Mechanisms Of Arsenic Accumulation And Tolerance In Rice And Arabidopsis

Arsenic is a prevalent toxic and non-essential metalloid element in the nature.In recent decades,large areas of farmland soils,especially in south and southeast Asia,are contaminated with arsenic due to human activities,such as mining,smelting,spraying with high-arsenic herbicides and insecticides,irrigation with high-arsenic groundwater.Arsenic in contaminated farmland soil not only affects the growth of crops,but also increases the accumulation of arsenic in the edible parts of crops.Rice as a staple food is the main source of inorganic arsenic intake in Asian countries.It is very important to reduce the accumulation of arsenic in rice.The species of arsenic in soil are mainly the inorganic forms of arsenate[As(?)]and aresnite[As(?)].Arsenate is the main species in soil under aerobic conditions and is taken up by phosphate transporters in plant roots.Under the anaerobic conditions arsenite is the main species.A number of the aquaporin nodulin 26-like intrinsic proteins(NIPs)are able to transport arsenite.Rapid reduction of arsenate to arsenite by plant roots is catalyzed by arsenate reductase.Reduced arsenite is partially excreted into the medium by roots,sequestered in the vacuole after chelating with phytochelatins in roots,or transported to the aerial part of plants via the xylem.However,the mechanisms of arsenic accumulation and tolerance in plants are not fully understood.Rice varieties vary in arsenic accumulation and tolerance,but the genes involved in these processes are still unknown.The main results are as follows:1.In this study,quantitative trait loci(QTLs)affecting the concentrations of 15 elements in rice grain and straw of recombinant inbred lines were identified.To increase the opportunity to detect and characterize QTLs,recombinant inbred lines were grown under three field conditions and analyzed by QTL Cartographer ver.2.5 software.Fifty-six,64 and 8 QTLs were identified for the concentrations of 15 elements in grain and straw,and heading date,respectively.They were clustered into 21 genomic regions.Three QTLs were found to be common in all three experiments,whilst 14 QTLs were found for 6 elements in grain and straw in two of the three experiments.Nearly all of the grain and straw element loci were linked to QTLs affecting other elements in the study and reported QTLs.2.Furthermore,the physiological mechanisms underpinning the difference between rice cultivars in arsenic accumulation and tolerance were studied.We investigated genotypic variation in As(?)and As(?)tolerance among 26 rice cultivars.Screening results showed that there was difference in As(?)and As(?)tolerance between the tolerant cultivar(W0)and the sensitive cultivar(W31).We physiologically characterized these two cultivars by determining arsenic uptake,uptake kinetics of arsenite,and tolerance of L-buthionine-sulphoximine(BSO,an inhibitor of y-glutamylcysteine synthetase)in combination with arsenite exposure.The results showed that the rice cultivar W31 was more sensitive to As(V)and As(?)than W0,and W31 accumulated lower arsenic content than WO in both roots and shoots.We found that W31 had lower capacity to synthesize non-protein thiols compounds than W0.Furthermore,A significant positive correlation was found between total arsenic content and thiol compounds in the roots of the F2 population from the cross between WO and W31.These results showed that the capacity to synthesize non-protein thiols was the main reason for the difference of arsenic tolerance and arsenic accumulation between the two rice cultivars.3.Cysteine is the precursor for the synthesis of phytochelatins(PCs).We cloned the cysteine synthase gene OsOASTL-A1 from rice and studied its function.We also analyzed the accumulation and tolerance of arsenic in Osoastl-al mutant and wild type.We cloned the OsOASTL-A1 gene from rice cultivar Nipponbare.Heterologous expression of OsOASTL-A1 restored the growth of the E.coli mutant lacking the endogenous cysteine synthase in the M9 medium in the absence of cysteine.The OAS-TL activity was quantified as the release of Cys from O-acetyl-serine(OAS)and Na2S.The recombinant OAS-TL showed typical Michaelis-Menten kinetics in the catalytic activities with respect to the substrates sulphide and OAS.OsOASTL-A1 was predominantly expressed in roots,with the cortex and xylem parenchyma in the mature zone of roots,and in the vascular tissues of leaves and stems.Based on the experimental data described above,we concluded that the OASTL-A1 functions as a cysteine synthase in cytoplasm.The results of real-time quantitative PCR indicated that OsOASTL-A1 expression in rice roots was up-regulated after As(?)and As(V)exposure and showed highest level after As(?)exposure.Knockout of OsOASTL-Al resulted in decreased thiol content and decreased arsenic accumulation in rice roots.Sulfur-sufficient Osoastl-al mutants were more sensitive to arsenate than wild type.Knockout of OsOASTL-A1 had no significant effect on grain As or Cd accumulation in soil-grown rice.OsOASTL-A1,like AtOASTL-A1 in Arabidopsis,plays a role in the detoxification of As and Cd.Taken together,our study showed the OASTL-A1 functions as a cysteine synthase that mediates the synthesis of non-protein thiols and As accumulation in rice roots and As tolerance.4.To identify the function of Arabidopsis arsenate reductase AtACR2 and AtHAC1 gene.Physiological experiments were performed on the acr2,hac1,acr2 hac1 mutants of Arabidopsis thaliana.The ACR2 arsenate reductase in A.thaliana was found to play no detectable role in arsenic metabolism.Furthermore,ACR2 does not interact epistatically with HAC1.HAC1 functions to reduce arsenate to arsenite.5.Arabidopsis thaliana is a model plant.Arabidopsis was used to further study the mechanism of arsenic accumulation and tolerance.In the present study,we tested the effects of different combinations of gene knockout and heterologous expression(hac1 PvACR3?hac1 pho1?hac1 cad2-1?hac1 cad1-3)on As accumulation,especially the mobility of As during root-to-shoot translocation,and As tolerance in A.thaliana.Our study demonstrates that As hyperaccumulation can be engineered in A.thaliana by knocking out the HAC1 gene and expressing PvACR3 which encodes an arsenite vacuolar efflux protein in Pteris vittata.Non-protein thiols play an important role in arsenic tolerance in Arabidopsis.Taken together,the capacity to synthesize non-protein thiols in roots of rice and Arabidopsis was the important factor for arsenic tolerance and arsenic accumulation in roots,but it had limited effect on the arsenic accumulation in the shoots.