The role of nitric oxide (NO) signaling within the cold acclimation of forage legumes was investigated in this study. enzymes in the Calvin-Benson cycle of photosynthesis under low temperatures, which reduces the utilization of assimilated light energy for CO2 assimilation and results in an increased photosynthetic electron flux to O2 . The antioxidant defense system includes superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), glutathione reductase (GR), and non-enzyme antioxidants, such as ascorbate (AsA) and glutathione (GSH). Numerous investigations reveal that this antioxidant defense system protects plants against oxidative damages induced by chilly stress . Substantial evidence reveals that NO participates in chilly acclimation and freezing tolerance [7,8,9,10,11]. Cold induces NO production in plants, which is Kif15-IN-1 considered a general response in plants . NO accumulates rapidly in and when plants are exposed to low temperatures [8,9], while treatment with exogenous NO donor increases the chilly tolerance of maize by affecting antioxidant enzymes . Nitric reductase (NR) is considered to be the most important enzymatic source of NO from nitrite reduction  and plays Kif15-IN-1 a key role in controlling NO levels in plants . NR-dependent NO levels are positively correlated with chilly acclimation and freezing tolerance in [8,12,16]. Treatment with exogenous NO donors increases antioxidant enzyme activity in tobacco , and NO depletion diminishes the cold-induced expression of and CBF regulons, such as in . Cold induces S-nitrosylation of some proteins. NO-mediated S-nitrosylation of iron-containing SOD is essential for chilling tolerance in  also. Lately, NR was discovered to provide NADH electrons to some molybdo enzyme called NOFNiR (nitric oxide-forming nitrite reductase) that catalyzes the NO creation from nitrite in in the current presence of nitrate . NR is certainly encoded by two genes, and [19, and 20]. is more linked to NO creation than in [19,20]. NR-derived NO creation with induced appearance is involved with frosty acclimation in ; nevertheless, it is unidentified whether NR-derived NO is certainly mixed up in legislation of antioxidant enzymes during frosty acclimation in legume vegetation. Alfalfa (L.) may be the most significant forage legume, with high biomass efficiency and a fantastic nutritional profile. is really a legume model with low cold tolerance for the investigation of other leguminous plants . It VAV1 is interesting to analyze the differential response to chilly between and to understand the chilly tolerance mechanisms in with higher activities of sucrose phosphate synthase and sucrose synthase during chilly acclimation. Transcripts of ((are managed in than in . A series of genes responsive to chilly, such as myo-inositol phosphate synthase (in our laboratory [25,26,27,28,29,30]. NO is usually involved in cold-induced expression of [25,26,29]. However, it is unknown whether NO signaling is usually associated with the differential chilly tolerance between and with those in than in and during chilly treatment at 5 C, except for that in at 21 d, and lower TEL50 was observed in than Kif15-IN-1 in throughout the chilly treatment (Physique 1), indicating that experienced higher chilly tolerance than indicates a decrease in the chilly tolerance. To understand whether this case was associated with chilling injury as a result of the long time exposure to low temperatures, the ion leakage and after 21 d of chilly treatment, while they were not altered in (Physique 2A,B). The results indicated that plants were damaged by long time exposure to low temperatures as compared with (Mf) in comparison to (Mt) as affected by tungstate and 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO) during 21 d of chilly treatment. Ten-week-old and cv. A17 plants were irrigated with 15 mL of 1 1 mM tungstate or 100 M PTIO answer or H2O as a control, followed by exposure to 5 C in.