Uptake, Metabolism And Physiological Responses Of Pakchoi Under Glycine-nitrogen Nutrition Conditions

The classical paradigm of the terrestrial nitrogen cycle has considered that organicnitrogen must be converted into inorganic nitrogen (NO3–-N, NH4+-N) by soil microorganisms,prior to becoming available to plant root, therefore, more attention has often been focused onthe behavior of inorganic nitrogen in agricultural soils and overlooked the roles of organicnitrogen in soil nitrogen cycles. Up to now, little information is known about organic nitrogenabsorbed by plants and its significance may be underestimated than predicted in some naturalecosystems, soil management systems and other special environments. Studies on plantorganic nutrition can enrich plant nutrition theory and reconsider the soil nitrogen cycle. Inthis study, we first investigated the size and composition of soluble organic nitrogen (SON)pool in horticultural soil, then studied the pakchoi (Brassica campestris ssp. chinensis.Makino. var. communis Tsen et Lee) glycine (Gly) uptake by isotopic tracer method; at last, ahydroponic experiment was conducted to determine the nutritional effect of Gly-N and thephysiological responses of pakchoi under Gly-N nutrition conditions.1. Assessing the soluble organic nitrogen pools in horticultural soilsThe aim of this study was to investigate the abundance and composition of SON and itslabile components (protein and total free amino acids) in horticultural soils managed byorganic (ORG) or conventional (CNV) practices under greenhouse or open field conditions.SON was one of the major N forms in horticultural soil, accounted for54.23%of total solublenitrogen (TSN). Protein concentration accounted for a high proportion of the SON fraction inall sampled sites (47.36%), while free amino acids (FAA) only constituted a small proportion(4.61%). The contents of soil SON, protein, total FAA and nitrate-nitrogen (NO3–-N)increased in organic soils more than those under conventional practice. Soils cultivated in greenhouse conditions contained greater pools of TSN and SON as well as NO3–-N andammonium-nitrogen (NH4+-N) compared to open fields. Seasonal changes also affected thetotal abundance of SON, protein and FAA in all the treatments, and the seasonal variationdepends on the N form being measured. Electrophoretic patterns of soil proteins ingreenhouse soils were clearly different from those in open feld soil, but these signifcantdifferences were not observed between ORG and CNV soils. The soil amino acid pools weredominated by the same five amino acids, glycine (Gly), serine, asparagic acid, threonine andtyrosine, among four treatments. Greenhouse cultivation significantly affected the FAAprofiles, while ORG management did not have the same effect. We propose that themanagement practice (ORG vs. CNV) has an effect on the quality of the SON labile pools,while it plays a lesser role in the SON quality compared to cultivation method (greenhouse vs.open feld).2.15N-glycine uptake of pakchoi in horticultural soilIsotopic tracer method was used to study the pakchoi Gly uptake in soil conditions.Pakchoi were grown in pot experiment with two soil management types (organic vs.conventional) for15days prior to labeling with13C and15N-Gly. Uptake of Gly relative tonitrate and ammonium was tested in two pakchoi (Brassica campestris ssp. chinensis. Makino.var. communis Tsen et Lee) cultivar (‘Huawang’ and ‘Wuyueman’) and two soil managementtypes under two added nitrogen concentrations (0.05and15μg N g-1dry soil, as low N andhigh N treatment). The15N uptake rates,15N recoveries and proportions of15N recovery from15NO3–-N were greater than those from15N-Gly and15NH4+-N, indicated NO3–-N was themost important nitrogen form for pakchoi. The15N from15N-Gly constituted a significantlygreater proportion of total15N in organic and high N-treated soil than in conventional and lowN-treated soil. The15N-Gly recovery of ‘Huawang’ was greater than that of ‘Wuyueman’. Asthe13.63%of Gly-derived15N acquisition was due to uptake of intact Gly molecules, thecontribution of organic N in the form of intact Gly to total N uptake was about3.60%。Theseresults show that the organic N in horticultural soil can be a direct nitrogen source forvegetable, and the its uptake efficiency can be affected by soil management type and N supplylevel. 