Effect Of Momilactone B Of Rice Key Allelochemical On Seed Germination And Root Development Of Arabidopsis Thaliana And The Related Mechanisms

Weeds in farmlands are important factors that affect the farm crop yield and qualities.At present,the main ways to control farmland weeds is the use of chemical herbicides.The residual pollution caused by the excessive use of chemical herbicides has seriously threatened the safety of organisms,and the indirect consequences of the production of resistant weeds and the destruction of biodiversity resources is destroying ecological balance.Allelopathy is a new way to control weeds,which can avoid the great harm caused by chemical herbicide to the environment.It is a sustainable agriculture measure.In the research on rice allelopathy,Momilactone B has a strong inhibitory effect on barnyard grass and other weeds,which is the main allelopathic chemical of rice,but its mechanism is seldom reported.In this study,the effects of Momilactone B on seed germination and seedling root growth of Arabidopsis thaliana were studied systematically,and the mechanism of Momilactone B of inhibiting seed germination and seedling root growth of Arabidopsis thaliana was investigated from the perspective of hormone metabolism.In addition,with the Arabidopsis thaliana activation-tagging mutant library as the material,this study also screened the insensitive mutants of Arabidopsis thaliana,so as to investigate whether there are other regulatory mechanisms of Momilactone B.The main results are as follows:(1)2 ?M Momilactone B significantly inhibited the germination rate of Arabidopsis thaliana seeds,and the inhibitory effect was more significant with the increase of concentration of Momilactone B.(2)Momilactone B significantly induced the up-regulation of the transcription level of key transcription factors in ABA signaling pathway and ABA synthesis pathway in Arabidopsis thaliana seeds,among which ABI4 was up-regulated by 1162 times,suggesting that Momilactone B may induce the dormancy and inhibit germination through promoting the ABA synthesis and activating the ABA signaling pathways.After the treatment with Momilactone B,the germination rate and the rate of cotyledon greening of ABA mutant abi4 were significantly faster than that of wild-type,and the inhibitory effect on seedling root growth had no difference with the wild-type Arabidopsis thaliana,suggesting that ABA mutant abi4 can weaken the inhibitory effect of Momilactone B on the germination of Arabidopsis thaliana seeds.In addition,Momilactone B significantly induced the up-regulation of DELLA protein gene transcription level in GA signaling pathway in Arabidopsis thaliana seed,among which the major DELLA protein RGL2 that inhibited the seed germination was up-regulated by 46 times,suggesting that Momilactone B promoted the high expression of DELLA protein gene in GA signaling pathway to inhibit the seed germination.(3)cps4 is a key gene in the synthesis of Momilactone B.Arabidopsis thaliana Col-0 and ABA mutant abi4 were co-cultured with mutant rice cps4 and wild-type rice.The germination rate and root length of abi4 were always larger than those of Col-0,suggesting that abi4 weakened the allelopathy of Momilactone B on Arabidopsis thaliana.(4)Momilactone B inhibited the root growth of barnyard grass,and the inhibitory effect was more significant with the increase of Momilactone B concentration.Momilactone B inhibited the length of main root of Arabidopsis thaliana and the number of lateral roots,and the inhibitory effect was more significant with the increase of Momilacton B concentration.Confocal microscopy showed that the length of apical meristem of Arabidopsis thaliana was significantly shorter and the number of cells was significantly decreased after the treatment with Momilactone B,suggesting that Momilactone B inhibits the growth of apical meristem and cell division.The treatment with Momilactone B significantly reduced the fluorescence of Arabidopsis thaliana J2341,label by stem cell ecological niche.In addition,the treatment with Momilactone B significantly reduced the apical GUS activity of Arabidopsis thaliana CYCB1::GUS reflecting the cell division activity,suggesting that Momilactone B inhibits the root growth by inhibiting stem cell activity and cell division activity in apical meristem.(5)ABA mutant abi4 can weaken the inhibitory effect of Momilactone B on the germination of Arabidopsis thaliana seeds,but cannot weaken the inhibitory effect of Momilactone B on root growth of Arabidopsis thaliana.Exogenous administration of GA could alleviate the inhibitory effect of Momilactone B on the main roots and lateral roots.Exogenous administration of low-concentration NAA could alleviate the inhibitory effect of Momilactone B on the main roots,lateral roots and root hair.After treatment with Momilactone B,the fluorescence of auxin rapid response Arabidopsis thaliana pro DR5-GFP was significantly weakened,and the fluorescence of Arabidopsis material DII-VENUS that monitored the auxin distribution system in the plant was significantly enhanced,suggesting that Momilactone B reduces the accumulation of auxin in the root tip.After treatment with Momilactone B,the transcript levels of auxin early response genes,IAA gene family,GH3 gene family and SUAR gene family,were significantly up-regulated,suggesting that MB enhances the response of Arabidopsis thaliana to Auxin.After treatment with Momilactone B,the fluorescence of Arabidopsis thaliana of auxin transport,pro PIN1:PIN1-GFP,pro PIN2:PIN2-GFP and pro PIN3:PIN3-GFP,was significantly weakened,and the protein expression level of auxin transporter was down-regulated,suggesting that Momilactone B inhibits the activity of apical meristem and tissue elongation through inhibiting the auxin transport,finally inhibiting the root growth of Arabidopsis thaliana.(6)Momilactone B screened the insensitive mutants of Momilactone B in T-DNA activation-tagging mutant bank CS31100,and nine mutants with significant resistance genotype were screened out: M16,M17,M25,M26,M28,M29,M31,M32 and M33,and the T-DNA insertion sites were as follows: M17: At4g23980(ARF9),M25: At5g58360,M26: At4g58370,M28: At1g03620,M29: At4g00580(COP1-interacting protein-related),M31: At1g21480,M33:At4g29840.After the homozygous lines of M17 and M29 were screened,Momilactone B was used to treat the seedlings of mutants M17 and M29,and it was found that the inhibition degree of Momilactone B on root growth of M17 and M29 was significantly smaller than that of wild-type Arabidopsis thaliana,suggesting that the mutants M17 and M29 have the resistance phenotype to Momilactone B.The results of quantitative analysis showed that the expression levels of At4g23980(M17)and At4g00580(M29)were significantly up-regulated after T-DNA insertion,suggesting that the resistance phenotype of M17 and M29 is related to these two genes.