Effects Of Nitrogen Nutrition On Oxalate Accumulation In Different Spinach Genotypes And Corresponding Mechanism

Oxalate is an ubiquitous of many plants, where it is implicated to play various functional roles in plants, including Ca2+regulation, detoxification of aluminum and heavy metal, plant protection etc.. However, oxalate acts as an antinutrient by binding minerals, such as calcium and magnesium, generally resulting in reduction of the elements’bioavailability. Excessive intake of oxalate may lead to formation of renal calcium stones in human body. It is therefore important to develop some effective strategies to reduce oxalate contents in vegetables. Spinach has been identified as high-oxalate vegetables. However, oxalate content differs among genotypes even within a given species for many vegetables, which is also true for spinach. But the genotypic variation in oxalate content of spinach cultivated in China and feasibility of selecting spinach for a low level of oxalate remains unclear. Nitrogen nutrition is one of the most important factors affecting biomass production and oxalate accumulation in plants, whereas little is known about the mechanisms involved in the association of nitrogen nutrition with oxalate accumulation in spinach. In this study, thirty-one cultivars of spinach native to China were employed to select high and low oxalate genotypes of spinach. Afterwards, kinetics studies and incubation experiment were performed under different nitrogen conditions to investigate the differences of oxalate accumulation in two spinach genotypes and its relationships with nitrogen uptake, reduction and assimilation, also the effect and mechanisms of nitrogen levels on oxalate synthesis pathway in difference spinach genotypes. The present study aims to investigate the effect and mechanism of nitrogen on oxalate content within spinach by using two different genotypes, as to provide scientific basis for the production of spinach with low oxalate by combination of nitrogen fertilization and cultivar selection. The main results were listed as follows:(1) Thirty-one spinach cultivars currently used in China were employed to investigate the genetic variations of oxalate content under different nitrate levels (2,10,18mmol·L-1). Among the30genotypes, there were significant differences in total and soluble oxalate contents. Oxalate content of31spinach cultivars were in normal distribution, with values ranging between8-21mg·g-1FW. Blade oxalate contents in high oxalate-accumulated cultivar (Heizhenzhu) were20%higher than of low oxalate-accumulated cultivar (Weilv). Spinach with lobate blade generally contained more oxalate than those with round blade in our collections. In addition, blade oxalate content was positively correlated with SPAD values, but negatively correlated with nitrate content under different nitrogen levels. Thus, it is feasible to select spinach with low-oxalate accumulation. The results also indicated that the effect of nitrogen levels on oxalate content may be associated with nitrate metabolism.(2) Two spinach genotypes (Heizhenzhu and Weilv), which differ in oxalate accumulation, were employed in absorption kinetics experiment to investigate whether genetic variations of blade oxalate accumulation in spinach correlated with their ability to uptake nitrate and ammonium by roots. Dose-dependent increase of blade oxalate contents and root nitrate uptake rate were observed in both spinach genotypes after exposure to increasing nitrate levels. In contrast, increasing ammonium did not result in elevation of blade oxalate. Correlation analysis confirmed that blade oxalate accumulation was positively associated with root uptake of nitrate but not ammonium. Treatments of mixed nitrate and ammonium (1:1) solution caused a decrease of oxalate accumulation in the both genotypes of spinach when compared with those treated with equal nitrate levels. Meanwhile, the value of Vmax for nitrate in mixed nitrogen solution decreased significantly when compared with sole nitrate, but the two treatments exhibited similar Km values. When exogenous ammonium (1.0mM) added to nitrate solution, both oxalate content and nitrate uptake was inhibited compared with that grown in nitrate alone. When compared with the controls, the values of Vmax for nitrate were reduced by55.36%and67.15%with addition of ammonium for Heizhenzhu and Weilv, respectively, whereas no significant change of Km values was observed. High-oxalate accumulated cultivar Heizhenzhu had a higher nitrate uptake rate when compared with the low oxalate accumulated cultivar Weilv when supply nitrate or mixed nitrogen solution. The results suggest that oxalate synthesis in spinach blades is associated with its root uptake of nitrate, and ammonium is able to reduce oxalate accumulation by inhibiting uptake of nitrate. Genotypic difference in oxalate accumulation are primarily relate to root nitrate uptake rate(3) Two spinach genotypes (Heizhenzhu and Weilv) differing in oxalate accumulation was employed in hydroponic experiments by addition of nitrogen metabolism inhibitor to explore the possible association of nitrate uptake, reduction and assimilation in spinach plants with its blade accumulation of oxalate. Increased nitrate levels in solution resulted in elevated root uptake rate of nitrate in Heizhenzhu and Weilv. Similarly, NR and GS activities in blades of both genotypes increased when nitrate levels in solution increased. Regardless of the treatments, the high-oxalate accumulating genotype Heizhenzhu