| Isoquinoline alkaloids,in a variety of plants,are widely used in medicine and pesticides.Protoberberine alkaloids,the most important class of isoquinoline alkaloids,usually include Berberine,Jatrorrhizine,Palmatine and Tetrahydropalmatine etc.In this paper,berberine and its structural analogues were used as the research object to study the herbicidal activity on Mikania micrantha Kunth and Ageratum conyzoides L.and to preliminarily study their mechanism of herbicidal activity,providing a theoretical basis and scientific basis for the application of Isoquinoline alkaloids as new botanical herbicides and the control of Mikania micrantha Kunth and Ageratum conyzoides L.Using small glass method,Mikania micrantha Kunth and Ageratum conyzoides L.were treated with untested compounds at a concentration of 10 mg·L-1for 7 days.Worenine chloride,Tetrahydroberberine,Coptisinechloride,Dehydrocorydaline,D-Tetrahydropalmatine,Dehydrocavidine,Rotundine,Tetrahydrocoptisine,berberine,Dihydroberberine,palmatine,Corydaline and Tetrahydropalmatine showed good herbicidal activity on Mikania micrantha Kunth.Their root length inhibition rates were 89.19%,88.65%,88.27%,84.36%,76.21%,75.38%,71.24%,71.07%,70.20%,67.20%,66.15%,65.02%and 62.52%,respectively;and the fresh weight inhibition rates were 87.93%,84.00%,85.92%,84.38%,75.78%,78.01%,70.97%,77.41%,74.90%,78.16%,74.48%,70.52%and 63.89%,respectively.with partly rotted main roots;Worenine chloride,Coptisine chloride,Dihydroberberine,Dehydrocavidine,Berberine,Palmatine,Rotundine,Dehydrocorydaline,Tetrahydroberberine,Columbamine,Tetrahydrocoptisineand Chelerythrine chloride showed relatively good herbicidal activity on Ageratum conyzoides L.Their root length inhibition rates were 96.7%,94.45%,92.74%,91.18%,89.38%,87.8%,85.51%,83.97%,83.19%,77.86%,77.23%and 71.01%,respectively;and the fresh weight inhibition rates were 85.88%,84.87%,78.35%,78.86%,76.25%,83.85%,78.93%,73.43%,68.86%,66.11%,71.33%and 61.69%,respectively;with partly rotted main roots.After Mikania micrantha Kunth and Ageratum conyzoides L.were treated with untested compounds for 7 days,the median inhibitory concentration(IC50)of Worenine chloride,Tetrahydroberberine,Coptisinechloride,Dehydrocorydaline,D-Tetrahydropalmatine,Dehydrocavidine,Rotundine,Tetrahydrocoptisine,Berberine,Dihydroberberine,Palmatine,Corydaline and Tetrahydropalmatine on the root length of Mikania micrantha Kunth was 2.03,2.42,2.36,6.64,5.84,3.84,5.30,2.57,5.73,4.50,3.78,6.52 and 5.36 mg·L-1,respectively;and their IC50 values of fresh weight were 2.39,3.56,2.34,4.96,6.21,3.08,5.76,2.80,5.72,3.85,4.48,4.80 and 8.74 mg·L-1,respectively.the IC50values of Worenine chloride,Coptisine chloride,Dihydroberberine,Dehydrocavidine,Berberine,Palmatine,Rotundine,Dehydrocorydaline,Tetrahydroberberine,Columbamine,Tetrahydrocoptisine and Chelerythrine chloride on the root lengths of Ageratum conyzoides L.were 0.37,0.50,0.88,0.39,0.89,2.98,5.54,1.25,2.67,12.02,2.40 and 12.37 mg·L-1,respectively;and the IC50values of fresh weight were 1.33,1.63,1.27,0.46,1.70,2.08,7.83,2.63,1.77,8.00,1.55 and 11.39 mg·L-1,respectively.The seedlings of Arabidopsis mutants DR5:GFP were treated with several effective herbicide compounds.The results showed that all of the tested compounds could affect the localization of auxin in Arabidopsis root.The seedlings of Arabidopsis mutants PIN1:GFP,PIN2:GFP and AUX1:YFP were treated with the above compounds.The results showed that all of the tested compounds could affect the localization of these proteins in Arabidopsis root.Using vertical plate method,after four hours in the 10 mg·L-1 berberine treatment,the root cells of Arabidopsis thaliana seedlings that had been growing for seven days appeared holes on the surface of cells,indicating that berberine at 10 mg·L-1 would destroy the root cells and tissues of Arabidopsis thaliana.The transcriptome sequencing results showed that compared with the control group,the root gene expression of Arabidopsis thaliana seedlings treated with 0.25 mg·L-1 berberine for three hours and 6 mg·L-1 berberine for an hour,namely the expression level of phenylalanine ammonia lyase?PAL4?and 4-coumarate:CoA ligase?4CL3?genes related to lignin?a cell wall composition?synthesis significantly declined.After the treatment of Arabidopsis thaliana with 10 mg·L-1 berberine for 0,0.5,1,2,4,and 6 hours,the expression levels of key enzyme gene PAL4,cinnamate-4-hydroxylase?C4H?gene and 4CL3 during lignin biosynthesis in the root were detected by Quantitative Real-time PCR?qPCR?,showing that the expression level of 4CL3 gene significantly decreased with the an increase of treating time;the expression level of PAL4 gene significantly decreased at the 6th hour and the expression level of C4H gene didn't significantly decreased.When the treatment time of berberine was prolonged,the lowest expression level of 4CL3 genes was at the 6th hour.The qPCR results showed that after Arabidopsis thaliana treated with 10 mg·L-1 berberine for six hours,the expression levels of lignin synthesis-related genes significantly declined compared to the control group,and the expression of some genes related to cellulose synthesis partly declined while with no significant difference in part.The experimental results showed that most of the tested compounds exhibited higher activity of PAL enzyme and lower activity of 4CL enzyme than those of the control group after treating Mikania micrantha Kunth and Ageratum conyzoides L..It is suggested that the4CL enzymes may be the initial targets of the tested compounds,and the commercial herbicides acting on this targets have not been reported. |
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