| Soil pollution has become a global environmental problem.Most contaminated soils contain multiple contaminants.Especially,the problem of cadmium(Cd)and arsenic(As)compound pollution has attracted the general attention of scholars in the world.The characteristics of perennial ryegrass(Lolium perenne L.)responses to Cd and As stress and varieties differences are the theoretical basis for the remediation and utilization of Cd and As contaminated soils.The research on the physiological and molecular mechanism of Cd and As absorption and transport in plants has important theoretical and practical significance.In this study,31 perennial ryegrass varieties were used as plant materials.Soil culture experiment was carried out under the treatments of different concentrations of Cd and As to understand four varieties with differences in Cd and As accumulation capacity and study the concentration and time effects,subcellular distribution and ultrastructure,antioxidant enzyme activity,key substances contents,related enzyme activity,and transporter activity of different varieties under Cd and As combined stress.The molecular mechanism of varieties difference was probed with transcriptome RNA-seq technology.The results showed that:(1)Perennial ryegrass was one of plants with high Cd accumulation capacity.The root was the main Cd accumulated site with the BCF(Bio-concentration factors)> 1.The BCF and TF(transport coefficient)of Cd of 31 perennial ryegrass varieties were higher than those of As.In the ranking of comprehensive membership function values,the accumulation capacity of ‘Bay’,‘Nicaragua’ and‘Holly Green’ was the first group with the membership function values 2.31,1.89 and 1.81,respectively.Through comprehensive evaluation combined with cluster analysis,four different varieties with different Cd and As accumulation capacity were screened out,namely ‘Nicaragua’with high Cd and high As,‘Venus’ with low Cd and low As,‘Excellent’ with high Cd and low As and ‘Monroe’ with low Cd and high As.(2)With the increase of Cd and As treatment concentration,the plant height,root length,shoot and root biomass of perennial ryegrass increased first and then decreased,in which there was a phenomenon of promoting in low treatment concentration and inhibiting in high treatment concentration.With 1 mg/kg or 3 mg/kg Cd and 40 mg/kg or 80 mg/kg As treatments,the plant height,root length,shoot and root biomass of perennial ryegrass increased significantly,which the maximum increased 26.82%,53.29%,2.98% and 17.78% compared with the control.However,With 12 mg/kg Cd and 160 mg/kg As treatment,the plant height,root length,shoot and root biomass of perennial ryegrass decreased by 42.89%,56.25%,53.43% and 70.73%.Compared with single stress,the contents of Cd and As in perennial ryegrass decreased significantly under the combined stress,in which the contents of Cd and As in shoots decreased by 63.39% and 32.69%,while in roots were decreased by 61.26% and 45.98%.(3)Cd and As in perennial ryegrass were mainly distributed in cell wall and soluble components.Under the combined stress,the highest contents of Cd and As in the cell wall occupied60.47% and 65.25% of the total,in the soluble components 66.10% and 55.84% of the total.Chloroplasts were also important sites for the accumulation of Cd and As.The distribution of Cd in cell wall and soluble components was scattered and extensive.The distribution of As in cell wall and chloroplast had obvious regional characteristics,which was consistent with the distribution of P.(4)Compared with the control,the peroxidase(POD)activity and phytochelatin(PCs)content of perennial ryegrass under As stress increased with the peak of 271.63% and 567.98%,respectively.Under the combined stress of Cd and As,the content of malondialdehyde(MDA)and the activities of POD,superoxide dismutase(SOD)and catalase(CAT)of low accumulation variety ‘Venus’ were28.31%,86.83%,81.46% and 46.26%,which were higher than those of high accumulation variety‘Nicaragua’,respectively.The activities of γ-glutamylcysteine synthetase(γ-ECS),glutathione synthetase(GSS),phytochelatin synthases(PCSase),the contents of glutathione(GSH)and PCs were 10.14%,10.87%,7.23%,18.00% and 293.89%,which of ‘Nicaragua’ were higher than those of ‘Venus’,respectively.However,the arsenate reductase(AR)activity of ‘Venus’ was greater than that of ‘Nicaragua’.Under combined stress,antioxidant enzyme activities and the parameters of NPT,GSH,PCs,γ-ECS,PCSase and AR were significantly correlated with the contents of Cd and As,except for GSS activity(P<0.05).(5)Under Cd and As combined stress,the activities of CAX,HMA,NRAMP and PHT in shoots of four perennial ryegrass varieties increased.‘Nicaragua’ showed the highest increase,which increased by 53.19%,79.08%,53.82% and 39.94% compared with the control,respectively.Transporter protein CAX,NRAMP and PHT activities in shoots were higher than those in roots of perennial ryegrass.The highest shoots/roots ratios were up to 1.44,1.39 and 1.23,respectively.(6)A total of 1.75 GB of clean data were obtained by RNA-seq.A total of 105243 Unigene sequences were obtained after assembly with Trinity software.Annotation information was obtained for 55428 sequences,accounting for 73.23% of all Unigene.And 594 homologous species information were obtained.The number of Unigene annotations to Nr,Swiss-Prot,KOG and KEGG were 66234,60062,31229 and 40275,respectively.The number of differential genes between‘Monroe’ and ‘Excellent’ with combined stress was higher that with single As or Cd stress,which were mainly down-regulated.The differential genes were mainly belonging to glutathione metabolism,MAPK signaling pathway-plant and protein export.‘Excellent’ was mainly focused on oxidative phosphorylation,glutathione metabolism and selenium compound metabolism.‘Monroe’was mainly focused on oxidative phosphorylation,photosynthesis,cysteine and methionine metabolism.The glutathione metabolic pathway was an important differential metabolic pathway.(7)Under Cd and As combined stress,the significantly different genes of transporter protein CAX of perennial ryegrass roots were dominated by Lp CAX1 a and Lp CAX1.The significant differences genes of transporter HMA were Lp HMA4、Lp HMA3、Lp HMA5 and Lp HMA2,among which Lp HMA4 genes were widely observed among different treatments.The significant differential genes of NRAMP was dominated by Lp NRAMP6.The genes significant differences of transporter PHT were Lp PHT1,Lp PHT1-4,Lp PHT1-5,Lp PHT1-6,Lp PHT1-8,Lp PHT1-9,Lp PHT1-13,Lp PHT2,Lp PHT3,Lp PHT4;3 and Lp PHT4;7.Gene Lp PHT1 had the highest number of differential genes and Lp PHT1 and Lp PHT1-4 genes differences existed widely.In conclusion,the results indicate that the responses of perennial ryegrass to Cd and As stress have great differences in resistance physiology and related gene expression.Glutathione metabolism and transporters Lp CAX1,Lp HMA4,Lp NRAMP6 and Lp PHT1 in perennial ryegrass roots are important molecular mechanisms for the response and distribution of Cd and As. |
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