The Mechanism Of Coumarin Inhibiting The Germination And Growth Of Lolium Perenne L

Coumarin is an important allelochemical substance widely distributed in many higher plants and microbial metabolites such as Rutaceae and Umbelliferae.Its biological activity is diverse and has been extensively researched in various fields.It has good application prospects in the fields of medicine and plant protection.In the field of plant protection,it has been applied as an insecticide and fungicide.At present,we mainly focus on weeding.Most studies have showed that it is have good inhibitory activity on many weeds,but their specific mechanism of action and target are not clear.Based on the studies on the inhibition activity of various weed seeds,the ryegrass with the best inhibitory activity（Lolium perenne L）were selected as a target,to study its mechanism of inhibition on germination of ryegrass seeds,and that was provided a theoretical basis for the study of coumarin compounds as a new herbicide.1.The results of biological activity assay showed that coumarin had good inhibitory activity on germination,radicle and germ growth of ryegrass,and its concentration on germination inhibition(IC₅₀)was 67.69 mg/L.The results of the action mode of coumarin by stem and leaf spray method showed that the stem and leaf spray method had no significant effect on the growth of ryegrass,while the hydroponic treatment of 10 mg/L significantly inhibited the growth of ryegrass roots and stems.The root activity of ryegrass after hydroponic treatment was further determined.With the increase of treatment concentration and the treatment time,the root activity of ryegrass decreased significantly.It is indicated that coumarin mainly acts on the roots of ryegrass and further inhibits the growth of ryegrass by inhibiting the root activity of ryegrass.2.The changes of soluble protein,total starch and soluble sugar content in ryegrass seeds treated with 50 and 100 mg/L coumarin during germination were determined.The results showed that coumarin had no significant effect on soluble protein in ryegrass seeds.After 60 hours of treatment,100 mg/L coumarin significantly inhibited starch decomposition and soluble sugar synthesis.The alpha-amylase activity of ryegrass seeds treated with different concentrations was further determined.Compared with the control group,the treatment group significantly inhibited the activity of alpha-amylase.The results showed that coumarin affected the metabolism of substance and energy during seed germination by inhibiting the activity of alpha-amylase,and then affected seed germination.3.Determination of superoxide dismutase（SOD）,peroxidase（POD）,catalase（CAT）activity and activity under concentration gradient during germination of 50 and100 mg/L coumarin seeds.Oxygen hydroperoxide（H₂O₂）,superoxide anion(O^2-)content and membrane lipid peroxidation index malondialdehyde（MDA）,lipid peroxide（LPO）content;treatment concentration at 100mg/L at all time points The activity of CAT was significantly inhibited,and the activity of SOD and POD increased significantly after 60h treatment.The content of reactive oxygen,malondialdehyde and lipid peroxide increased significantly with the increase of treatment concentration.It indicated that the coumarin treatment destroyed the protective enzyme system inside the ryegrass seeds,resulting in the accumulation of reactive oxygen species and causing oxidative damage of the cells and inhibiting the germination of the seeds.4.The results of hormone content determination during seed germination showed that coumarin had no significant effect on the content of abscisic acid（ABA）during seed germination.When the concentration was 100 mg/L,it significantly inhibited gibberellic acid（GA₃）during seed germination.Synthesis;indicates that coumarin treatment affects hormone metabolism during seed germination.5.The luciferin-treated ryegrass seeds were transcribed by the Illumina Hiseq high-throughput sequencing platform,and a total of 41,683 Unigenes were obtained after assembly.There are 17,306 Unigenes with a length of 1 kb or more.Functional annotations for Unigene,including comparisons with NR,Swiss-Prot,KEGG,COG,KOG,GO,and Pfam databases,resulted in a total of 27,919 Unigene annotation results.The differentially expressed genes were screened.After treatment with 100 mg/L for 24h,there were 1814 differentially expressed genes.After 50 and 100 mg/L treatment for48 h,there were 3675 and 1848 differentially expressed genes,respectively.Through the functional enrichment analysis of differentially expressed genes,it is shown that the differential genes are in the BP class,the most representative of which are metabolic processes,cellular processes,single biological processes,and stimulatory responses;in the CC class,cells and cellular components The membrane,membrane fraction and organelle are the most representative;in the MF class,the activities of binding,catalytic activity,transporter activity and antioxidant activity are the most representative.The enrichment analysis of differentially expressed genes in KEGG pathway indicates that the significantly enriched pathways include TCA cycle,oxidative phosphorylation,ribosome,proteasome and carbon metabolism.Further analysis showed thatα-amylase,SOD,POD,CAT and other related genes showed different degrees of up-and-down regulation after coumarin treatment for 24 h and 48 h,and further confirmed coumarin inhibited ryegrass seed germination at the molecular level.