Effect Of Nitrogen Forms On The Growth And Organic Acid Metabolism Of Lettuce

Nitrogen is the essential nutrient for all organisms,which is one of the key factors that determine the biomass and yield.The use of nitrogen fertilizer greatly promoted the crop yield.However,the large application of nitrogen fertilizer not only increases the cost of agricultural production,but also causes a series of environmental problems,which including climate change,soil acidification and eutrophication of water.Therefore,it is of great significance to seek new ways to increase nitrogen use efficiency.In addition,organic acid plays an irreplaceable role in the absorption and metabolism of nutrients in plants or microorganisms,as well as the detoxification of plants to metals,propagation of rhizosphere microorganisms,decomposition of soil minerals,etc.In order to find a new way to increase nitrogen utilization and reduce environmental pollution.In this study,Lactuca sativa L.was used as material,five different ratios of ammonium nitrogen and nitrate nitrogen were set:T1(NO3--N:NH4+ N=10:0),T2(NO3--N:NH4+-N=7:3),T3(NO3--N:NH4+-N=5:5),T4(NO3N:NH4+-N=3:7),and T5(NO3--N:NH4+-N=0:10),which were used to study the effect of nitrogen forms on organic acid composition and physiological metabolic pathway.The research was aimed at revealing the effect of nitrogen forms on the growth of lettuce,the coupling regulation relationship between key enzymes in organic acid metabolism,which can provide a theoretical basis on revealing the regulation mechanism of organic acid metabolism.The main results were as follows:1.Effect of nitrogen forms on organic acid compositions of lettuceIn order to clarify the effect of nitrogen forms on the metabolism of organic acid in lettuce,the extraction of total organic acid was optimized by orthogonal test technique,and the total organic acid content and organic acid components were analyzed.The results showed that:The optimum technological conditions for extracting the total organic acid from lettuce were:extraction time 45 min,temperature 75℃,ethanol concentration 75%,material liquid ratio 1:30,extraction rate was 0.18%.The lettuce was treated with different nitrogen forms and its total organic acid content showed:The total organic acid content secreted by leaves and roots was the highest in T5 and T1 was the lowest,indicating that NH4+-N was more conducive to the secretion of total organic acids and different nitrogen forms have relatively obvious influence on the organic acid components in lettuce leaves and roots.The higher concentration of NO3--N was beneficial to the accumulation of citric acid,malic acid and succinic acid in leaves,while the higher concentration of NH4+-N was beneficial to the accumulation of tartaric acid.In the root,T2 was beneficial to citric acid,tartaric acid and succinic acid accumulation,the higher concentrations of NH4+-N was conducive to the accumulation of malic acid and malonic acid.2.Physiological effects of nitrogen forms on organic acid metabolism in lettuceIn order to study the effect of nitrogen forms on the growth of lettuce and the absorption of mineral elements,the growth of lettuce with different nitrogen forms at different growth stages was investigated.The content of mineral elements in the whole plant was measured.The Pearson method was used to analyze the correlation between organic acid,growth situation and absorption of mineral elements.The results showed that:T1 was beneficial to lengthen the root system,promote the rooting,increase the number of root tip,as well as the absorption and utilization of kalium;T2 and T3 were conducive to root density,stem diameter,root volume,root topological area,and root surface area,as well as conducive to the growth of leaves,lateral leaf growth,total chlorophyll synthesis,and the absorption and utilization of Zn,Ca,Mg,B,Fe,P,Mn and N.Ammonium nitrogen was beneficial to the synthesis of chlorophyll,but in high concentration,the roots of the plants were thinner and friable,and the color burned,the total chlorophyll content was decreased,the plants were short and the leaves were yellowing,as well as showed the phenomenon of ammonium poisoning.Which sequentially affected the absorption of water and mineral elements,and then inhibit the growth of plant parts.All nitrate nitrogen could easily lead to the spindling on the overground part and the decrease of chlorophyll content,the utilization of zinc,ferrum and nitrogen would decrease,and excessive nitrate could threaten human health.Therefore,in a comprehensive comparison,T2 was recommended as the best nitrogen ratio for hydroponic lettuce.3.Expression analysis of key genes in the organic acid metabolism of lettuceOrganic acid metabolism is a complex process,which regulated by a series of enzymes.In order to reveal the metabolic mechanism from physiological and molecular level,the activity of related enzymes in the metabolism of organic acid in the lettuce was measured,and the related enzyme genes were cloned and the expression level of related enzyme genes in different nitrogen forms and different growth stages of Lactuca sativa L.were analyzed by RT-qPCR.The results showed that:Different nitrogen forms had a significant effect on the activity of related enzymes in lettuce.In the leaves,T1 was conducive to the improvement of NADPIDH,MDH,PEPC activity,and with the increase of ammonium nitrogen ratio,the activity would get decrease.But high concentration of ammonium nitrogen was more conducive to the increase of ACOm and ACOc activity.In the root,all nitrate nitrogen was conducive to the increase of NADPIDH activity,and the activity of MDH was the highest in T4,PEPC was the highest in T2;ACOm was the highest in T5,ACOc was the highest in T3 and T4.The relative expression of related genes showed that:The relative expression levels of NADP-IDH,ACO and MDH in leaves were highest at the high concentration of ammonium nitrogen,while that of CS,H+-PPase and PEPC were higher at T1 and higher concentration of nitrate nitrogen was higher.In the root,higher concentrations of nitrate nitrogen increased the expression of ACO,CS,H+-ATPase and PEPC.The expression of CS and H+-PPase reached the highest level in T2,while NADP-IDH and MDH were highly expressed in high concentration of ammonium nitrogen,but NADP-IDH and MDH in roots were almost unchanged at different periods.The results showed that the activities of NADP-IDH and MDH were negatively correlated with the changes of NADP-IDH and MDH expression in the leaves,while ACO,PEPC activity and ACO,PEPC expression trends consistent;In the root,only PEPC,MDH activity and PEPC,MDH expression trend consistent.The results showed that the activities of enzymes related to organic acid metabolism of lettuce were not exactly the same with the different nitrogen forms,which indicated that the nitrogen treatment of lettuce was not only affected by enzyme activity but also related to other factors.