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Spinach (Spinacia oleracea L.) is one of most important leafy vegetables because of its high nutritional value and high oxalate content, which can be toxic with negative effects on human nutrition. Ammonium and nitrate can effectively regulate oxalate accumulation, although the mechanisms underlying the oxalate biosynthesis and regulation are still undetermined in plants. In the present study, we identified 25 putative genes that are involved in the oxalate biosynthetic and degradation pathway, before analyzing the oxalate content and the expression levels of the corresponding proteins under normal growth conditions, with or without ammonium and nitrate treatments, using high and low oxalate-accumulated spinach genotypes. The two cultivars exhibited different profiles of total oxalate and soluble oxalate accumulation. The high oxalate concentrations in spinach were as a result of the high transcription levels of the genes that are involved in oxalate biosynthesis under normal growth conditions, such as SoGLO2, SoGLO3, three SoOXACs, SoMLS, SoMDH1, SoMDH2, and SoMDH4. The results revealed that the ammonium and nitrate were able to control the oxalate content in leaves, possibly because of the different transcription levels of the genes. The oxalate content is regulated by complex regulatory mechanisms and is varied in the different varieties of spinach. The results from this research may be used to assist the investigation of the mechanism of oxalate regulation and breeding for reduced oxalate content in spinach. [ABSTRACT FROM AUTHOR]