Map-based Cloning And Functional Analysis Of Grain Arsenic Accumulation Gene HAG1 In Rice

Arsenic（As）,is an important environmental pollutant,generates a strong toxic effect on animals and human through the enrichment and amplification of food chain.Rice is one of the staple foods of mankind,compared with other cereal crops,the high accumulation of As in rice makes it a major source of arsenic in the human food chain.Therefore,it is crucial to solve the problem of As contamination in rice grains and improve rice quality and safety by understanding the absorption,translocation and detoxification mechanisms of As in rice.In this study,a mutant hag1 with high grain arsenic accumulation and arsenic sensitivity was identified from the EMS-generated mutant rice library.The physiological and genetic mechanism of hag1 were further studied.The main results are as follows:（1）The total arsenic concentration in grains of hag1 and wild-type planted in paddy field were detected.The result showed that the arsenic in grains of hag1 was 5 to 6 times higher than that of wild-type,which was also exceed the limit of arsenic in rice（0.2 mg/kg）.However,the total arsenic concentration in nodes and leaves of hag1 were significantly lower than those of wild-type.Analysis of five arsenic species in grains showed that arsenic trivalent（Arsenite,As（III））was dominant,accounting for 62-87%of the total arsenic.（2）Hydroponic test was conducted to analyze the tolerance of hag1 and wild-type seedlings to arsenic stress.The results exhibited that the growth of roots and shoots of hag1 was significantly inhibited with the increasing of arsenic treatment concentration,while the wild-type maintained strong arsenic tolerance.Analysis of As concentration in hag1 and wild-type at seedling stage indicated that hag1 displayed lower transfer factor than that of wild-type,resulting in higher arsenic in roots under low As condition（<5μM）.However,at high As stress（>5μM）,there was significantly lower arsenic concentration in the roots than wild-type because of weak As sequestration ability and strong transfer to shoots.（3）Contents of H₂O₂and MDA in roots of hag1 and wild-type under different concentrations of As were analyzed.The results showed that the contents of H₂O₂and MDA in roots of hag1 were significantly higher than those of wild-type,indicating that As stress generate more serious oxidative damage to hag1.There were no significant differences in the activities of CAT,SOD,APX and other antioxidant enzymes between the hag1 and the wild-type under low As concentration,but the activities of CAT and SOD in the roots of hag1 were significantly lower than those of the wild-type at high arsenic level.In addition,a large number of biothiols（eg.Cys and PCs）were synthesized in the roots of hag1 under low concentration conditions,which reported to chelate with As and enhance the tolerance to arsenic.However,under high arsenic concentration,the concentration of biothiols in the hag1 was significantly lower than that of wild-type,resulting in poor detoxification ability and high sensitivity to arsenic.（4）According to the result of genetic analysis,the As high accumulation phenotype of hag1 was deduced to be caused by a single recessive gene mutation.Genetic linkage analysis was carried out by F₂hybrid population of hag1 and indica Nanjing 11.The HAG1locus on the long arm of chromosome 4 between indel9-Indel17 with a 233 kb physical distance.Genomic resequencing of the hag1 and the wild-type revealed that a single base substitution（G2661A）was found in the 10th intron of Os ABCC1（LOC＿Os04g52900）.The mutation caused an incorrect splice and truncated 16 bp in the coding DNA sequence（CDS）of Os ABCC1,which resulting in the loss and substitution of amino acid372-382 in Os ABCC1 protein.The translation of Os ABCC1 were premature termination at the 383 amino acid in hag1.These results may ultimately disrupted the transport function of As-PC.Transgenic complementing test showed that transferred Os ABCC1 from wild-type into the hag1 could reduce the As concentration in grains and improve the tolerance to arsenic at seedling stage.Above all,ABCC1 is the target gene of HAG1.（5）Transcriptome analysis of Node I of the wild-type and hag1 showed that there are amounts of differentially expressed genes in the hag1 compared with the wild-type.The function analysis of those differentially expressed genes revealed that the expression of Os HAC1;1 and Os ABCC1 were significantly down-regulate,which were reported as key genes involved in the regulation of arsenic accumulation in shoot and grain.To achieve self-detoxification and protection under arsenic stress,Os ABCC1 mutation may also influence the expression of glutathione S-transferase,the precursors of antioxidant enzymes（SOD,CAT,GR,APX）and other stress genes.In addition,compared with the wild-type,the expression of Os CCA1,Os MFT1 and other flowering regulation genes in hag1 were significantly different,which prolonged the growth period of the mutant and lead to the phenomenon of“late maturation”.It was found that the Os NR2,Os SULTR3;6and Os Pht1;8 and other genes in hag1 were up-regulated,which were identified as involving in nutrient element transport and may lead to grain enlargement.In present study,a large number of differentially expressed transcription factors（eg.b ZIPs,TCPs,MYBs,AP2）involved in the transcription regulation of arsenic transporters or related proteins were also excavated,thus providing more genetic resources for the cultivation of low arsenic accumulation materials.（6）Uptake and translocation of Arsenic in 55 rice cultivars were analyzed under arsenic stress.It was found that the arsenic uptake ability of indica rice were generally stronger than those of japonica rice,but transfer factor were lower than those of japonica rice.These results contribute to more arsenic in roots and lower arsenic in shoots of indica than those of japonica rice.The result of arsenic and cadmium concentration in grains indicated that the arsenic concentrations in japonica rice were generally higher than those of indica rice,while the cadmium concentrations were lower than those of indica rice.Based on the analysis of arsenic and cadmium concentration in grains of different cultivars,japonica rice Asominori and indica rice IR66 were screened to be genetic resources for breeding low arsenic and low cadmium cultivars.（7）Analysis of the arsenic uptake and translocation of Kasalath,Nipponbare and CSSL19（background of Nipponbare containing ABCC1 gene from Kasalath）showed that the arsenic uptake ability of CSSL19 was the same as that of Nipponbare,but the transport capacity was significantly lower than that of Nipponbare,which indicating that ABCC1was mainly involved in the regulation of arsenic transport from root to shoot,but did not affect the arsenic uptake of rice.The sequences of ABCC1 full-length gene and promoter between indica and japonica were analyzed.The results revealed that there were a large number of SNP and Indel variants in the promoter region of ABCC1 between indica and japonica rice,leading to different expression level of ABCC1 gene in the root and Node I between indica and japonica rice.The differential expression of ABCC1 gene mainly influenced on the transfer factors of arsenic from root to shoot and the redistribution of arsenic to grain,resulting in the difference of arsenic concentration in shoot and grain between indica and japonica rice.