Study On Regulation Of Exogenous Nitrogen Forms On The Growth,Nitrogen Uptake And Metabolism Of Cucumber

The growth of cucumber（Cucumis sativus L.）is associated with increasing nitrogen dependence.Deficient or excessive amounts of N have a significant negative impact on vegetables,suggesting that changing the forms of nitrogen supply without changing the amount of nitrogen is a very important pathway that influences the growth of plants.Some studies have shown that the mixed NO₃^-+NH₄⁺nitrogen forms could promote plant growth.However,few studies have been reported how to promote nitrogen uptake and metabolism of cucumber and enhance chilling tolerance of cucumber seedlings by the scientific supply of exogenous nitrogen,such as substitution amide nitrogen for other forms or increasing the proportion of ammonium,then it need further study to make clear the role of enzymes and genes in the pathway.In the study,cucumber seedlings were experimented by increasing ammonium nitrogen to replace nitrate nitrogen partly in nitrogen fertilizer under sub low temperature（control was NO₃^-,treatments were 25%NH₄⁺、50%NH₄⁺、75%NH₄⁺、100%NH₄⁺）,and adult plants of cucumber were experimented by adding equal amide to ammonium and adding equal amide to nitrate at optimum temperature（100%NH₄⁺、100%NO₃^-、100%CO（NH₂）₂、50%NH₄⁺+50%CO（NH₂）₂、50%NO₃^-+50%CO（NH₂）₂、50%NO₃^-+50%NH₄⁺）,in order to find the effects of exogenous nitrogen on cucumber growth,enzymes and genes of nitrogen absorption and metabolism.The findings were as follows:1.Urea addition increased biomass,and improved proportion of fruit weight,total content and protein content of cucumber.Treatment with both NO₃^--N with added equal CO（NH₂）₂ and NH₄⁺-N with added equal CO（NH₂）₂ enhanced glutamate,protein,chlorophyll,nitrogen contents,and improved dry matter weight.Furthermore,greater proportion of dry matter was distributed to the fruit,then led to significantly higher yields,especially treatments with 50%NO₃^--N+50%CO（NH₂）₂ as the recommended nitrogen form at adult stage in this study.Meanwhile,analysis of mineral elements showed that NH₄⁺-N promoted the absorption of P and Fe in cucumber,but inhibitted the absorption of K,Ca and Mg.On the contrary,NO₃^--N has adverse effect on the absorption of P and Fe in cucumber,but enhanced the absorption of K,Ca and Mg significantly.As a result,mixed nitrogen could promote the balanced absorption of P,K,Ca,Mg,Fe in cucumber to some extent.In addition,the Ca and Mg content in leaves were significantly higher than those in roots,stems and fruits.2.Urea addition promoted nitrate reduction,urea utilization and the GS-GOGAT cycle of nitrogen metabolism.After treatment NO₃^--N with added equal CO（NH₂）₂,UR-2,NR-2,NR-3,Ni R,GOGAT-1-1,and GS-4 were upregulated in response to these changes.After NH₄⁺-N treatment with added equal CO（NH₂）₂,UR-2,GOGAT-2-2,and GS-4 were upregulated in response to these changes.Furthermore,both NO₃^--N with added equal CO（NH₂）₂ and NH₄⁺-N with added equal CO（NH₂）₂ enhanced the activities of GOGAT,GS,UR and amino acid pathway involved in the urea metabolism.3.The treatment of 50%ammonium promoted the growth,increased the content of protein and proline,decreased MDA content,enhanced chilling tolerance of cucumber seedlings.The methods were to increase ammonium nitrogen to replace nitrate nitrogen from 0 to 100%of nitrogen fertilizer when cucumber seedlings were kept for 14 days under sub low temperature treatment.The treatment of increasing50%ammonium treatment promoted greatly the growth of cucumber seedlings under sub low temperature,it was higher optimum ammonium ratio than increasing 25%ammonium at suitable temperature.With the increase of ammonium ratio by step to step,the number of root volume,root length and root branching of cucumber seedlings increased firstly then decreased gradually under sub low temperature,and the treatment of 50%ammonium manifested the maximum value.As the greater the proportion of ammonium nitrogen,the smaller the root-shoot ratio,but the higher the root activity.Furthermore,through the analysis of protein,proline and malondialdehyde,it was found that the proline and protein of 7 days stress,and the protein of 14 days stress all increased firstly then decreased with the ratio of increasing ammonium,but MDA content decreased firstly then increased at 7 days stress and 14 days stress,and the treatment of 50%ammonium showed the highest proline and protein content and lowest MDA content.The results suggested that 50%ammonium nutrition enhanced chilling tolerance of cucumber seedlings.4.The mechanism was that ammonium enhancing could promote uptake and metabolism of nitrogen to enhance chilling tolerance of cucumber seedlings by genes analyzing of nitrogen uptake,nitrate reduction gene,glutamate cycle and PM H⁺-ATP.The treatment of 50%ammonium up-regulated the expression of nitrogen uptake CSNRT1.3,CSNRT1.5 and CSAMT1.1,which promoted the uptake and transport of nitrogen.Meanwhile,up-regulation expression of glutamate cycle genes Cs GOGAT-1-2,Cs GOGAT-2-1,Cs GOGAT-2-2,Cs GS-2-2,Cs GS-2,Cs GS-3,Cs GS-4,Cs GDH-2 and Cs GDH-3 promoted metabolism of nitrogen by 50%ammonium addition.Furthermore,ammonium increasing induced up-regulation of PM H⁺-ATP genes CSHA2 and CSHA3 in roots,which maintained nitrogen transport and membrane at sub low temperature.In addition,16 genes preferred expression of roots in treatments of increasing ammonium under low temperature after 14 days,they thereby promoted nitrogen assimilation in roots to enhance chilling tolerance of cucumber seedling.To sum up,the adaptive nitrogen forms of cucumber varied with temperature and growth stage.The replacement could promote the metabolism of nitrogen,then improved the biomass and yield of cucumber,which substituted equal amide nitrogen for 50%nitrate nitrogen at adult stage.Furthermore,the replacement could upregulate the expression of genes including in nitrogen genes and PM H⁺-ATP genes,and promote the absorption and metabolism of nitrogen,then enhanced chilling tolerance of cucumber seedling,which substituted equal ammonium nitrogen for 50%nitrate nitrogen at seedling stage of cucumber under sub low temperature.