The Mechanism Of Cold Tolerance In Alfalfa (Medicago Sativa L.) As Affected By Water Deficit And Abrrupt Chilling

Alfalfa(Medicago sativa L.)is one of the most important forage grass.In China,alfalfa often suffers from freezing damage in winter and spring,which seriously affects the planting and popularization of the grass.Previous studies have shown that the water condition of soil has an important effect on alfalfa winter survival,but the related mechanism is still unclear.In present study,the water condition in cold acclimation and winter dormancy periods and the abrupt chilling environment in de-acclimation period of alfalfa were simulated,and the mechanism of alfalfa survive winter as well as the difference among cultivars was analyzed.1.In the water control experiments during cold acclimation phase,cultivar “WL353LH” was selected,and alfalfa seedlings were grown under two soil water regimes of 80% and 25% of water-holding capacity,respectively.All the seedlings were subject to low temperatures at 4/0°C(day/night)and then at-2/-6°C(day/night).Results showed that the semi-lethal temperatures of alfalfa which grown in water-deficit treatment were lower than those in well-watered treatment.Combined with the determination of biochemical compounds and metabolomic analysis,we found the pool sizes of total soluble sugar,amino acids & peptides and analogues(e.g.proline and proline betaine),and lipid and lipid-like molecules(e.g.fatty acids,unsaturated fatty acids,glycerophospholipids)were upregulated in response to the water deficit treatment.KEGG analyses showed that 18 pathways were significantly enriched involved in the biosynthesis and metabolism of carbohydrate,unsaturated fatty acids,amino acids and glycerophospholipid.Water deficit significantly enhanced the freezing tolerance of alfalfa which was related with the enhancement of soluble sugars,amino acids,lipid and lipid-like molecules,and some others.These compounds act as osmotic regulation and cryoprotectant and involves in the synthesis,fluidity and stability of cellular membrane.Our study provides a reference gist for improving alfalfa winter survival through water management.2.In the water control experiments after the soil was frozen,“WL440HQ” and “ZhaoDong” were selected.Alfalfa seedlings were grown in soil with moisture of 75% water holding capacity and were cold-acclimated at 4/0?(day/night)for one week.After acclimation,half of seedlings were selected out randomly and changed the surface 5-cm soil into dried sandy soil which could not hold water,and the soil water content of the other half of seedlings were maintained at 75% water holding capacity.Subsequently,all the plants were treated at-2? without light to simulate freeze-drying environment and normal freezing,respectively.Comparing with “ZhaoDong”,alfalfa crown of “WL440HQ” accumulated lower content of betaine and solulble sugars(total soluble sugar,sucrose,fructose,galactose and stachyose)during cold acclimation,and which act as osmotic regulators.Under the subsequent freeze-drying environment,“WL440HQ” had higher water loss rate and higher relative electrolyte leakage,which caused irreparable damage to crown cells and strongly influenced the capacity of resisting with cold.Wherears,alfalfa “ZhaoDong” had lower water loss rate and the cold hardiness was not influenced significantly under the freeze-drying environment.3.To assess the responses of alfalfa to abrupt chilling environment in physiological,biochemical and molecular aspects.“WL440HQ” and “ZhaoDong” were subjected to five experimental phases including two abrupt chilling events.Comparing with alfalfa “WL440HQ”,“ZhaoDong” had higher relative expression in genes GaS and StaS and higher accumulation of raffinose and stachyose in response to the abrupt chillings,and thus owned higher freezing resistance to cope with the adversed environment conditons.In summarry,the effect of abrupt chillings on “ZhaoDong” alfalfa was lower based on the results of our study.Present study will provide a theoretical reference for wintering water management and for alfalfa cultivars selection in abrupt chilling high incidence area,and also lay a foundation for effective management measures to reduce the damage of abrupt chilling to alfalfa.