Effects Of Phosphate On The Resistance And Speciation Transformation Of Arsenic In Chlamydomonas Reinhardtii

Arsenic(As)is a toxic metalloid widely distributed in the marine,freshwater and soil environment.A large number of As compounds are cause increasingly serious arsenic pollution in marine ecosystems,introduced into the environment from both natural and anthropogenic sources,and ultimately with rivers moving from the land to the ocean.Microalgae are a class of primary producers in marine ecosystems.Then have strong As accumulation ability,and can mediate the speciation transformation of As,also play an important role in the As biogeochemical cycle.Research have shown that Chlamydomonas reinhardtii has a strong ability to accumulate As and have the potential in removing the As pollutions from water.However,the effect of phosphate(PO43-)on As resistance and speciation transformation of C.reinhardtii has not been studied systematically.Therefore,we first studied the effect of As and phosphorus content and their ratios on arsenic tolerance of arsenate(As(?))and arsenite(As(?))in C.reinhardtii.Then we compared the differences between wild-type and mutants of arsenate reductase gene and arsenite efflux gene on the absorption,transformation of As(V)under different PO43-levels.Besides,the arsenate reductase activity and gene expression between differet algae species,and its relationship with As speciation and intracellular phosphorus content were further investigated.The major results were listed as follows:1.Effects of intracellular arsenic and phosphorus content and ratio on the tolerance of As(?)and As(?)in C.reinhardtiiTwo phosphate(PO43-)levels and a series of As(?)and As(?)concentrations were set up to investigate their impact on the growth of C.reinhardtii after 72-h treatment.We explored the effects of culture medium and algal cell phosphorus content on arsenic absorption and tolerance.Results showed that As(?)-EC50(2090.3,21183.6 ?g-As/L)was obviously higher than As(V)-EC50(162.1,2358.3 ?g-As/L)under two phosphate(PO43-)levels,C.reinhardtii has a strong tolerance to As(?)and is sensitive to As(?).Higher level of PO43-in the culture did not affect the intracellular arsenic content([As]dis)under As(?)treatment,but significantly decreased that under As(V)treatment,so when EC50 is characterized by[As]dis,the data of[As]intra EC50 showed that PO43-level did not affect As(?)-EC50(123.9,125.0 ?g-As/g-dw),but significantly affected As(V)-EC50(7.4,58.6 ?g-As/g-dw).From the EC50 derived from intracellular As and P content ratio([As:P]intra),we found that the effect of PO43-on the tolerance of two As species was opposite,The As(?)-EC50 were 21.1 and 6.1 mol-As/mol-P,while As(V)-EC50 were 1.3 and 3.4 mol-As/mol-P.Taken together,these data indicated that As(V)was more toxic to the microalgae than As(?).The tolerance of As(?)and As(?)by C.reinhardtii were not only affected by the PO43-concentration in the culture,but also affected by intracellular As and P content and their ratios.2.Effects of different phosphate levels on arsenate metabolism of C.reinhardtiiThe dynamic changes of As(?)absorption and tolerance were studied in wild-type and mutants of arsenate reductase gene and arsenite efflux gene of C.reinhardtii under two differents PO43-levels(0.315,3.15mg·L-1,respectively).Results showed that As(?)absorption,accumulation and the reduction of As(?)to As(?)in C.reinhardtii was more remakable under the absence of phosphorus.The reduction ability and rate of As(?)and the efflux rate of As(?)in C.reinhardtii were different under different phosphate levels.As(?)reduction,As(?)efflux were found to be the main arsenic detoxification path of C.reinhardtii.Compared with the wild-type,the mutant ?ars B which absence of arsenite efflux arsB,was almost the same as the absorption rate of As(V)and the efflux rate of As(?).While the As(?)reduction ability of the mutants AHAC-1,?HAC-2 and ?ACR2.1,?ACR2.2-1,?ACR2.2-2,in which HAC and ACR2 were deleted was significantly reduced,indicating that arsenate reductase genes play an important role in the regulation of As(V)reduction process.The function of gene ACR2.1 was greater than gene ACR2.2.3.Arsenate reductase activity and gene expression of C.reinhardtii under different phosphate levelsBased on the above results,arsenate reductase activity and the relative expression levels of ACR2.1 and ACR2.2 genes in wild-type and mutants of C.reinhardtii under different phosphate levels were analyzed.The results showed that As(V)treatment significantly enhanced arsenate reductase activity,and phosphorus deficiency also increased arsenate reductase activity.The expression of arsenate reductase gene ACR2.1 and ACR2.2 plays an important role in the process of As(V)reduction of C.reinhardtii,the expression levels of ACR2.1 and ACR2.2 were up-regulated at phosphorus deficiency levels.Compared to ACR2.2,ACR2.1 showed a stronger As(V)reduction capability.Taken together,we systematically investigated the the As metabolisms of As in C.reinhardtii under different phosphorus levels.The effects of intracellular As and phosphorus content and ratio on the tolerance of As(V)and As(?)in C.reinhardtii.We also show that the As(V)reduction,As(?)efflux during the arsenate absorption,speciation transformation under different phosphate levels.The results showed that arsenite arsB was not a key gene to regulate As(?)efflux.The role of ACR2 is greater then HAC in the process of As(V)reduction of C.reinhardtii.At the same time,the expression of arsenate reductase gene ACR2.1 and ACR2.2 in the As(V)reduction process was regulated by phosphorus in the culture medium.The gene ACR2.1 showed stronger As(V)reduction ability than ACR2.2.