3. Nutritional effects of Gly nitrogen on pakchoiThe pakchoi responses to Gly concentration were evaluated by dry weight, root to shootratio, total N uptake, total carbon content and soluble sugar content, with4concentrationgradients (2.5，5.0，10.0，20.0mM) set up and non-nitrogen as the contrast. We also tested theeffects of different forms of N (NO3–-N, Gly-N) under same N concentration (5.0mM) on thegrowth, accumulation of nitrogen and carbon, and the nitrogen metabolism related enzymeactivities of two pakchoi cultivars (‘Huawang’ and ‘Wuyueman’) using a sterile hydroponicssystem. Compared with non-nitrogen, the fresh weight, total N content, total carbon contentof two cultivars increased at2.5and5.0mM Gly treatment, while they showednon-significant increases under10mM Gly treatments compared with2.5and5.0mM Glytreatment. The dry weights, total contents of N and carbon of two cultivars under20mM Glytreatment showed no significant difference with or even lower that those in non-nitrogentreatment. The contents of soluble sugar and protein showed no significant difference between2.5,5.0,10.0and20.0mM Gly concentration treatments. Compared with nitrate treatment,the Gly supply decreased the fresh weight, total nitrogen and nitrate content of pakchoi, butsignificantly increased the contents of shoot protein, free amino acid, and soluble sugar. Nosignificant changes of root protein, total carbon and chlorophyll contents were found betweennitrate and Gly treatment. The activities of nitrate reductase (NR), glutamate pyruvatetransaminase (GPT), glutamate oxaloacetate transaminase (GOT) in shoot and glutamatedehydrogenase (GDH) in root in the two cultivars were higher under Gly treatment than thoseunder nitrate treatment, while the activities of glutamine synthetase (GS) in root showed asignificant decrease in Gly treatment. No significant differences of the glutamate synthase(GOGAT) activity were found between nitrate and Gly treatment. The superior Gly tolerancecultivar ‘Huawang’ had obvious higher activities of GDH and aminotransferase than‘Wuyueman’. Our results clearly demonstrated the intrinsic capacity for pakchoi plants todirectly use Gly as a sole source of nitrogen. Furthermore, GDH and aminotransferase mayplay a critical role in pakchoi adaptation to Gly-N.4. Metabolic response of pakchoi leaves and roots to glycine nitrogen The main objective of this study was to identify primary metabolites associated with Glythrough metabolic profile analysis using gas chromatography–mass spectrometry (GC-MS).The metabolic analysis of pakchoi polar extracts revealed37amino acid N-responsivemetabolites in the two pakchoi cultivars (‘Huawang’ and ‘Wuyueman’), mainly consisting ofsugars, amino acids, and organic acids. Changes in the carbon metabolism of pakchoi leavesunder Gly treatments occurred via the accumulation of fructose, glucose, galactose, xylose,and arabinose. Disruption of amino acid metabolism resulted in accumulation of endogenousAsn, Gly, Ser and Thr in Gly treatments. By contrast, the levels of endogenous Gln decreased.Gly-N supply also affected the citric acid cycle, namely, the second stage of respiration,where leaves in Gly, Gln, and Ala treatments contained low levels of malic acid, citric, andsuccinic acid compared with leaves in NO3–-N treatments. The metabolites that mainly serveas intermediates in carbon metabolism, amino acid biosynthesis, TCA cycle, photorespiration,cell growth/division and involved in other stress defense pathways could play important rolesin pakchoi adaptation to amino acid-N.5. Proteomic analysis of pakchoi leaves and roots under glycine nitrogen conditionsThe differential proteomic responses of pakchoi leaves and roots to glycine-nitrogen(Gly-N) treatments were determined. Two pakchoi cultivars,‘Huawang’ and ‘Wuyueman’,were grown under sterile hydroponic conditions with different N forms (Gly and nitrate).Atotal of23differentially regulated proteins were identified following Gly-N treatment using2-DE and MALDI-TOF MS/MS. These spots are involved in several processes, such asenergy synthesis (ferredoxin-NADP(H)-reductase and cytochrome c), N metabolism (GS andS-adenosylmethionine synthase isozyme), photosynthesis (ribulose-1,5-bisphosphatecarboxylase/oxygenase and carbonic anhydrase), and active antioxidant defense (copperchaperone, Gly-rich RNA-binding protein and putative pathogenesis-related protein)mechanisms, that could enhance plant adaptation to Gly-N. The superior Gly tolerance of‘Huawang’ was predominantly associated with a less severe down-regulation of proteins thatare involved in the electron transport chain and N metabolism. Other factors could includeless ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) turnover or a higherup-regulation of stress defense proteins. These characteristics demonstrated that maintaining ATP synthesis, N metabolism, photosynthesis, and active defense mechanisms play a criticalrole in pakchoi adaptation to Gly-N.In summary, Gly as a model organic N in sampled soil, can be a direct N source forpakchoi. The Gly uptake by pakchoi varied with different N supply levels and soilmanagement types. High N supplied or organically managed soils increased15N recovery andpercentage of15N recovery from15N-Gly. Suitable Gly concentration promoted the growthand accumulation of carbon and nitrogen of pakchoi. GDH and aminotransferase may play acritical role in pakchoi adaptation to Gly-N. Maintaining ATP synthesis, N metabolism,photosynthesis, and active defense mechanisms play a critical role in pakchoi adaptation toGly-N.