Evaluation Of Rhizobium Symbiosis And Melatonin Application For Salinity,Heat,and Cold Stress Tolerance In Medicago Truncatula

In recent years,global warming has become more pronounced,and severe extreme climates have also increased.These factors have led to an increase in the incidence of crop abiotic stresses,reducing crop yields and threatening global food security.Leguminous crops,as an important food and economic source worldwide,are strongly affected by abiotic stresses notably salinity,heat and cold,which hinder their production and cause huge economic losses.Therefore,there is an urgent need to improve the resistance of legume crops to abiotic stress.Legumes are stress-sensitive plants,and only few of them can withstand stresses environments.However,the interaction of legumes with plant growth promoting bacteria boosted the growth and stress tolerance of legume plants.Rhizobia are important symbiotic microorganisms that can form nodules on the roots of legumes and release molecules such as plant hormones that can protect plants from stress.At the same time,as a plant hormone naturally produced in plants,melatonin can significantly improve the ability of plants to cope with adversity.Therefore,we speculate that the combined application of rhizobia and melatonin can alleviate the destructive effects of abiotic stress on Medicago truncatula,thereby maintaining stability and increasing the yield of Medicago truncatula in terms of yield and quality.The main research results and conclusions are as follows:1.The effects of the active nodulation process on salt stress tolerance of Medicago truncatula were evaluated by quantifying the compatible solutes,soluble sugars,and antioxidants enzymes,as well as growth and survival rate of plants.Eight weeks old plants,divided in three groups:（i）no nodules（NN）,（ii）inactive nodules（IN）,and（iii）active nodules（AN）,were exposed to 150 m M of Na Cl salt stress for 0,8,16,24,32,40,and 48 h in hydroponic system.AN plants showed a higher survival rate（30.83%and 38.35%;p≤0.05）,chlorophyll contents（37.18%and 44.51%;p≤0.05）,and photosynthesis compared to IN and NN plants,respectively.Improved salt tolerance in AN plants was linked with higher activities of enzymatic and nonenzymatic antioxidants and higher K⁺（20.45%and 39.21%）and lower Na⁺accumulations（17.54%and 24.51%）when compared with IN and NN plants,respectively.Additionally,higher generation of ROS was indicative of salt stress,causing membrane damage as revealed by higher electrolyte leakage and lipid peroxidation.All such effects were significantly ameliorated in AN plants,showing higher compatible solutes（proline,free amino acids,glycine betaine,soluble sugars,and proteins）and maintaining higher relative water contents（61.34%;p≤0.05）.2.The combined effects of Rhizobium inoculation（RI）and foliar application of melatonin（MT）were studied to evade heat-induced damages in Medicago truncatula.In the present study,eight weeks old seedlings were divided into two groups:（i）non-inoculated plants（NI）and（ii）Rhizobium-inoculated plants（RI）,both groups were treated with 60μM MT one week prior to inducing moderate heat（MH;32/26°C light/dark）and severe heat（SH;40/32°C light/dark）stress for 24 h.The response of plants to stress was measured through antioxidant metabolism and osmolyte accumulation.The results showed that RI and exogenous MT pretreatment improved plants resistance to heat stress indicated by higher photosynthetic rate（74.29%and 182.75%;p≤0.05）,chlorophyll（59.58%and 77.07%;p≤0.05）,and carotenoid contents（47.86%and 88.97%;p≤0.05）under MH and SH stress,respectively.The heat-induced membrane damages were prevented by declining electrolyte leakage（EL;52.27%;p≤0.05）and malondialdehyde contents（MDA;26.43%;p≤0.05）,minimum release of ROS(superoxide anion radical;O₂^–·and hydrogen peroxide;H₂O₂)and regulation of the activities of antioxidant enzymes:catalase（CAT）,superoxide dismutase（SOD）,ascorbate peroxidase（APX）,and peroxidase（POD）.The heat-mediated damages were significantly ameliorated in RI plants of MT-MH and MT-SH treatments,showing higher compatible solutes（glycine betaine,proline,proteins,and soluble sugars）and maintained higher activities of lipoxygenase（LOX）,nitrate reductase（NR）,and nitric oxide（NO）.3.Lastly,combined use of Rhizobium inoculation and melatonin pretreatment were investigated to ameliorate cold stress in Medicago truncatula.Eight weeks old seedlings were divided into eight groups:（i）CK（no stress,non-inoculated）,（ii）RI（Rhizobium-inoculated）,（iii）MT（75μM of melatonin）,（iv）RI+MT（v）CS（cold stress at4°C）,（vi）CS+RI,（vii）CS+MT,and（viii）CS+RI+MT for 3 days.RI and MT pretreatment significantly enhanced biomass accumulation and root activity of Medicago truncatula plants under control conditions and alleviated the cold-induced decline.Relative to control,declined in photosynthesis pigments and increment in oxidative stress were observed under stress conditions.Interestingly,compared to CS treatment,chlorophyll contents were significantly increased by 242.81%（p≤0.05）and MDA levels were dramatically decreased by 34.22%（p≤0.05）in CS+RI+MT treatments.Moreover,RI and MT pretreatment significantly enhanced the anti-oxidation ability by increasing the activities of superoxide dismutase（SOD;50.36%;p≤0.05）,catalase（CAT;140.26%;p≤0.05）,peroxidase（POD;8.45%;p≤0.05）,and ascorbate peroxidase（APX;42.63%;p≤0.05）over CS treated plants,under stress conditions.It was also observed that increased osmolytes accumulation,nutrients uptake and nitrate reductase activity due to RI and MT pretreatment helped plants to counteract the cold-mediated damage by strengthening the non-enzymatic antioxidant system.Therefore,these data indicated that combined application of RI and MT-pretreatment can attenuate the cold damage via enhancing anti-oxidation ability.Conclusively,abiotic stresses are hostile to plant growth and development,leading to great crop yield penalty worldwide.Among salt,heat,and cold stresses we found that heat stress is the most harmful stress which not only strongly decreased plants growth and yield,but consistent rise in global temperature due to global warming will further worsen its effects.Among Rhizobium inoculation（RI）and exogenous application of melatonin（MT）,we found that foliar application of melatonin was better at improving stress tolerance in Medicago truncatula.Nevertheless,combined application of both RI and MT is the best way forward to enhance the stress tolerance capacity in Medicago truncatula plants.