Accumulation,Distribution And Transformation Of Arsenate By Chlamydomonas Reinhardtii

Arsenic(As)is a toxic metalloid widely distributed in the marine,freshwater and soil environment.In the natural environment,As has much speciation,which have.different toxicity.The transformation of As from high toxic speciation to low toxic one is one of the important biological detoxification mechanisms.As a class of primary producers in marine ecosystems,microalgae has complicated As absorption and detoxification processes.Microalgae can detoxify As through a series of processes such as adsorption,oxidation of arsenite(As(Ⅲ)),reduction of arsenate(As(Ⅴ)),methylation of As(Ⅲ),synthesis of organic arsenicals,efflux,etc.It’s also affected by phosphate in the environment.It is known that Chlamydomonas reinhardtii can transform absorbed inorganic As into different speciation.However,As distribution in various cellular components(water-soluble,lipid-soluble,and residual)is not clear.Under different As(Ⅴ)treatments,the influence of genes related to As accumulation and detoxification in C.reinhardtii is also unknown.Therefore,the differences in the growth of wild type(WT)and multiple phosphate transporter mutants of C.reinhardtii were first explored,and the effect of PHOX on phosphate absorption was confirmed to be the greatest.The impacts of PHOX deletion on the As(Ⅴ)accumulation and transformation by C.reinhardtii were also studied.Then,the differences in As accumulation,component distribution and speciation transformation were compared between WT and the mutants of As(Ⅴ)reductase gene(ACR2.1,ACR2.2),As(Ⅲ)methylase gene(arsM)and As(Ⅲ)efflux gene(arsB)exposed to different As(Ⅴ)concentrations.The results of this study are as follows:1.Differences in the accumulation,distribution and speciation transformation of As(Ⅴ)by WT and phosphate transporter gene mutants of C.reinhardtiiWT and phosphate transporter mutants were cultured for 7 d under different phosphorus(P)nutrient conditions to identify the main transporter gene controlling phosphate entry into cells.Then,As(Ⅴ)treatment was set up to explore the influence of the mutation of the main phosphate transporter gene on the accumulation and transformation of As and P absorption in C.reinhardtii.The results showed that under different P conditions(3150,315,157.5,63,31.5 μg·L1),WT was dominated by the absorption of P,with less adsorption.With the decrease of P concentration,the accumulation and absorption of P by WT and various mutants were significantly reduced,indicating that the accumulation of P by C.reinhardtii was related to the concentration of P in the medium,and the absorption of P by various mutants was significantly different.Under different P conditions,the P absorption of △PHOX was significantly lower than WT,with the maximum decrease of 41.49%,which was far more than other mutants.It was showed that PHOX played a leading role in the absorption of P by C.reinhardtii,especially under low P conditions.In the 315 μg-1·L-1 P condition,WT and △PHOX were treated with 10 μg·L-1 As(Ⅴ),and the results showed that the As accumulation of WT was more than △PHOX.The As content in the medium was just the opposite,indicating that PHOX could regulate the absorption of As(Ⅴ)by C.reinhardtii.The content of lipid-soluble As was higher than that of water-soluble and residual As by both △PHOX and WT.The content and proportion of △PHOX lipidsoluble As were significantly lower than WT,while the contents of water-soluble and residual As were not significantly different from WT.In addition,compared with WT,the lack of PHOX promoted the reduction of As(Ⅴ)and affected the formation of arsenosugars and other As compounds,indicating that PHOX had an important effect on As transformation of C.reinhardtii.2.Differences in the accumulation,distribution and speciation transformation of As by WT and various As metabolism-related gene mutants of C.reinhardtii under different As(Ⅴ)treatmentsWT and various As metabolism-related gene mutants were cultured for 7 d under different As(Ⅴ)concentrations to study the As(Ⅴ)accumulation,distribution and speciation transformation by C.reinhardtii.The results showed that,upon the As(Ⅴ)exposure,absorption dominated the As accumulation of WT and mutants.The content of lipid-soluble As was also higher than that of water-soluble and residual As.As(Ⅲ)were the main species in the water-soluble As by WT,which also contained a small amount of DMA and two unknown As compounds,possibly arsenosugars.With the increase of As(Ⅴ)concentration,the proportion of lipidsoluble As in the total intracellular As decreased first and then increased,while that of watersoluble As showed an opposite trend.It showed that there were differences in the As detoxification pathways.Therefore,under low As stress,As(Ⅴ)was reduced to As(Ⅲ),followed by methylation to DMA and arsenosugars,and efflux occurred in WT cells.With the increase of As concentration,in addition to the above processes,WT detoxified As through decrease of As(Ⅴ)absorption and generation of more lipid-soluble As.Compared with WT,As accumulation in △ACR2.1 was significantly reduced,while△ACR2.2 increased.Under different As(Ⅴ)concentrations,the content of water-soluble As by △ACR2.1 was lower,but there was no significant difference in the proportion of each component As,while the As distribution change by △ACR2.2 were similar to WT.Although the As(Ⅴ)reduction ability of △ACR2.1 and △ACR2.2 were declined,as the concentration of As(Ⅴ)increased,the proportion of As(III)in △ACR2.1 water-soluble As decreased significantly,DMA and arsenosugars increased significantly,and the degree of methylation and efflux increased,indicating that the As(Ⅴ)reductase gene played an important role in the regulation of As(Ⅴ)reduction,distribution and speciation transformation.Compared with WT,As accumulation in △arsM was significantly increased under 50100 μg·L-1 As(Ⅴ).With the increase of As(Ⅴ)concentration,the arsM mutation led to a decrease in the synthesis of water-soluble methyl As compounds by C.reinhardtii,which promoted the formation of complex lipid-soluble As compounds.In addition,under As(Ⅴ)stress,although △arsM produced methyl As compounds such as DMA and arsenosugars,its content was reduced and unknown As specie was produced.It indicated that arsM had a certain effect on As methylation,however,it was not a key enzyme for the synthesis of complex methyl As compounds such as arsenosugar.Thus the lack of arsM does not completely cause C.reinhardtii to lose its methylation ability.Compared with WT,the accumulation of As in △arsB was reduced.The lack of arsB had no significant effect on the distribution of different As components of C.reinhardtii.Under As(Ⅴ)stress,△arsB used As(Ⅴ)reduced to As(III)and efflux as the main detoxification mechanism,indicating that arsB was not a key gene that regulated As(III)efflux in C.reinhardtii,and there were other ways of As release.In addition,the arsB mutation only affected the methylation ability of C.reinhardtii under low As treatment,but had no significant effect under high As treatment.In summary,this study clarified the effect of the phosphate transporter gene PHOX on the As(Ⅴ)absorption and transformation of C.reinhardtii.It also systematically explored the differences in As(Ⅴ)absorption,distribution and speciation transformation of WT and various As metabolism-related gene mutants under different As(V)treatments,and further revealed the metabolic regulation of As in microalgal cells.