Nitric oxide enhances the nitrate stress tolerance of spinach by scavenging ROS and RNS

• Excess nitrate induces accumulation of NO, which was regulated by NR, Hb, and GSNOR.
• Exogenous SNP increased antioxidant enzyme activities, combating ROS accumulation.
• Exogenous SNP enhanced GSNOR activities, which removes over accumulated RNS.

In this study, the role of nitric oxide (NO) in response to excess nitrate stress in spinach was investigated. The results showed that excess nitrate stress provoked a significant reduction in the seedling growth and an increase in lipid peroxidation and H2O2 content in spinach. The gene and protein expression of nitrate reductase and non-symbiotic hemoglobin were up-regulated, while S-nitrosoglutathione reductase (GSNOR) was down-regulated with the accumulation of NO and total S-nitrosothiols (SNOs) after 160 mM nitrate treatment for 24 h. Nitrate stress treatment increased the S-nitrosylation level of spinach. Using biotin-switch coupled with LC–MS/MS method, several proteins were identified as targets of S-nitrosylation including ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit, phosphoglycerate kinase, 23 kDa oxygen-evolving protein (OEC), 16 kDa protein of the photosynthetic OEC, and ribulose-1,5-bisphosphate carboxylase oxygenase. We further demonstrated that exogenous NO donor (SNP) increased the superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), glutathione reductase (GR) and GSNOR activities, which would reduce reactive oxygen species (ROS) and reactive nitrogen species (RNS) contents and thereby enhance spinach tolerance to nitrate stress. Suppressing NO accumulation by NR inhibitor, NOS inhibitor, and NO scavenger aggravated the damage to spinach under nitrate stress. These results suggest that NO acts as an essential signal to enhance spinach tolerance to nitrate stress via reducing ROS and RNS toxicity.