Physiological And Molecular Mechanisms Of Response To Autotoxicity Courmain In Alfalfa(Medicago Sativa L.) Varieties

Alfalfa(Medicago sativa L.)is an excellent perennial leguminous plant that is the main grass species for cultivated grassland.Crop rotation and other methods improve the soil environment of the subsequent stubble to increase the productivity of the re-established alfalfa grassland.Meanwhile,alfalfa also has important ecological functions,they are often used to restore ecological vegetation.However,alfalfa autotoxicity limits the next generation seeds germination and seedlings growth,which seriously affects its stability in the ecological grassland community,resulting in a cliff-like decline phenomenon after several years of alfalfa ecological grassland establishment.The coumarin is main autotoxic substance for alfalfa.Why does the coumarin have an inhibitory effect on the root development and seedlings growth?In this study,we chosen the high autotoxicity alfalfa 3105c(Medicago sativa cv.3105c)and low autotoxicity alfalfa WL656HQ(Medicago sativa cv.WL656HQ)as test materials and the autotoxic substance coumarin was used to simulate autotoxicity.Comparing the differences in photosynthetic physiology,root morphology morphological characteristics,and root stress-resistant physiological response to autotoxicity in WL656HQ and 3105c seedlings under different concentrations of coumarin and coumarin at different times.To clarify the mechanism of coumarin inhibiting signaling pathway regulating cell growth and proliferation,to construct the key metabolic network of alfalfa seedling development and the physiological and molecular mechanism of alfalfa in response to coumarin.It is important and significant for creating low autotoxicity alfalfa material.It is vital to expand the natural reproductive capacity of alfalfa community and its application in ecological restoration.The main results are summarized as follow:(1)The autotoxicity of 22 alfalfa varieties had significantly different effects on seed germination.We used TOPSIS analysis to determine that WL656HQ and 3105c were the most tolerant and sensitive varieties to autotoxicity,respectively.WL656HQ and 3105c had different trigger concentrations and reaction thresholds for autotoxicity.We found that the SVI and radicle length were mainly affected by SE in both WL656HQ and 3105c.Moreover,WL656HQ responded to autotoxicity by increasing antioxidant enzyme(POD,APX and GR)activities and the proline content to maintain membrane stability and oxidation scavenging.In 3105c,ROS production was greater than the capacity of the antioxidant defense system,resulting in oxidative damage.In conclusion,the positive and negative indicators to autotoxicity in the two alfalfa varieties were ASA and OH~·,and Pro and DHA,respectively.(2)Coumarin treatment determined for 7th day was the sensitive time for affecting photosynthesis and the 3rd day was the sensitive time for affecting root morphological characteristics and physiological processes of alfalfa according to cluster analysis.The autotoxicity threshold of WL656HQ is higher than that of 3105c.The Growth of WL656HQ was promoted below the threshold,but was slowed above the threshold,while inhibiting seed germination.Non-stomatal factors were the main reason that coumarin inhibited the photosynthetic efficiency of WL656HQ and 3105c seedlings.3105c and WL656HQ have two different strategies to cope with coumarin stress.3105c responded to coumarin treatment by causing oxidative damage,while it cannot maintain the oxidative balance and cause oxidative damage.WL656HQ has a stronger ability to scavenge ROS to maintain its redox homeostasis.The increase of H2O2 content and the inhibition of C4H activity were the main reasons for the autotoxicity of WL656HQ under coumarin stress.The increase of H2O2 content and the inhibition of GR and PAL activities were the main reasons for the autotoxicity of 3105c.(3)After coumarin treatment for 3 days,RNA sequencing-based transcriptomics analysis was performed to compare the global transcriptional profiles in the roots of different alfalfa varieties.Among these DEGs,218 up-and 103 down-regulated genes were present in 3105c,and 429 up-and 389 down-regulated genes were present in WL656HQ.Most of these DEGs were involved in carbohydrate metabolism,lipid metabolism,amino acid metabolism and secondary metabolism,signal transduction,cell defense pathway.Compared with 3105c,WL656HQ mainly changes carbohydrate metabolism(pentose phosphate pathway and phosphoinositide metabolism),lipid metabolism(linoleic acid metabolism),amino acid metabolism,secondary metabolism(tropane piperidine and pyridine and isoquinoline and morpholino alkaloid biosynthesis),signal transduction(phosphoinositide signal transduction),and cellular defense(peroxisome)activates the antioxidant metabolic pathway in vivo and alleviates the stress effect of coumarin.(4)After coumarin treatment for 3 days,metabolomic analysis was used to compare the roots of 3105c and WL656HQ alfalfa varieties.194 differentially expressed metabolites(DEMs)(61 up-regulated,133 down-regulated)and 186 DEMs(29 up-regulated,157down-regulated)were identified in 3105c and WL656HQ roots,respectively.The differences between alfalfa 3105c and WL656HQ in response to autotoxicity are manifested in primary metabolism:galactose metabolism-ascorbic acid metabolism,purine metabolism,glutathione metabolism and secondary metabolism:shikimic acid metabolism(secondary metabolic pathway-aromatic amino acid metabolism),phenylalanine metabolism and tyrosine metabolism.(5)When WL656HQ responded to coumarin treatment,it showed low seed germination promotion and high inhibition,strong osmotic regulation ability,low membrane lipid peroxidation,low ROS accumulation level,high ROS scavenging rate,and high antioxidant defense ability;At the molecular level,The main differences of WL656HQ were manifested in carbohydrate metabolism,lipid metabolism,amino acid metabolism pathway,secondary metabolite biosynthesis pathway,signal transduction,cellular defense,and nucleotide metabolism.Coumarin can significantly induce phosphoinositide signaling in the root system of WL656HQ,initiate cellular defense responses in vivo,alter pentose phosphate pathway,phosphoinositide metabolism pathway,arginine and proline metabolism,and induce anti-inflammatory effects.Oxidative metabolism pathway(tropane piperidine and pyridine metabolism and isoquinoline alkaloid biosynthesis),peroxidase pathway activates the antioxidant system in the body,up-regulates xanthine synthesis in purine metabolism,thereby improving its antioxidant capacity and effectively resisting Oxidative damage due to autotoxicity.