Addressing the challenge of cold stress resilience with the synergistic effect of Rhizobium inoculation and exogenous melatonin application in Medicago truncatula.

Cold stress is an adverse environmental condition that limits the growth and yield of leguminous plants. Thus, discovering an effective way of ameliorating cold-mediated damage is important for sustainable legume production. In this study, the combined use of Rhizobium inoculation (RI) and melatonin (MT) pretreatment was investigated in Medicago truncatula plants under cold stress. Eight-week-old seedlings were divided into eight groups: (i) CK (no stress, noninoculated, no MT), (ii) RI (Rhizobium inoculated), (iii) MT (75 μM melatonin), (iv) RI+MT, (v) CS (cold stress at 4 °C without Rhizobium inoculation and melatonin), (vi) CS+RI, (vii) CS+MT, and (viii) CS+RI+MT. Plants were exposed to cold stress for 24 hrs. Cold stress decreased photosynthetic pigments and increased oxidative stress. Pretreatment with RI and MT alone or combined significantly improved root activity and plant biomass production under cold stress. Interestingly, chlorophyll contents increased by 242.81% and MDA levels dramatically decreased by 34.22% in the CS+RI+MT treatment compared to the CS treatment. Moreover, RI+MT pretreatment improved the antioxidative ability by increasing the activities of peroxidase (POD; 8.45%), superoxide dismutase (SOD; 50.36%), catalase (CAT; 140.26%), and ascorbate peroxidase (APX; 42.63%) over CS treated plants. Additionally, increased osmolyte accumulation, nutrient uptake, and nitrate reductase activity due to the combined use of RI and MT helped the plants counteract cold-mediated damage by strengthening the nonenzymatic antioxidant system. These data indicate that pretreatment with a combined application of RI and MT can attenuate cold damage by enhancing the antioxidation ability of legumes.