Nitrogen Uptake And Metabolism Of Spinach (Spinacia Oleracea L.) Under Nitrate Or Glycine Supply

Organic nitrogen for plants,as one of the important direction of plant nutrition research,prolonged and improved the traditional nitrogen theory in plant.A lot of studies have confirmed plants can absorb organic nutrition in vary ecosystems.Especially due to the continuous development of organic agriculture,it is emergency to study the organic-nitrogen nutrition for agriculture crops further and in-depth.In the study,we first selected the suited ones to organic fertilizer from 18 spinach varieties?Spinacia oleracea L.?according to thier morphological and physiological indexes.Then with some technical methods,such as dual label amino acid,Gas chromatography-mass spectrometer?GC-MS?,amino acid analysis,metabonomics,fluorescence quantitative real-time PCR,we studied the effects of different concentrations or different proportions of NO₃-N and Gly-N on spinach growth,the absorption of carbon and nitrogen,metabolism in spinach leaves and related enzyme activity,etc.The absorption and utilization of spinach on amino acid,reveals the the important role of amino acids as organic nitrogen source for horticultural crops,and further improved the theory of organic-nitrogen nutrition of plants.The main results were as follows:1.Selection of spinach varieties suited to amino acids fertilizerSpinach plants were cultivated with or without mixed amino acids fertilizer.The organic fertilizer promoted shoot growth.The average values for fresh mass,dry mass,and carbon and nitrogen contents of spinach varieties cultivated with organic fertilizer were greater than their corresponding values in the no-fertilizer control.Shoots and roots showed opposite responses to organic fertilizer in terms of morphological traits.The values for shoot-related traits were higher while those for root-related traits were lower in the organic fertilizer treatment than in the control.There were significant variations in responses among the spinach varieties.The main differences were in root trait and nitrogen accumulation responses.The varieties were grouped according to their sensitivity to organic fertilizer to identify those best suited to cultivation in organic systems.2.Nitrogen uptake by spinach supplying with nitrate or GlyThrough a hydroponic experiment using two spinach cultivars?Littlefield and Blackwood?,with the C,N double labeled glycine(¹³C-¹⁵N-Gly)and ¹⁵N labeled nitrate(¹⁵N-NO₃^-)as nitrogen sources,we studied the different nitrogen absorption rate and nitrogen recovery rate of spinach under different concentrations of nitrate or Gly.According to the results,for both of the two varieties,¹⁵N contents in ¹⁵N-NO₃^-treated plants were significantly higher than that in Gly treated plants.After treated with Gly,¹⁵N contents of root showed significantly higher than that of shoot.While ¹⁵N-NO₃^-treatments did not show such difference.¹⁵N recovery rate of the two varieties exhibited that in low nitrogen concentration is greater than that in the high nitrogen concentration and¹⁵N-NO₃^-is greater than Gly treatments.Under the treatment of Gly,¹⁵N recovery rate of Littlefield?high nitrogen concentration 15.0%,low nitrogen concentration 31.7%?is significantly higher than that of Blackwood?high nitrogen concentration 9.9%,low nitrogen concentration 22.5%?.Using the relationship between ¹³C and ¹⁵N,calculated that the highest complete absorbtion rates of glycine in Littlefield and Blackwood were 22.1%and 19.6%?low nitrogen concentration?,13.8%and 8.2%?high nitrogen concentration?,respectively.3.Metabolism of spinach treated with different concentrations of Gly or nitrateWith the aim of elucidating the different effects of inorganic and organic nitrogen sources on primary metabolic pathways in spinach,a gas chromatography–mass spectrometry?GC-MS?based metabolic approach was used to identify differentially abundant compounds in spinach leaves after 2days hydroponic-culture in one of three concentrations of glycine or nitrate.Principal component analysis?PCA?revealed distinct clusters for different treatments.The first and second components of PCA accounted for 63.6%and13.6%of total variance,and seemed to be mainly influenced by the nitrogen concentration and amount of Gly input,respectively.Compared to control treatment,most identified metabolites increased in dose-dependent manners as nitrogen availability increased;however,distinct patterns were observed for NO₃^--and Gly-treated leaves.When nitrogen was deficient,NO₃^--treated leaves had lower relative contents of carbohydrates,organic acids and amino acids than Gly treated leaves,this trend reversed under N-sufficient conditions.Furthermore,the metabolites identified were profiled to highlight the overall metabolic shifts between NO₃^-and Gly as N sources at different concentrations.4.Enzymes in nitrogen metabolism of spinachWe chose organic nitrogen?Gly?and inorganic nitrogen?NO₃^-?as nitrogen sources,and set three different nitrogen concentrations?1 mM,5 mM and 10mM?to cultivated spinach for 2 days,then took spinach root and leaf as materials.We measured the activities of NR,GS,GOGAT and GPT in spinach roots or leaves under different nitrogen treatments,and compared the relative expression of SoNR1 and SoGS1 by fluorescence quantitative PCR technology.The results showed that the nitrate increased the activities of NR,GS,GOGAT and GPT,and the activities were increased with increasing the nitrate input.Glycine did not influence NR activity significantly,and only increased the activities of GS,GOGAT and GPT in spinach.NR activities in roots were higher than that in leaves.On the contrary,GS,GOGAT and GPT showed the opposite conditions.The qPCR expression analysis showed SoNR1 mainly expressed in spinach leaves,while SoGS1 mainly expressed in the roots.5.Nitrate/Gly ration influence spinach growth and amino acidsA hydroponic experiment was conducted to study the effects of fertilization with different nitrate/Gly ratios on spinach growth and composition of amino acids.Results showed partly replacing nitrate by Gly supplied both nitrogen and carbon nutrition for spinach.Spinach accumulated the equal biomass.Total nitrogen in plant had a positive relationship with nitrate/Gly ratios in nutrition.There was more than 2-fold increase in N uptake from all glycine to nitrate,and plant nitrate contents increased from 0.97 to 3.13 mg g^-1.All of 17 proteogenic amino acids in mature leaves exhibited a similar trend,the content in leaf tissue increased with an increasing ratio of nitrate/Gly,whereas relative change rate differed for each individual amino acid.In self-organizing map?SOM?,17amino acids were divided into three clusters according to responds to nitrate/Gly ratios.Considering all above,we can draw a conclusion that spinach can absorb either inorganic nitrogen source as nitrate or organic nitrogen source as glycine to meet the need to grow and nitrogen nutrition.And the absorption of nitrogen source was influenced by spinach varieties,nitrogen forms and levels of nitrogen input.Different forms of nitrogen sources affected spinach seedlings on growth,accumulation of carbohydrates,amino acid,organic acid enzymes and so on.In particularly,different concentrations of nitrogen sources influence the spinach differently.The findings of this thesis may enrich the theory of plant organic nitrogen nutrition,and give some advices on varieties selection or nutrients supply in organic farming.The N cycle theory should be revisited with a new sight.