showed a much greater enhanced root uptake rate of nitrate, blade NR and GS activities as compared with Weilv, which is a low-oxalate accumulated genotype. Correlation analysis showed that both total and soluble oxalate contents in blades of the two spinach genotypes were positively associated with its root uptake rate of nitrate, blade NR and GS activities. Significantly reduced accumulation of both total and soluble oxalate was observed in blades collected from the two spinach genotypes after treatments of Na3VO4. Meanwhile, treatment of Na3VO4significantly inhibited root nitrate uptake, blade NR and GS activities in both spinach genotypes. The presence of Na2WO4and MSO in solution significantly reduced total and soluble oxalate content in both Heizhenzhu and Weilv. Exposure of Na2WO4and MSO also resulted in substantial inhibition of GS activities in blades, as well as reduced root uptake rate of nitrate and NR activity in blades for the both spinach genotype. In controls, the plants of Heizhenzhu showed significantly higher root uptake rate of nitrate, blade NR and GS activities when compared with low-oxalate genotype Weilv. Total and soluble oxalate accumulation increased markedly in blades of both spinach genotypes as the solution pH changed from6.0to8.0. In contrast, there is no significant effect of solution pH on NR activities in blades of both genotypes. The present study provides clear evidence that oxalate accumulation in spinach is positively associated with both root uptake of nitrate, nitrate reduction and its assimilation within the plants. The description of oxalate accumulation between different spinach genotypes may be cause by nitrate uptake, reduction and assimilation.(4) Two spinach genotypes (Heizhenzhu and Weilv) differing in oxalate accumulation were employed by addition of oxalate precursor and oxalate metabolism inhibitor to estimate the effect of nitrogen levels on oxalate synthesis pathway. Increased nitrate levels in solution resulted in elevated total oxalate, soluble oxalate, glyoxylate and GO activity in Heizhenzhu and Weilv. Blade oxalate content, glyoxylate content and GO activity in high-oxalate accumulating genotype Heizhenzhu was higher than that of Weilv (low-oxalate accumulated genotype). Correlation analysis showed that oxalate content in blades of the two spinach genotypes were positively associated with its glyoxylate content and GO activity. Glycolate and glyoxylate exposed resulted in significantly increase of oxalate contents, and the increase was even more effective under higher nitrogen levels. In addition, Heizhenzhu showed higher increase of oxalate content compared with Weilv by addition of glycolate and glyoxylate. However, oxalate accumulation in both spinach genotypes was inhibited by GO inhibitor (HPMS and NaHSOs). Blade As A content was increased with increasing nitrogen levels from2to8mmol L-1, and then decreased as nitrogen levels were further increased. No significant correlation was observed between oxalate accumulation and AsA content in blade. Exogenous AsA enhanced the accumulation of oxalate, and the increase of oxalate content was similar between different nitrogen levels or genotypes. Exogenous glycine and glycine oxidase inhibitor enhanced the accumulation of oxalate in blades of spinach, but the increase was much more dramatic under higher nitrogen level or in Heizhenzhu spinach. Spinach fed with compounds from tricarboxylic acid (TCA) cycle also had a great increase in oxalate accumulation, but the increases were similar under different nitrogen levels. In addition, the increase of oxalate was higher in Heizhenzhu than in Weilv. Results clearly showed that glycolate and AsA pathway are responsible for oxalate synthesis in spinach. These results also suggest oxalate accumulation affected by nitrogen levels mainly through glycolate pathway. Difference of oxalate content between two spinach genotypes mainly cause by glycolate pathway and TCA cycle.(5) Two spinach genotypes (Heizhenzhu and Weilv), which differ in oxalate accumulation, were employed by addition of oxalate precursor, oxalate metabolism inhibitor to estimate the effect of nitrogen forms on oxalate accumulation. Exposure of mixed-nitrogen (nitrate and ammonium) significantly decreased blade oxalate and glyoxylate contents, nitrate uptake rate, NR and GO activities. Blade oxalate content was enhanced in both spinach genotypes by the addition of glycolate and glyoxylate, but the enhancement was even more effective as nitrate proportion was elevated. The accumulation of oxalate in different spinach genotypes were inhibited by GO inhibitor HPMS and NaHSO3. Glycine and glycine oxidase inhibitor exposure had a significantly incremental effect on oxalate accumulation. We also found that nitrate-fed plants had a greater oxalate accumulation than those fed with mixed-N. Exogenous AsA enhanced the accumulation of oxalate in blades of spinach under different nitrogen forms, and the increase was similar under different nitrogen forms. Compounds from TCA cycle significantly promoted oxalate accumulation in blades of spinach, and the increase of oxalate was similar under different nitrogen forms. TCA cycle inhibitor (malonate) markedly decreased oxalate accumulation in blades of spinach. Oxalate content was reduced by mixed nitrate and ammonium, which may be due to the lowed nitrate uptake and reduction caused by ammonium. These results also suggested that glycolate pathway was the predominant pathway for oxalate regulation by nitrogen forms.