| Pontederia cordata,a perennial herb with developed roots and large biomass,belongs to the genus Pontederia,displaying a great potential advantage in the remediation of heavy metal-contaminated water and wetland environments.And meanwhile,it has a high ornamental value and is believed to be an excellent material for water afforestation and beautification,and also for wetland restoration and reconstruction.Solution culture was adopted to investigated(i)effects of Cd2+at different concentrations(0,5,25,50,75 mg/L)on the growth,physiological and biochemical characteristics of P.cordata\(ii)variations in photosynthetic and chlorophyll fluorescence parameters in leaves of the plant cultivated with various Cd2+concentrations;(iii)Cd2+contents accumulated in the roots,stems and leaves of the plant,chemical forms and variations in non-protein thiol peptide contents in the leaves;(iv)Illumina HiSeqTM 2000 platform was used for leaf transcriptome sequencing of the plant exposed to 50 mg/L Cd2+for Oh,1h,3h,6h,12,24h and 36h.Main results are as follows:1.According to morphological characteristics of the plant that we observed,it can grew well in the solution with 5 mg/L Cd2+cultivated for 21d,showing no toxicity symptoms.With increasing Cd2+concentration in the solution and the extensed treatment time,the plant showed obvious toxicity symptoms,including foliage chlorosis,dried stems,waterlogged roots and even the entire plant death.2.The biomass of the plant decreased with Cd2+concentrations in the solution increased,and plant death was induced by 50 mg/L and 75 mg/L Cd2+cultivated for 15d and 21d,respectively.Significant reduction in biomass of roots,stems and leaves were observed with 25 and 50 mg/L Cd2+exposed for 15d compared those in the control,and the death of the entire plant was induced by the highest Cd2+concentration(75 mg/L).With increasing Cd2+concentration in the solution and the extensed treatment time,Malondialdehyde(MDA)contents accumulated gradually in the leaves increased,enhancing membrane peroxidation,and therefore resulting in aggravated the degree of membrane system damage.The activities of superoxide dismutase(SOD),catalase(CAT)and peroxidase(POD)showed different variation trends,and SOD activity increased,while CAT activity showed an opposite tendency,and POD activity rose in the first stage and than desreased.Soluble sugar and proline contents showed increased first and than decreased,while soluble protein content in leaves showed a decreased tendency with increasing Cd2+concentration in the solution and the extensed treatment time.With increasing Cd2+concentration and the extensed treatment time,ascorbic acid(AsA)content in the leaves decreased,and dehydroascorbic acid(AsA-DHA)content increased first and than decreased.In summary,higher concentration of Cd2+and long time treatment are not in favor of the plant growth,and will suppress its physiologically and biochemically metabolic activities.And more,the plant can adapt to Cd2+stress by increasing antioxidant enzyme activities,osmotic regulatory substance contents,as well as AsA and AsA-DHA contents,but this regulatory has limitations to some extent.3.With Cd2+concentration increased in the solution and the treatment time prolonged,Chlorophyll biosynthetic precursors including protoporphyrin IX(Proto IX),Magnesium protoporphyrin IX(Mg-proto IX)and Pchlide contents decreased,and similar changes were also found in chlorophyll a(Ch1 ?),chlorophyll b(Ch1 b),total chlorophyll(Ch1 T),carotenoid(Car),lutein as well as total carotenoid(Car T)contents,which were responsible for toxic symptoms such as chlorisis induced by Cd2+.Photosynthetic physiological parameters including net photosynthetic rate(Pn),ntercellular carbon dioxide concentration(CG),Transpiration rate(Tr),stomatal conductance(Gs)and carbon dioxide utilization efficiency(CUE)showed a generally descended trend,which were contrary to those in vapor pressure deficient(VPD)and stomatal limitation(Ls),and water use efficiency(WUE)increased by 24.93%?105.54%with 5 mg/L Cd2+addition in the solution during the trial period and than decreased induced by higher Cd2+concentrations compared to that in the control,indicating that stomatal factors were responsible for the decreased Pn in P.cordata leaves with Cd2+exposure.According to the variations in chlorophyll fluorescence parameters,we argued that higher Cd2+concentration and prolonged long treatment time will have a negative impact on photosynthetic apparatus in P.cordata leaves,and induced partial closure in PSII reaction center and reduced photosynthetic activity,making PSII reaction center less efficient,thereby inducing photoinhibition.However,the PSII reaction center can relieve inhibited effects induced by Cd2+by increasing the non-photochemical dissipated energy,reduceing the electron transfer efficiency and enhancing the PS reaction center load,which may be one of the important mechanisms by which the plant protects the photosynthetic apparatus from Cd2+damage.4.Cd2+contents accumulated in the roots,stems and leaves of P.cordata increased with Cd2+concentrations increasing in the solution.Cd2+contents accumulated in the roots,stems and leaves were 112.98?528.98 times,77.04?193.49 times and 80.30?192.98 times as many as the control groups after the plants were exposed to 5,25,and 50 mg/L Cd2+for 15d.Roots of P.cordata were demonstrated to be an important organ for accumulating Cd2+,and Cd2+contents accumulated in the roots increased by 3.93?25.82 times and 1.59?4.04 times than