Effects Of Nitrogen Nutrition On Ascorbate Content And Metabolism In Spinach (Spinacia Oleracea L.)

L-Ascorbic acid (vitamin C, AsA) is a major antioxidant and plays multiple roles in plant growth and development. Fruits and vegetables are the main source of AsA supply to human because humans are incapable of synthesizing AsA and must secure by means of dietary uptake. The accumulation of ascorbate in the vegetables depends upon their biological characteristics and many environmental factors, e.g. amount and form of N application, light intensity, temperature, water supply and photoperiod, and so on. However, nitrogen nutrition is one of the most important factors affecting ascorbate content in vegetables. There were a few reports available on the effect of nitrogen nutrition on ascorbate contents in vegetables. However, most of previous reports were focused mainly on the total ascorbate in plants, the effects of nitrogen nutrition on the ascorbate of different forms in edible parts of plants were scarcely reported. Up to now, mechanism of the effect of nitrogen on ascorbate contents in vegetables is still not well understood. In this paper, AsA-rich spinach (Spinacia oleracea L.) was selected as the tested plants and hydroponics was carried to investigate the effects of nitrogen levels, nitrogen forms and pre-harvest nitrogen forms on AsA contents and its related metabolic activity in spinach leaves. The objective of this study was to provide scientific basis for the nitrogen management in the production of quality vegetables rich in the Ascorbic acid. The main results of this research were summarized as follows:1. A hydroponics was carried out to determine the effects of different N levels (2, 10, 18, 26 mmol/L) on ascorbate contents and its related metabolic enzymes activities in spinach. The results showed that biomass of spinach and ascorbate content increased markedly with nitrogen increasing from 2 mmol/L to 10 mmol/L, while biomass of spinach was not affected, ascorbate contents dropped and nitrate increased with N level further increasing. Besides, comparing other nitrogen levels, AsA,dehydroascorbate (DHA) and total ascorbate (AsA+DHA) got the highest content when N level was 10 mmol/L. The activities of L-galactono-1,4-lactone dehydrogenase (GalLDH) increased significantly with nitrogen increasing from 2 mmol/L to 10 mmol/L and dropped with N level further increasing. N levels have no significant effects on the ascorbate oxidase (AAO) activities, while monodehydroascorbate reductase (MDHAR) and dehydro ascorbate reductase (DHAR) and ascorbate peroxidase (APX) activities raised markedly with nitrogen increasing from 2 mmol/L to 10 mmol/L. Therefore, The effects of nitrogen levels on GalLDH,MDHAR,DHAR and APX has the same trends as well as AsA, which showed that the effects of nitrogen levels on AsA content realized mainly through the regulation of synthesis enzyme and regeneration enzymes.2. The effects of NH₄⁺:NO₃^- ratios (0:100, 25:75, 50:50, 75:25, 100:0) in the nutrient solution on AsA contents and its related metabolic enzymes activities in hydroponically grown spinach were studied. Results showed that both fresh and dry weights were signicantly decreased when NH₄⁺:NO₃^- ratio exceeded 50:50, and highest fresh and dry wheight was recorded at the NH₄⁺:NO₃^- ratio of 25:75. AsA and (AsA+DHA) contents gradually incresead while the ratios of AsA/(AsA+DHA) had no significant change with increasing of NH₄⁺:NO₃^- ratio. The activities of GalLDH were not decreased until the ratio of NH₄⁺/NO₃^-was over 50/50 and AAO had no significant changes with increasing NH₄⁺ ratio. The APX activity was decreased firstly and then increased when the NH₄⁺-N ratio was elevated. The activities of two enzymes related to AsA regeneration, DHAR and MDHAR were increased as well as the trends of ascorbate contents when elevated NH₄⁺:NO₃^- ratio. The results suggested that increased AsA contents with increasing NH₄⁺:NO₃^- ratio in the nutrient solution could be attributed to the enhanced DHAR and MDHAR activities in AsA metabolism, could not be significantly related to the changes of GalLDH and AAO activities.3. A water culture experiment was carried out to study the effects of different patterns of nitrogen supply before havesting (100% NO₃^-,0% NO₃^-,50% NO₃^-,50% NO₃^-+50% NH₄⁺,50% NH₄⁺,100% NH₄⁺) on ascorbate contents and its related metabolic enzymes activities in spinach. Results showed that the treatments of 50% NH₄⁺ and 100% NH₄⁺ before harvesting markedly increased AsA and ASA+DHA content, however, decreased the biomass accumulation..nitrate dropped obviously with low nitrogen levels (0% NO₃^-,50% NH₄⁺) but the AsA content and biomass decreased significantly with the treatment of 0% NO₃^-. Moreover, the mixed nitrogen forms (50% NO₃^-+50% NH₄⁺) increased obviously the ascorbate contents and reduced the nitrate while has no effects on the biomass. The enzymes activities of GalLDH dropped while MDHAR,DHAR,APX activities keeped a higher activities with ammonium alone.Besides, the MDHAR and APX activities lowed markedly with the reduction of nitrogen levels (0% NO₃^- 50% NO₃^-) while GalLDH,MDHAR,DHAR and APX activities enhanced notably with 50% NO₃^-+50% NH₄^-. AAO activities has no significant changes. Therefore, the treatment of 50% NO₃^-50% NH₄^- before harvesting increased ascorbate contents of spinach might had relationship with the higher activities of GalLDH,MDHAR,DHA and APX ,which enhanced the synthesis and regeneration of AsA.