Physiological And Genetic Toxicity Of Copper Stress In Hydrilla Verticillata(L.f.) Royle

With the development of social and economy,soil and water pollution becomes more and more serious.It has been reported that heavy metal becomes a major pollutant for water.Phytoremediation has some advantages,such as less investment,high efficiency,easy operation and no damage to the environment,which makes it widely used in the remediation of heavy metal contaminated water.Hydrilla verticillata(L.f.)Royle,which is widely distributed submerged macrophyte in freshwater lakes,is considered as one of the potential hydrophyte for the remediation of heavy metal-contaminated water.In this study,the accumulation and distribution of Cu in H.verticillata,as well as Cu-induced physiological and genetic toxicity to H.verticillata,were studied by using physiological,proteomic and genome epigenetic methods.These results provide a basis for monitoring heavy metal pollution in freshwater lakes and subsequent remediation by a proper hydrophyte plant.In this study,excessive Cu significantly reduced the biomass of H.verticillata.With the increase of Cu concentration,the Chla/Chlb ratio in leaves decreased,while no significant changes of such ratio were observed in stems.Under Cu treatment,the accumulation of O2·-,H2O2 and MDA increased in leaves;the activities of POD,APX and GR increased,but AsA/DHA decreased significantly in leaves.In addition,only the activity of SOD increased significantly in stems.Thus,these results indicated that the leaves of H.verticillata were more sensitive to Cu stress,and the antioxidant responses between leaves and stems were different.Compared to the control leaves,0.05 and 0.1 mg L-1 Cu treatment reduced the contents of both Chl a(Chlorophyll a)and Chl b(Chlorophyll b)as well as the ratio of Chl a/Chl b in mature and young leaves in both mature and young leaves,and the chlorophyll contents decreased more in mature leaves than in young leaves.We found that Cu accumulated mainly in the leaves of the plants,and these Cu was mainly deposited in the cell wall.Compared with young leaves,the mature leaves accumulated significantly higher amount of Cu,and the chloroplast microstructure and the structure of the leaf surface were damaged more seriously.The Cu contents in cell wall cellulose,hemicellulose 1 and pectin of mature leaves were significantly higher than those in young leaves,which was consistent with the fact that the galacturonic acid contents of cell walls cellulose,hemicellulose 1 and pectin were higher in mature leaves,which demonstrated that the carboxyl and hydroxyl in cell wall polysaccharides play key roles for absorption and complexation of Cu2+.Besides,these studies also indicated that there was more Cu transport from young to mature tissues than from mature to young tissues.To investigate proteins involved in responses to Cu stress,two-dimensional electrophoresis and MALDI-TOF mass spectrometry techniques were used to isolate and identify the differentially expressed proteins in mature leaves and young leaves of H.verticillata following Cu treatment.The results showed that,after 5 days of Cu treatment,abundance of a total of 44 protein had a?1.5-fold change each in mature leaves and young leaves compared to the control.The aundances of 27 protein spots were up-regulated,and 4 spots were down-regulated in mature leaves.Furthermore,the abundances of 35 protein spots were up-regulated,and 3 protein spots were down-regulated in young leaves.Besides,the abundances of 21 protein spots were both up-regulated in young leaves and mature leaves,of which 5 spots were more significantly up-regulated in mature than in young leaves,and 8 spots were more significantly up-regulated in young than in mature leaves.These proteins are important enzymes involved in the cell wall synthesis pathway,such as alpha-1,4-glucan-protein synthase,multicopper oxidase LPR1-like,pectinesterase-like,UTP--glucose-1-phosphate uridylyltransferase-like,UDP-glucose 6-dehydrogenase and UDP-D-apiose/UDP-D-xylose synthase 2.In addition,these differentially expressed proteins are involved in a variety of physiological processes,such as photosynthesis,energy metabolism,antioxidant defense,anabolism metabolism,methylation,transcription regulation,protein folding and stability maintenance,cell structure and signal transduction.More importantly,the proteins involved in chloroplast repair(such as Rieske iron-sulfur protein precursor and ATP-dependent zinc metalloprotease FTSH 2)and antioxidant defense(such as ascorbate peroxidase)were significantly up-regulated in young leaves than in mature leaves.It has been reported that heavy metal stress is able to cause plant genomic DNA damage.We used AFLP(Amplified Fragment Length Polymorphism)technique to analyze the genomic DNA damage of H.verticillata leaves.The results showed that the AFLP diversity in leaves of H.verticillata increased gradually with the increase of Cu concentration.Under 0.1 mg L-1 Cu treatment,AFLP polymorphism and the contents of 8-OHdG were significantly higher in mature leaves than in young leaves of H.verticillata.Heavy metal stress can also lead to abnormal methylation patterns of plant genomic DNA.In response to Cu stress,epigenetic modifications occurred in leaves genome of H.verticillata.,MSAP(Methylation Sensitive Amplification Polymorphism)revealed that the percentage of unmethylated 5'-CCGG-3' sites of genomic DNA of mature leaves significantly increased,and the total methylation levels decreased significantly after these leaves were treated with 0.1 mg L-1 Cu.And the methylation and demethylation of 5'-CCGG-3' sites in mature leaf genome,and the demethylation in young leaf genome significantly increased.Somemethylation-modified fragments of H.verticillata were isolated,which were identified as genes closely related to methylation and physiological activities,and the regulatory sequences of non-coding genes.There are many types of promoter cis-acting elements in MSAP detection specific band sequences above.In addition,the results also showed that the CpG dinucleotide-rich region in H.verticillata genome is the potential site for DNA cytosine methylation.Changes in DNA methyltransferase activity are one of the ways to regulate DNA methylation levels.Using proteomic analysis techniques,we found that the protein abundances of three DNA methylation-related enzymes(chromomethylases,domains rearranged methyltransferases and su(var)3-9 homologue 6)were significantly upregulated in the leaves of H.verticillata under Cu stress,which may lead to increased levels of methylation.The pretreatment of the NADPH oxidase inhibitor reduced the Cu-induced ROS(Reactive oxygen species)production,which is probably assocaited with a change in the DNA demethylation pattern,but had little effect on the methylation pattern.It is likely that under Cu treatment,accumulation of ROS induced DNA damage,which may prevent DNA methyltransferase binding and thus result in demethylation of some sites.Taken together,this study demonstrated that the mature leaves had significantly higher Cu accumulation than young leaves,and the chloroplast microstructure and the structure of the mature leaf surface were relatively vulnerable to Cu.There was more Cu transport from young tissues to mature tissues than from mature tissues to young tissues,which may result from the fact that the cell wall of mature tissues has more D-galacturonic acid.Using proteomics techniques,we found that proteins involved in chloroplast repair(such as Rieske iron-sulfur protein precursor and ATP-dependent zinc metalloprotease FTSH 2)and antioxidant defense(such as ascorbate peroxidase)were significantly up-regulated in young leaves than in mature leaves.Under Cu stress,the expression changes of DNA methyltransferase and the ROS accumulation are two ways wherein the genomic DNA methylation level was changed.The results of these studies can provide important theoretical basis for elucidating the mechanism of Cu toxicity to aquatic plants.