Comparative Physiological Analysis Reveals the Role of NR-Derived Nitric Oxide in the Cold Tolerance of Forage Legumes.

The role of nitric oxide (NO) signaling in the cold acclimation of forage legumes was investigated in this study. subsp. (L.) Arcang. (hereafter ) is a forage legume with a higher cold tolerance than , a model legume. Cold acclimation treatment resulted in increased cold tolerance in both and , which was suppressed by pretreatment with tungstate, an inhibitor of nitrate reductase (NR), and 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO), a scavenger of NO. Likely, (), but not transcript, NR activity, and NO production were increased after cold treatment. Treatments with exogenous NO donors resulted in increased cold tolerance in both species. Superoxide dismutase (SOD), catalase (CAT), and ascorbate-peroxidase (APX) activities and , , (), and () transcript levels were induced in both species after cold treatment, which was suppressed by tungstate and 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO). Treatment with exogenous NO resulted in enhanced activities of SOD, CAT, and APX. Moreover, higher levels of transcript, NR activity, NO production, and antioxidant enzyme activities and transcripts were observed in as compared with after cold treatment. The results suggest that NR-derived NO production and upregulated antioxidant defense are involved in cold acclimation in both species, while the higher levels of NO production and its derived antioxidant enzymes are associated with the higher cold tolerance in as compared with .