The Underlying Mechanism Of Allelopathic Interaction Between Rice And Rhizosphere Microbes Mediated By PAL2-1

Allelopathy results from the interactions between rice and its rhizosphere microbes.Allelochemicals are released by allelopathic plants into the soil,bio-transformed into more active allelochemicals by microbes and then restrain the growth and development of the target plants(including microorganisms)in their vicinity.Therefore,it is important to study microbes colonized in the plant growth environment,and explore the interactions between plant secondary metabolites and rhizosphere microbial flora to further understand the allelopathy mechanisms,which provide theoretical basis and technical support for practical uses.Previous studies had shown that when the key gene PAL2-1 of phenylpropane metabolism regulation was over-expressed,the rice root exudates promoted myxobacteria abundance,and increased allelopathic inhibition rate of rice.However PAL2-1 gene suppressed line showed the opposite trend.It meant that gene mediated the interactions between plant metabolites and soil microbes,and determined the allelopathy.However,little is known about the synthesis of the allelochemicals that are regulated by PAL 2-1 gene,and the interactions between soil microbes and these allelochemicals released into soil by roots.In this study,wild type rice accessions-(i.e.allelopathic rice PI312777,nonallelopathic rice Lemont),and transgenic rice accessions –(i.e.PAL2-1 over-expressed PI312777 rice PO,PAL2-1 over-expressed Lemont rice LO and PAL2-1 interference PI312777 rice PR)were used as the experimental material.Firstly,we determined the differences of microbial communities in rhizosphere,rhizoplane and endosphere among different treatments,as well as the amounts of phenolic acids,terpenoids,and momilactones in the rhizosphere soils of different allelopathic-potential rice seedlings at 5-leaf stage.Then,the metabolomics technology was applied to further analyze the differences of metabolites in different rice rhizosphere soils,in order to explicit the action mode and mechanisms of rice allelochemicals.The main conclusions are as follows:1,High-throughput sequencing results showed that there were no differences in microbial abundances in-rhizoplane and endosphere of different allelopathic potential rice accessions at 5-leaf stage.However,microbial diversity was significantly different in the two rhizocompartments.Moreover,significant differences were observed in microbial abundance and diversity in the rhizosphere soil among different allelopathic potential rice accessions.Furthermore,the microbial community structure of rhizosphere soil was more similar to that of rhizoplane,and far different from that of endosphere.It is suggested that the shape process of rhizosphere microbiomes was preceded in the order of rhizosphere-rhizoplane-endosphere.From this study we concluded that rhizosphere mainly plays a role in the agglomeration of specific microorganisms,but rhizoplane plays a selective gating role to control entry of the microbes into the root endophere.Besides,Myxococcales abundance of allelopathic rice PI312777 was higher than the nonallelopathic rice accessions in the rhizosphere,rhizoplane and endosphere.Similarly,the results of quantitative PCR demonstrated that Myxococcales abundance in soil significantly increased when PAL2-1 gene was over-expressed,and was two times higher than the wild type rice.The opposite was true for the PAL2-1 suppressed rice accessions.The above mentioned results confirmed the root secretion played a leading role in shaping the rhizosphere microorganisms.2,This study applied LC-MS/MS method to analyze the contents of phenolic acids in rhizosphere soil of different allelopathic potential rice at5-leaf stage.The results showed that total phenolic acids in rhizosphere soil of allelopathic rice PI312777 were significantly higher than that of Lemont,and PAL 2-1 suppressed and over-expressed transgenic lines.As PAL2-1gene was over-expressed in PI312777 and Lemont rice accessions,the amount of total phenolic acids in rhizosphere soil significantly decreased,about half of the wild type rice.Especially,p-hydroxy benzoic acid content in rhizosphere significantly decreased as PAL2-1 gene was over-expressed in PI312777 and Lemont.Similarly,the amount of total phenolic acids in rhizosphere greatly decreased in PAL2-1 suppressed line as compared to the wild-type rice.In particular,the content of ferulic acid significantly decreased,but it was still higher than PAL2-1 over-expressed transgenic lines.This may be due to the increase of the phenolic-acids-utilizing microbes,especially Myxococcales abundance,in the rhizosphere soil of PAL2-1 over-expressed transgenic lines,which might decreas the phenolic acids content in soils.3,In order to further explore the effects of terpenoids on microbial communities in rice rhizosphere soil,this study applied GC-MS method to analyze the amounts of terpenoid in rhizosphere soil of different allelopathic potential rice accessions at 5-leaf stage.The result showed that the kinds of monoterpenes was hightest,especially oxygenated monoterpenes,followed by sesquiterpenes,and polyterpene showed the least in rhizosphere soil.It was found that non-allelopathic rice Lemont and PAL2-1 suppression transgenic line PR had 29 different kinds of terpenoids;allelopathic rice PI312777 had 26 kinds of terpenoids;PAL2-1overexpressed transgenic line PO and LO had 27 and 24 kinds of terpenoids,respectively.Furthermore,it was found that the total amounts of terpenoids in rhizosphere soil,sharply increased when PAL2-1 gene was suppressed in allelopathic rice PI312777(PR),far different from the wild type;allelopathic rice