Selenium Biofortification In Buckwheat And Characterization Of ATP Sulfurylase 2(ATPS2) Related To Selenium Assimilation In Plant

Selenium(Se)is an essential microelement for human and animals which plays an importance of role on human and animal health.Se deficiency in daily diet occurs commonly in the world,and it is also a serious issue in China,because most of cultivated soil conditions are Se deficient.Crop Se biofortification is an alternative approach to solve the above problem.Thus,Se biofortification in buckwheat is attracting an increasing focus since buckwheat products could be used as a kind of functional food.Additionally,ATP sulfurylase(ATPS)is a rate-limiting enzyme in Se metabolic pathway of higher plants,its characterization in plants is significance of crop Se biofortification and Se phytoremediation.In this study,the effects of Se application as selenite and selenate on buckwheat production and Se biofortification under field condition were determined from 2014 to 2016.Se tolerance,Se uptake as well as Se translocation of common buckwheat and tartary buckwheat were investigated in the plants cultured under nutrient solution.Furthermore,the expression level of ATPS gene family,the subcellular localization and protein structure of ATPS2 from Stanleya pinnata(Se hyperaccumulator)and Stanleya elata(Se non-accumulator)were determined,the characterization of enzyme activity and inhabitation of purified SpATPS2 were detected in vitro.The main results were as follows:In the 3-year field experiment,'15 g Se ha-1 and 30 g Se ha-1 applied as selenite and selenate increased the biomass and grain yield of common buckwheat significantly(P<0.05),the highest grain yield were observed from plants with 15 g Se ha-1 supplement,with 1908.6 kg ha-1,1622.9 kg ha-1 and 1567.0 kg ha-1,and no interact effects were found between Se species and Se levels treatments on plant biomass,grain yield as well as yield components.Both Se species application improved the Se concentration in plant and grain significantly(P<0.05),Se concentration in shoot and root reduced gradually with plants growing,Se content in grain from plants supplied with selenate were increased 3-,10-fold higher than those from plants treated with selenite.Seed Se use efficiency under 90 g Se ha-1 treatment was lower than those with 15-30 g ha-1 Se supply(P<0.05).The performance of common buckwheat under 40 g Se ha-1 treated with selenite,selenate as well as the combination of both selenite and selenate was investigated in a 2-year filed experiment.The results shown that the highest grain Se were found from plants treated with selenate,with 256 ?g kg-1.The grain yield were increased by the combination of selenite and selenate application,with 10.9%and 10.3%in 2015 and 2016,respectively.Additionally,the seed Se use efficiency increased with 2-,3-fold and 2-,5-fold by respective selenate application or selenate combined with selenite application compared with that treated with selenite only(P<0.05).2-week-old plants of common buckwheat and tartary buckwheat were incubated with a range of Se levels supplied as selenite(0,10,20,30,40,50 ?M),selenate(0,15,30,45,60,75 ?M)to determine the Se tolerance of both species.The results shown that Se content in shoot and root increased with the increasing Se levels supplied(P<0.05),root Se was 10-,20-fold higher than shoot Se when plants were supplied Se as selenite with a range of Se levels,conversely,shoot Se was 2-,4-fold higher than root Se when plants were fed with a series of selenate levels.The Se concentration that buckwheat seedling were tolerant was up to 30 ?M of selenate during one week,the seedling growth was inhibited when theSe concentration was over 15?M of selenate in the nutrient solution.The Se tolerance index of tartary buckwheat was higher than that from common buckwheat.Moreover,the S content in shoot increased by selenate application(P<0.05),which resulted in an increasing Se/S in shoot.P contents in shoot of both plant species were increased with increasing selenate supplement,tartary buckwheat tend to accumulate more P in shoot than common buckwheat plants,generally,the S and P content in plants were improved by selenate treatment.2-week-old plants of common buckwheat and tartary buckwheat were incubated with 20 ?M of Se supplied with selenite,selenate and plant extract Se(MeSecys)over 2h.The results indicated that the plants supplied with plant extract Se accumulated 4-,6-fold more Se in shoot and 2-,3-fold more Se in root of both buckwheat species compared with those plants applied with selenite and selenate,0.5 mM of phosphate or sulphate did not influence the inorganic Se uptake in plants in 2h(P>0.05).Moreover,Se uptake by plants under plant extract Se treatment were found with 2-,5-fold higher Se uptake rate of root than that from plants treated with inorganic Se,but with lower Se translocation factor(TF),and significantly influenced by 0.5mM of sulfate and phosphate supply(P<0.05).30-day-old plants of common buckwheat and tartary buckwheat were incubated with 20 ?M of Se supplied with selenite,selenate and plant extract Se(MeSecys)over 24h.The results indicated that the Se content in shoot and root was significantly increased from plants treated with plant extract Se compared with those from plants under selenite and selenate treatments(P<0.05),with 1.2-5-fold higher.The shoot Se and root Se in tartary buckwheat were increased with 20%-60%and 40%-80%compared with the Se content in common buckwheat,respectively.Additionally,MeSecys was found in the xylem sap from both plant species under plant extract Se supplement,the concentration was 2.5 ?M and 5.8 ?M in the respective xylem sap from common buckwheat and tartary buckwheat.In the Se biofortification study with nutrient solution application,the Se content in leaves is similar to the grain Se,the stem shown the lowest Se content among the Se concentration among organs of buckwheat,there is not significant difference in the grain from plant extract Se treatment compared with those treated with selenite and selenate.The comparison of ATP sulfurylase from Stanleya pinnata(SpATPS2)and Stanleya elata(SeATPS2)indicates that the expression level of ATPS2 in the root of S.pinnata is 5-fold higher than that in root of S.elata,ATP sulfurylase enzyme activity in S.pinnta root is 2-fold higher than that in S.elata;The protein of SpATPS2 and SeATPS2 contain 480 and 488 amino acid residuals,respectively,and the difference of amino acid between SpATPS2 and SeATPS2 is up to 4%.A stop codon was found in the transit peptide of SpATPS2 resulting in the cytosolic localization of this enzyme,whereas SeATPS2 shown dual localization in cytosol and chloroplast;the modelling of ATPS2 suggested that 16 amino acid residuals were used for the enzyme active site,and there is no amino acid difference located in either active site,flexible loop or conserved amino acid sequences.Furthermore,200 mM of both sulfate and selenate shown inhabitation on the purified SpATPS2 suggesting that SpATPS2 could catalyze the selenate assimilation reaction in vitro.