main veins of tobacco leaves were measured by using the Western-blot technology combined with the gray analysis.At the same time,the characteristics and differences of nitrogen metabolism in leaves of flue-cured tobacco varieties with different nitrogen use efficiency at mature stage were studied by observing the ultrastructure of chloroplast and determining chlorophyll content,enzyme activity related to nitrogen metabolism,nitrogen metabolite content in tobacco leaf,physiological parameters related to apoplast and ammonia volatilization,to study the relationship between GS isoenzyme activity and nitrogen metabolism related indicators.The optimum amount of nitrogen for three new varieties of flue-cured tobacco in Nanyang tobacco area was investigated by using the dynamic changes of GS isozyme subunits and related physiological indexes in the mature period of tobacco leaves.The results show: 1.The expression of Nt GS1,the gene encoding the GS1 isozyme of main cultivars and mesophylls of all varieties,was significantly increased after entry into senescence.After 45 days of leaf age,they all showed NC89>K326>Yuyan 10.The expression of GS2 isoenzyme gene Nt GS2 decreased significantly at the onset of aging,and then gradually decreased with the increase of leaf age.The Nt GS2 expression abundance of the inefficient nitrogen variety Yuyan 10 was lower than that of the medium nitrogen efficiency variety K326 and the nitrogen efficient variety NC89.Native-PAGE and Blue-native PAGE combined with activity staining showed that the isozymes of GS1 and GS2 in flue-cured tobacco leaf had the same aggregation pattern and the relative molecular weight was about 440 k D.The results of Western-blot indicated that the GS2 isoenzyme was the dominant species in the leaf mesophyll of flue-cured tobacco leaves,and the protein subunit content gradually decreased with the leaf age,which was consistent with the chloroplast degradation.The GS1 isoenzymes in the veins dominated,and their protein subunits increased significantly from the 45 th leaf stage and then increased first and then decreased.The dynamic changes of GS isoenzyme protein subunits in the mesophyll and main veins and the differences between the varieties and gene expression abundances were similar.The GS isozymes in leaves and main veins of flue-cured tobacco showed significant or highly significant correlations with nitrogen metabolism-related enzyme activities,nitrogen metabolite content,apoplastic-related physiological indicators,and ammonia volatilization,respectively.This showed that the differences in the expression of GS isoenzymes in different flue-cured tobacco varieties may lead to a higher “source-sink intensity difference” of nitrogen-efficient varieties in mature stage than to nitrogen-inefficient varieties.The degradation rate of chloroplasts in nitrogen-efficient varieties was slow,and the amount of degraded nitrogen was reused.The amount of NH4+ transferred to the apoplastic was small,and the ammonia gas was less volatile.However,the activity of two GS isoenzymes in leaves of inefficient nitrogen cultivars at maturity was weak at each leaf stage,and the transport efficiency and assimilation capacity of nitrogen metabolites in the source and sink were low.As a result,the aged and degraded nitrogen was transferred to the apoplast in a large amount and volatilized in the form of ammonia to achieve its own nitrogen deficit.The difference in pressure between the source and sink and the physiologically different synthesis of the apoplastic body resulted in differences in the nitrogen use efficiency of different genotypes of flue-cured tobacco.2.There were significant differences in nitrogen metabolism characteristics among the three flue-cured tobacco varieties in Nanyang tobacco-growing area.NC71 had lower activity of GS1,GS2 and nitrate reductase(NR)during the maturation stage,and had a higher activity of glutamate dehydrogenase(GDH)and lower nitrogen assimilation and reuse ability,at the same time,the apoptotic NH4+ concentration and ammonia volatilization are both high,which showed that the species can achieve nitrogen deficit through ammonia volatilization and maintain the same trend in the case of high nitrogen.Therefore,the content of total nitrogen and free amino acids in leaves during mature period was low,which proved that it had a lower nitrogen efficiency in mature stage.The ratio of potassium and chlorine was higher in NC71's toasted tobacco leaves under high nitrogen conditions.The ratio of sugar to nicotine was in a suitable range,and the ratio of total nitrogen to nicotine was also close to 1;its combustibility,momentum,softness and economic traits were significantly better than other treatments,therefore should be planted under higher nitrogen conditions.Yunyan 99 had higher GS isoenzyme and NR activity at maturity,and the nitrogen degraded due to aging was heavily assimilated and reused.The contents of nitrogen metabolites in leaf were higher and the ammonia volatilization was lower;the total quality of Yunyan 99 was best when it was roasted under low nitrogen conditions.At this time,the ratio of total sugar and nicotine was higher,the ratio of potassium to chlorine was also higher,and the ratio of total nitrogen and nicotine was the closest to 1;the grey,flammability,momentum and softness in sensory quality assessment and the economic traits were significantly better than those in medium and high nitrogen treatments.Therefore,it is appropriate to reduce the amount of nitrogen fertilizer.The nitrogen metabolic characteristics of Qushou 1 were between the other two varieties,and the conventional chemical composition,sensory evaluation,and economic traits were all optimal under medium-nitrogen use conditions,and therefore should be planted under medium-nitrogen conditions.