PI312777(PI).There is no distinct difference in the total amounts of terpenoids in rhizosphere soil between PAL2-1 overexpressed transgenic lines PO and LO,but total amounts of terpenoids of PO and LO were lower than the corresponding wild-type rice accessions PI312777 and Lemont,respectively.Therefore,the allelopathy potential of the tested rice accessions is irrelevant to the amounts of total terpenoids.4,The amounts of momilactones in allelopathic and nonallelopathic rice accessions were determined and analyzed in this study.The results showed that PAL2-1 over-expressed transgenic line PO had the highest content of momilactone A in root,Lemont roots had the lowest.The content of momilactone A in the roots of allelopathic rice PI312777 was about 4 times higher than that of nonallelopathic rice Lemont.However,the opposite was true for the content of momilactone B.Nonallelopathic rice Lemont had the hightest content of momilactone B in roots,which was two times higher than the allelopathic rice PI312777.PAL2-1 overexpressed transgenic line LO had the lowest content of momilactone B.As for the content of momilactone A in rhizosphere soils,it was found that PR had the highest content of momilactone A,PAL2-1 over-expressed transgenic line LO had the lowest,and the other four lines(i.e.CK,PO,Lemont and PI312777)showed no differences in the content of momilactone A.Besides,momilacton B was not detected in rhizosphere soil in this study.These results further illustrated that the allelopathy potential of PI312777 rice had no correlation with the content of terpenoids including momilacton A and B.5,In order to explore the differences in rhizosphere microecology of the rice accessions with different allelochemical potential,this study applied the metabonomics method to further analyze the chemical ecological characteristics in rhizosphere soil of different rice accessions.The results showed that 21 differential metabolites detected by ADS-8resin extraction in rhizosphere between PI312777 and Lemont,of which,acetic acid showed the most obvious difference.Likewise,21 differential metabolites detected by ADS-21 resin extraction in rhizophere between PI312777 and Lemont,of which phenol showed the most obvious difference with the highest VIP value.Besides,based on the results of OPLS-DA analysis,there were 26 differential metabolites between PI312777 and PAL2-1 suppressed transgenic line PR(for ADS-8 resin extraction),and 12 differential metabolites(for ADS-21 resin extraction),of which,(by ADS-8 resin extraction)and 12(by ADS-21 resin extraction),of which,hexanoic acid and Di-n-octyl phthalate showed the most obvious difference.There were 18 differential metabolites between PI312777 and PAL2-1 over-expressed transgenic line PO(by ADS-8 resin extraction),and methoprene showed the most obvious difference with the highest VIP value.Likewise,17 differential metabolites detected by ADS-21 resin extraction in rhizosphere among different rice accessions,of which Di-noctyl phthalate showed the most obvious difference with the highest VIP value.Besides,there were 24 differential metabolites between Lemont and PAL2-1 overexpressed transgenic line LO for ADS-8 resin extraction,and27 differential metabolites for ADS-21 resin extraction,of which,9-octadecenoic and phenol showed the most obvious difference.There were30 differential metabolites between Lemont and PAL2-1 overexpressed transgenic line LO for ADS-8 resin extraction,and 24 differential metabolites for ADS-21 resin extraction,of which,cucurbitacin b and Din-octyl phthalate showed the most obvious difference.KEGG annotation for those differential metabolites showed that several metabolic pathways were involved in the response of rice to PAL2-1 gene expression,such as microbial metabolism in diverse environments,fatty acid biosynthesis,and endocrine and other factor-regulated calcium reabsorption and lysine degradation,ABC transporters,purine metabolism etc.Most of these pathways were closely related to nutrition metabolism,secondary metabolite synthesis,signal substance synthesis,and toxins degradation.The results were consistent with the results of root exudates,by which a suitable micro-ecological environment was created for the interactions between rice and rhizosphere microorganisms.6,The results based on the interactions between specific rhizosphere microorganisms and main allelochemicals showed that phenolic acids could promote the growth of Myxococcus xanthus,which momilactone A,and B had a inhibitory effect on Myxococcus xanthus.It was also found that phenolic acids exuded from allelopathic rice PI312777 showed a promoting effect on Myxococcus xanthus,especially when exposed to compared to Lemont.Based on the suppression bioassay of phenolic acids and momilactone on target weed,it was found that the inhibition rate of single phenolic acid on weed growth was lower,however,the inhibitory effect of momilactones on weeds was higher,and momilactone B showed higher suppression rate on weeds than momilacton A.Besides when the target weeds were treated with phenolic acids and Myxococcus xanthus,the inhibitory rate on weeds increased.However,no significant effect,even the decreased inhibitory rate was observed when the target weeds were treated with momilactione and Myxococcus xanthus.Therefore,the suppression of allelopathic rice on weeds is the results of the interactions between phenol acids and specific microorganisms.In conclusion,the degree of the suppression of allelopathic rice PI312777 on weeds is closely related to PAL2-1 gene expression,which regulated the synthesis of phenolic acids,mediated the chemotactic proliferation of specific microbes(i.e.Myxococcus)and then produced allelopathic inhibition effect.