those in stems and leaves,respectively.Translocation factors for Cd2+from roots to shoots(TFi)and from roots to stems(TF2)decreased with Cd2+concentrations increased,while translocation factor for Cd2+from stems to leaves(TF3)manifest a relative stable level.Bioaccmulation factors for roots and shoots to Cd2+ also decreased.The predominant forms of Cd2+were residue,water-extraction and ethanol-extraction in roots,residue,acetic acid-extraction,protein-extraction in stems,which is in favor of inhibiting Cd2+transportation from stems to leaves,and residue,hydrochloric acid-extraction and acetic acid-extraction in leaves that can alleviating the toxic effects of Cd2+to physiologically and biochemically metabolic activities such as respiration and photosynthesis.To some extent,higher Cd2+concentrations and prolonged treatment time can induce total non-protein thiol peptide,glutathione and other non-protein thiol peptide(such as PCs and Cys)contens increasing,and the content of other non-protein thiol peptide is far lower,probably demostrating that they may be not the major chelating peptides that reducing the toxic effects of Cd2+.5.Illumina HiSeqTM 2000 platform was used for transcriptome sequencing of P.cordata leaves at control(0h),1h,3h,6h,12h,24h and 36h Cd2+stress.We constructed 21 cDNA libraries,and obtained 139.02 GB clean reads after filtering 970,961,618 raw reads.After Trinity splicing,258,354 contigs with an average length of 1,542 bp were obtained,and 221,392 non-redundant Unigenes were obtained after further splicing,with an average length of 1,753 bp.6.GO functional classification showed that 221,392 uni genes were annotated into 55 GO items of biological process,cell component and molecular function.There are totally 25 GO items in biological process,mainly including cell process,metabolic process,single organism process and other functional groups,20 GO items in cell component with cell and cell part in predomination,and 10 GO items in molecular function with protein binding activity,catalytic activity and transport activity dominating.There were totally 54,087 uni genes annotated into 131 KEGG pathways mainly including carbon metabolism,ribosome,amino acid biosynthesis,plant-pathogen interaction,protein processing in endoplasmic reticulum,plant hormone signal transduction,endocytosis,spliceosomal,RNA transport and purine metabolism.Secondary metabolite biosynthesis plays a secondary role in the metabolic pathway,mainly including the of phenylpropanoid,carotenoid and terpenoid skeleton biosyntheses,demonstrating these secondary metabolites could play a vital role in the response of P.cordata to Cd2+stress.7.Differentially expressed gene(DEG)were identified by the standard of padj<0.05 and|log2 FoldChange|>1.Compared to the control,a plenty variety of DEGs were identified at different sampling dates.GO functional classification demonstrated that the up-regulated DEGs were mainly involved in the metabolic process,cellular process,primary metabolic process,organic metabolic process,catalytic activity,and binding process as well the organic process,which were similar to the down-regulated DEGs,and indicated that the plant could adapt to Cd2+exposure by changing cellular component,the catalytic activity and various metabolic processes.By analyzing of KEGG pathway,we found that there totally were 122 most enriched KEGG pathway of DEGs,and pathways related to glycolysis/gluconeogenesis,photosynthesis-antenna proteins,photosynthesis,starch and sucrose metabolism,amino and fatty acid metabolism,plant hormone signal,and ABC transporters as well as transcriptional factors were largely affected by Cd2+exposure.What's more,genes involved in aquaporins,antioxidant enzymes,heat shock proteins,metalloprotein,zinc finger proteins,and ABC transportproteins as well as transcriptional factor families were probably play vital role in the slow and rapid response of P.cordata to Cd2+stress.In summary,the response of P.cordata to Cd2+stress involves multiple genes and biological processes.8.There were 81 transcription factor familes including 10,782 transcription factors found in the transcriptome database of P.cordata leaves,and MYB,WRKY,Orphans,C3H and bHLH as well as the other familes probabaly were invovled in coping with Cd2+stress via regulation of transcription,ion transport,osmotic adjustment and amino acid metabolism as well as signal transduction,which could play improtant role in the tolerance of P.cordata to Cd2+stress.Our present investigations revealed the improtant roles of antioxidants(antioxidant enzymes,ascorbic acid and carotenoids)and osmotic regulation substances in the leaves in the response of P.cordata to Cd2+stress by employing physiological methods,made it clear the cause of the declined in photosynthetic capacity induced by Cd2+stress,and threw light on the detoxification mechanisms of the plant to Cd2+stress throught investigating the translocation and bioaccumulation,chemical forms of Cd2+and the contents of non-protein thiol peptide in the leaves,thus laying a foundation for the application of the plant in heavy-metal polluted water or wetlands.What's more,a large number of differentially expressed genes were identified in the leaves of the plant by transcriptome sequencing,and we revealed the molecular mechanisms of the plant to Cd2+stress,providing gene resouces for the future exploration and in-depth investigation on the genes involved in Cd2+tolerance and accumulation. |
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