Study On Regrowth Of Alfalfa Under Drought Stress And Nitrogen Supply

Alfalfa（Medicago sativa）is a perennial leguminous forage and regrowth is one of its important characteristics.The arid and semi-arid region of the Loess Plateau is one of the important bases for alfalfa cultivation in China,but the shortage of water resources,poor soils and the regrowth period of alfalfa mostly occur in the dry season in this region,which restricts the development of agriculture and animal husbandry,so exploring suitable water and fertilizer management modes is the key to improving alfalfa production in this region.In this study,we used a greenhouse pot experiment to simulate drought and fertilization by weighing and adding exogenous nitrogen,with normal water（CK）,drought stress（D）and added nitrogen under drought stress（ND）and isotope ¹⁵N labeling treatments.We investigated the effects of drought stress and nitrogen supply on the biomass accumulation,physiological changes and nitrogen uptake and transfer strategies of alfalfa at regrowth stage,and analyzed the molecular mechanisms of alfalfa leaves and roots in response to drought stress and exogenous nitrogen at different regrowth stages at the transcriptional and metabolic levels to provide a theoretical basis for high quality and high yield of alfalfa in the dry zone of Loess Plateau.The main results were as follows:（1）Drought stress reduced the regrowth biomass of alfalfa and increased the content of osmoregulatory substances such as soluble protein,proline,soluble sugar and sucrose,while increasing the carbon（C）,nitrogen（N）and phosphorus（P）contents of the leaves and roots,which was more obvious at 9 d of regrowth,indicating that alfalfa resisted drought stress mainly by increasing the content of osmoregulatory substances and C,N in the early regrowth period.Drought stress increased the MDA content of leaves at 9 d of regrowth and roots at 14 d of regrowth,indicating that drought stress has caused some damage to the plant.（2）Compared to drought stress,nitrogen application increased the biomass,plant height and proline content of alfalfa at 9 and 14 d of regrowth;the increase in osmoregulatory substances in leaves and roots mainly occurred at 9 d of regrowth;MDA content decreased in leaves at 9 d of regrowth and in roots at 14 d of regrowth,respectively.It also reduced the starch content of the root at 14 d of regrowth,indicating that nitrogen addition,to a certain extent,increased the content of osmoregulatory substances and reduced membrane lipid peroxidation in alfalfa at the regrowth stage by coordinating above and below ground to alleviate drought stress and promote regrowth growth.（3）Transcriptome analysis showed that differentially expressed genes were the least at 9d of regrowth with nitrogen application under drought stress,but the root genes were more responsive to exogenous nitrogen compared to the leaves at this time;differential genes expression were more obvious at 14 d of regrowth under drought and drought applied nitrogen conditions,and the leaf genes were more susceptible to drought and nitrogen compared to the roots.Most of the differential genes involved in the pathways encoding arginine and proline metabolism,starch and sucrose metabolism and plant hormone signal transduction identified in leaves and roots under drought stress and nitrogen addition during different regrowth periods,such as SAMDC,PAO,P5CS,P5CR,INV,BAM,endoglucanase,β-glucosidase,AUX/IAA,PP2C,Sn RK2,ABF,GID1,JAR1,JAZ,MYC2 and BRI1,whose expression s were up-regulated under drought stress and down-regulated after nitrogen application,and transcription factors showed the same trend of expression and were more pronounced in the leaves.Overall,leaf genes were more susceptible to drought stress and nitrogen compared to the root.（4）Metabolomic analysis showed that metabolites in leaves were more susceptible to drought stress throughout the regrowth period and nitrogen addition had a weak effect on metabolites during the regrowth period under drought stress,which was basically consistent with transcriptional level.Nitrogen application for 9 d under drought stress and drought mainly increased the content of amino acids and their derivatives and lipid metabolites in leaves and decreased the content of phenolic acid metabolites in roots;after 14 d of nitrogen application,the accumulation of metabolite content under these conditions changed in their opposite directions,respectively.Most of the metabolites in leaves and roots were related to amino acid biosynthesis.Furthermore,transcriptome and metabolome analyses showed that differential genes and differential metabolites were mainly involved in plant hormone signal transduction,biosynthesis of amino acids,arginine and proline metabolism,glutathione metabolism,carotenoid biosynthesis and valine,valine,leucine and isoleucine biosynthesis pathways.（5）Exogenous nitrogen at medium concentrations under different concentrations of isotope ¹⁵N labelling increased the reproduction of the second crop of alfalfa and there was a more pronounced advantage in various physiological parameters at this concentration,indicating that moderate concentrations of exogenous nitrogen could promote the regrowth of alfalfa plants after mowing.The isotopeδ¹⁵N content in the leaves was much higher than in roots,indicating that the alfalfa root is able to take up effective soil N and transfer it to the above-ground for plant regrowth.