| The "Novel Weapon hypothesis" expounded that allelopathy plays a very important role in the successful invasion of alien plants.Its underlying mechanism on the genetic and molecular biological levels is not sufficient.Allelochemicals can directly inhibit the growth of local plants or change the structure of underground microbial community indirectly,to affect the interaction with plants and promote the successful invasion of alien plants.Polyploidy exists widely in invasive plants and could have a significant impact on plant secondary metabolites.Solidago canadensis L.is a perennial herb of Asteraceae.It is native to North America and has escaped to become a malignant invasive weed all over the world.S.canadensis has rich chromosome variation,mainly including diploid(2n = 18),tetraploid(2n = 36)and hexaploid(2n = 54).Polyploids successfully invaded in East Asia are often accompanied by enhanced allelopathy.Phenolics,flavonoids and saponins are its main allelopathic chemicals.Polyploidization can promote the allelopathic effect of S.canadensis,but the regulation mechanism is not clear yet.Based on the commen garden experiment,this paper analyzes the differences of allelopathic potential among different geo-cytotypes of S.canadensis,and compares the temporal and spatial dynamic changes of the content of main allelochemicals and their allelopathic inhibitory effects.After that,the expression patterns,copy numbers and DNA methylation modification of six key genes in the biosynthetic pathway of flavonoids in different geo-cytotypes were also compared.This study attempts to clarify the physiological and molecular mechanism of polyploidy promoting the allelopathic potential of S.canadensis.The main research contents are as follows:Through the isolation,extraction and detection of the main allelochemicals of S.canadensis,the allelopathic potential of 70 different geo-cytotypes populations was evaluated by laboratory bioassay.It was found that the invasive tetraploid and hexaploid had the strongest allelopathic inhibition potential,followed by the native polyploid,and the diploid population had weak allelopathic activity.It was also found that flavonoids had the strongest allelopathic activity among the three main allelochemicals,and the relative content in different geo-cytotypes of S.canadensis was positively correlated with the inhibitory effect.The results showed that there was differentiation of allelopathic potential in different geo-cytotypes of S.canadensis population.In order to evaluate the allelopathic potential of different geo-cytotypes of S.canadensis in the whole growth and development cycle under natural conditions,we selected the vegetative stage,flowering stage and fruiting stage to determine the contents of three main allelochemicals in the aerial parts and rhizosphere soil of different populations in the field condition commen garden experiment.The results showed that the contents of three allelochemicals showed dynamic changes during the whole growth and development cycle.The contents of phenolics and flavonoids were at the highest peak in the vegetative period,while the contents of saponins were the highest in the fruiting period,reflecting the biological characteristics of the balanced allocation of S.canadensis resources.However,no matter in which development period,the invasive tetraploid and hexaploid populations always have the highest content of allelochemicals,followed by the native hexaploid population,while the content of the native tetraploid and diploid populations is lower.The results of bioassay showed that the allelopathic potential of different geo-cytotypes of S.canadensis was positively correlated with the content of allelochemicals.The extract of S.canadensis had a significant inhibitory effect on the seed germination and seedling growth of both Digitaria sanguinalis and Amaranthus retroflexus,and the inhibition rate increased with the ploidy,but Setaria viridis and Alternanthera philoxeroides were not sensitive to it.This phenomenon was consistent with the dynamic succession of species community in the five-year field common garden experiment,which proved that the allelopathy of S.canadensis was one of the important factors to change the aboveground community composition.Polyploidization significantly promoted the allelochemicals metabolism of polyploids,after successful invasion,the invasive polyploids may further enhance this internal advantage through rapid adaptive evolution.The above mechanisms jointly confer the invasive polyploids better competitive ability with local weeds than diploid and native polyploids.Polyploidy enhanced the metabolism of allelochemicals is one of the important mechanisms for the successful invasion of S.canadensis into China.After proving that flavonoids are the main kind of allelochemicals of S.canadensis,the biological metabolic pathway of flavonoids was selected as a representative to study the internal molecular mechanism of the increased allelopathy of S.canadensis conferred by polyploidization.The conserved region sequences of three structural genes ScCHS,ScCHS,ScF3 H and three transcription factors ScTTG1,ScPAP1 and ScGL3 in MBW transcription regulation complex were cloned from S.canadensis,and the full-length DNA sequence and flanking sequence of ScCHS and ScTTG1 were further cloned.By analyzing the expression patterns of the above genes in different geo-cytotypes of S.canadensis at different development stages,it can be seen that the gene expression level is positively correlated with the content of flavonoids in the corresponding population,which is significantly down regulated from vegetative stage to fruiting stage.The gene expression of invasive polyploids at each stage is significantly higher than that of diploids.It is proved that polyploidy promotes the expression level of key genes in flavonoid metabolic pathway of S.canadensis.In order to clarify the molecular mechanism that how polyploidization affected ScCHS and ScTTG1 expression levels,the copy number and DNA methylation were detected.The results showed that the copy numbers of ScCHS and ScTTG1 genes of invasive hexaploid were 3 ～ 3.2 times than that of diploid population,and the copy number of invasive tetraploid were 2 times than that of diploid population.DNA methylation results showed that demethylation of ScCHS promoter region and methylation of gene body jointly promoted gene expression in polyploid populations.While no methylation site was found in ScTTG1 gene body,but demethylation of ScTTG1 promoter region promoted gene expression in polyploid populations.In conclusion,polyploidization drives S.canadensis to enhance the metabolism of allelochemicals such as phenolics,flavonoids and saponins.The key genes in flavonoid metabolism pathway of polyploids are up-regulated by the increased gene copy numbers,the demethylation of their promoter region and the methylation of their coding region.It promotes the accumulation of flavonoids in polyploid population of S.canadensis,which makes them have stronger allelopathy potential,and strengthens the competitive capacity with local palnt species.This research firstly uncovers the molecular mechanism of the new weapon hypothesis that S.canadensis successfully invaded in China through allelopathy-enhancement. |
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