Study On Herbicidal Action Mechanism Of 4-hydroxy-3-Methoxycinnamic Acid Derivatives

4-Hydroxy-3-methoxycinnamic acid is a kind of metabolites of Pythium aphanidermatum with herbicidal activity.In the previous research,26 derivatives of this compound were synthesized.In order to determine the biological activity of these compounds,Arabidopsis thaliana was used as the test weed and the herbicidal activity of these derivatives was determined using the plate method.Ethyl 4-hydroxy-3-methoxycinnamate was selected as a representative compound and its herbicidal action site was studied using the omics technique.The results were as follows:?1?By using acetochlor as a positive control agent,26 compounds were screened according to their herbicidal activity.The results showed that a total of 22 compounds had inhibitory activity on the length of roots of A.thaliana.When the concentration of the compound was 200.00 mg/L,the inhibition rates of 9 compounds,named compound 2,6,13,15,16,18,19,25 and 26,were above 90%,respectively:94.48%±0.60%,95.36%±0.74%,95.64%±1.14%,97.87%±1.20%,91.55%±0.59%,93.82%±0.18%,96.63%±0.28%,92.88%±5.44%and 99.52%±0.84%;the inhibitory effect of compounds 1,3,8and 17 was close to the positive control,and the inhibition rates were 87.00%±0.74%,83.77%±1.41%,84.42%±0.94%and 86.93%±0.16%.?2?Further,the herbicidal activity of 22 compounds with good herbicidal activity was determined,among which the compounds with the best herbicidal activity were compound8,11,15,22,25,and 26,and the IC₅₀ of the six compounds were 9.90 mg/L,6.93 mg/L,14.67 mg/L,11.08 mg/L,7.53 mg/L and 14.38 mg/L;the compounds with the second herbicidal activity were compound 1,2,6,17,23,and the IC50 of these five compounds were25.82 mg/L,19.99 mg/L,22.93 mg/L,20.92 mg/L and 24.82 mg/L;six kinds of compounds with poor herbicidal activity were compound 13,14 and 16,respectively,and the IC₅₀ were 35.87 mg/L,47.51 mg/L and 40.62 mg/L.The symptoms of root damage in A.thaliana were observed under a microscope,and it was found that the root length of A.thaliana was shortened,the number of lateral roots increased,and the main root was bent and deformed after treatment with 4-hydroxy-3-methoxycinnamic acid derivative;the lignification of the root tip was obvious,the root hair increased,the tip of the root was curled and the root cap was partially peeled off.?3?In order to find out the site of action of ethyl 4-hydroxy-3-methoxycinnamate,the A.thaliana was used as a test weed,the mechanism of action of the compound was observed at protein level and transcriptome level,and the related regulation and correlation analysis were conducted.Two experiments were conducted with arabidopsis seedlings treated with ethyl 4-hydroxy-3-methoxycinnamate:whole seedlings were sequenced by transcriptome,roots were sequenced by proteome,and transcriptome and proteome sequencing results were analyzed.The results showed that the number of differentially expressed genes and proteins was 154,of which 109 were down-regulated and 45 were up-regulated,and about 39metabolic pathways were involved,such as plant sugar metabolism,amino acid metabolism,fatty acid metabolism and biosynthesis,tricarboxylic acid cycle,pyruvate metabolism,carbon fixation and chlorophyll metabolism,biosynthesis of secondary metabolites,oxidative phosphorylation in photosynthesis and so on.Among these metabolic pathways,there were 12 genes whose expression level were down-regulated,such as AT2G37040,AT5G46290,AT5G15530,AT1G43800,AT1G77590,AT5G48880,AT1G17890,AT5G65690,AT1G09780,AT5G01330,AT2G24980 and AT1G54970,might be a candidate genes for the herbicidal action site of the compound.Further,the expression level of the above genes was verified by real-time quantitative PCR?RT-qPCR?,and the results were consistent with the results of the combined analysis of proteome and the transcriptome sequence.?4?The herbicidal action site of ethyl 4-hydroxy-3-methoxycinnamate was preliminarily defined,which mainly affected the extension of fatty acid chains of plants,the elongation of fatty acid chains was affected,the biofilm synthesis was hindered,the Arabidopsis cell membrane destroyed by ethyl 4-hydroxy-3-methoxycinnamate could not be replenished in time,resulting in cell membrane shrinkage,cytoplasmic outflow and Arabidopsis wilting.By commenting on the GO database and analyzing the KEGG metabolic pathway of differentially expressed genes and proteins,we found that the gene AT5G46290 was involved in the synthesis and metabolism of fatty acids and encoded a?-ketoacyl carrier protein synthetase I??-ketoacyl-ACP-synthase I,also known as fatty acid condensing enzyme I,KASI?,i.e.FabB.The main function of the enzyme using fatty acyl-ACP as a substrate was to catalyze the extension of fatty acid chains.After treatment with ethyl 4-hydroxy-3-methoxycinnamate,both transcriptome and RT-qPCR results of A.thaliana showed that the expression of KAS I was down-regulated,and the down-regulation multiples were 1,2.58,and 2.92,respectively;the proteome results also showed that KASI expression was down-regulated,and the multiple was 1.25.It could be speculated that due to the down-regulation of the expression of KAS I during fatty acid biosynthesis,the elongation of fatty acid chains was affected,the biofilm synthesis was hindered,the Arabidopsis cell membrane destroyed by ethyl 4-hydroxy-3-methoxycinnamate could not be replenished in time,resulting in cell membrane shrinkage,cytoplasmic outflow and Arabidopsis wilting.