| Many vegetable crops, such as spinach and amaranth, are subject to accumulate high levels of oxalate. Ingestion of these oxalate-rich vegetables not only reduces calcium, magnesium and other micronutrients bioavailability resulting in mineral deficiency, but also increases the risk of urinary stones such as kidney stone in human body. The accumulation of oxalate 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 oxalate content in vegetables. There were a few reports available on the effect of nitrogen nutrition on oxalate content in vegetables. However, most of previous reports were focused mainly on the total oxalate in plants, the effects of nitrogen nutrition on the oxalate of different forms in edible parts of plants were scarcely reported. Up to now, mechanism of the effect of nitrogen on oxalate content in vegetables is still not well understood. In the present study, spinach was selected as the tested plants because of their accumulation of higher level of oxalate. The objective of this study was to investigate the effect of nitrogen nutrition on the oxalate concentrations of different forms in spinach in hydroponics and soil culture and to determine the optimal nitrogen management that would minimize the oxalate concentrations in edible parts of spinach, which provide scientific basis for nitrogen management in the production of low-oxalate spinach with high yield and nutritional quality. The main results obtained from the experiments were summarized as follows:(1). The results obtained from the hydroponics showed that biomass of spinach increased markedly with nitrogen increasing from 4mmol/L to 8mmol/L, while biomass of spinach was not affected with N level further increasing. Petiole was the main nitrate-accumulating organ, nitrate content varied little as nitrogen level increased from 4mmol/L to 8mmol/L, then increased with N levels increasing; while nitrate content in leaves increased as N levels were elevated. Leaf was the main oxalate storage tissue of spinach. Soluble oxalate content in leaves increased obviously with the increase in N levels, but decreased slightly as N level was 8mmol/L. Total oxalate content in leaves and soluble oxalate content and total oxalate in petioles increased as N levels were elevated and then decreased as N levels surpassed 12mmol/L. This indicated that appropriate N supply could effectively reduce nitrate and oxalate content of spinach. As biomass and nutritional quality of spinach were taken into account, 8mmol/L N is proper N level in hydroponics for spinach.(2) MDA content in leaves of spinach decreased drastically as nitrogen concentration increased from 4mmol/L to 8mmol/L, then increased with further increased N level. Increase in antioxidant activities in leaves, such as SOD and POD, was observed when N levels were increased from 4mmol/L to 8mmol/L, then antioxidant activities decreased markedly with further increased N level. CAT activities in leaves reduced drastically, while proline content increased as N levels were elevated. It was shown that spinach suffered less oxidative stress under proper N level (8mmol/L).(3). As N levels were increased in hydroponics, both oxalate content and glyoxylate-oxidizing activities in leaves increased first and then decreased, while glycolate-oxidizing activities increased first and then kept constant. Under low nitrogen supply, ascorbate content in leaves increased drastically then markedly decreased as N levels were elevated. Slow decrease in oxalate oxidase activities was found as N levels were increased in the nutrient solution. The significant positive correlations between total oxalate content and glycolate-oxidizing activity and glyoxylate-oxidizing activity were observed. aHPMS and Choline chloride, which are both the inhibitors of glycolate oxidase, markedly reduced GO activities and decreased oxalate content in leaves at the same time. It indicated that the glycolate pathway is one of the main pathways involved in oxalate biosynthesis in spinach. Significant negative correlation was found between oxalate contents and ascorbate contents, however, significant correlation was not observed between oxalate contents and OXO activities. Thus, the differences in oxalate contents in spinach leaves under different N supply mainly resulted from the effects of N levels on GO activities which were involved in glycolate pathway of photorespiration and the cleavage of ascorbate, but the effect of oxalate oxidase activities was minor.(4). As ammonium proportion in the nutrient solution increased from 0% to 50%, biomass of spinach increased drastically, then decreased markedly as ammonium was further elevated. With the increase in the ratio of ammonium, nitrate and oxalate content in leaves, petioles, tap roots and lateral roots all decreased drastically. Nitrate contents in different organs were in the following order: petioles>tap root=lateral root>leaf. Soluble oxalate was the main form with which oxalate occurred in different spinach organs and insoluble oxalate constituted less proportion of total oxalate. Oxalate concentrations in different spinach organs were shown from high to low as follows: leaf>tap root>petiole>lateral root. Thus, spinach with higher biomass and lower nitrate content, soluble oxalate content and total oxalate was achieved when the ratio of nitrogen to ammonium in nutrient solutions was maintained in 75/25 or 50/50.(5). As the ratio of ammonium increased from 0% to 50%, MDA content inspinach leaves decreased slightly, then increased markedly as the ratio of ammonium was 100%. SOD activities in leaves of spinach increased obviously as ammonium proportion increased from 0% to 50% and then decreased markedly when the ratio of ammonium was further elevated. While POD and CAT activities decreased drastically with the ratio of ammonium increasing from 0% to 50% then increased with the further increase of ammonium proportion, Proline content in leaves decreased obviously first then reduced markedly when the ratio of ammonium was elevated, as nitrate/ammonium ratio was 50/50, proline content was the lowest. It was shown that in present experimental condition, the degree of oxidation stress which spinach suffered was low, when nitrate/ammonium ratio in nutrient solution was kept in 50/50.(6). Oxalate content and activities of glycolate-oxidizing and glyoxylate-oxidizing in leaves of spinach decreased drastically as the ratio of ammonium was elevated in nutrient solution. Significant positive correlations were observed between oxalate contents and glycolate-oxidizing activity and glyoxylate-oxidizing activity, so the glycolate pathway in photorespiration might be an important pathway for oxalate synthesis in the leaves of spinach. Ascorbate content in leaves of spinach was increased while oxalate oxidase activities were decreased when ammonium proportion was elevated in nutrient solution. Markedly negative correlations were found between oxalate content and ascorbate content and oxalate oxidase activity, so the cleavage of ascorbate might be another pathway for oxalate synthesis in spinach leaves, wheras the increase of oxalate oxidase activities would reduce oxalate content in leaves. Thus, the decrease in oxalate contents in spinach leaves of with the increase of ammonium proportion in nutrient solution was due to the inhibition of glycolate oxidase activities and ascorbate cleavage, and the enhancement of oxalate oxidase activities.(7). Compared to the CK, both of the treatments of 50%NO3" and NO3"+ NH/ before harvesting markedly reduced nitrate content, soluble oxalate and total oxalate content in leaves and petioles and had no effect on biomass of spinach. However, nitrate content and oxalate content were further reduced markedly by the treatments of nitrate deprivation (0% NO3') and sole ammonium supply (50% NH4+and 100%NH4+) for 6 days, but spinach yields were drastically decreased at the same time. The objective of practical production was to achieve high quality spinach with high biomass and low nitrate and oxalate content, so the treatments of 50% NO3" and NO3'+ NH,+ are both appropriate patterns of nitrogen supply before spinach harvesting.(8). The results obtained from soil culture shown that spinach biomass was not affected when nitrogen rates were increased at lower nitrogen supply, while spinach...
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