Distribution And Speciation Of Exogenous Selenium In Pakchoi And Wheat

Dietary intake is an important source of selenium(Se)for humans.Se biofortification by applying Se fertilizers to crops can effectively level up the Se content in human body in Se-deficient areas.The beneficial effects of Se-enriched crops are related not only to their total Se content,but also the speciation of Se in crops.The analysis of Se speciation in crops also helps to clarify the transport and transformation mechanisems of Se in crops,contributing to the scientific production of Se-enriched crops.Therefore,this study enstablished the analytical method of Se speciation in plants using liquid chromatography coupled with atomic florescence spectrometer based on the previous studies.Pot experiment was carried out to explore the characteristics of Se distribution and speciation in pakchoi and wheat and the differences of Se transportation and transformation in the two plants by cultivated pakchoi(Brassica chinensis)and wheat(Triticum aestivum L.)and supplementing selenite and selenate by soil and foliar application,.The subcellular distribution of Se and its speciations in pakchoi and wheat leaves was also explored,aiming to promote the understanding of Se metabolism and fate in crops.The main results are as follows:1.The method of Se speciation in plants was established by using liquid chromatography coupled with atomic florescence spectrometer(including sample extraction and Se compounds determination).The detailed steps are as follow:plant sample is added to 10 mL of 20 mmol/L Tris-HCl solution containing 5 %(of sample weight)protease E and the mixture is shaken for 12 h at 37 ?.The mixture is then centrifuged and separated by filteration.The supernatant is carried by mobile phase(60 mmol/L diammonium hydrogen phosphate and adding formic acid to pH = 6.0)through anion exchange column(Hamilton PRP X100).The eluent from the column is mixed with 2.0 % potassium borohydrideand1.0 % potassium iodide and then pass through UV radiation device to transform all Se compounds into tetravalent state,which is responded by an atomic florescence spectrometer.External standard method is used to obtain the concentrations of Se-containing compounds.The dection limits of selenocystine,methylselenocysteine,selenomethionine,selenite and selenate are 6.40,6.03,8.50,2.52,6.22 ?g/L.This method has been proved to be able quantitative analysis of plant Se speciation after precision and accuracy test by theinternational standard material SELM-1 which has calibrated concentration of selenomethionine.2.Selenomethionine was the major organic Se compound in pakchoi and wheat after enzymatic extraction.Methylselenocysteine took the second place,and selenocystine had the least content,usually accounting for less than 10 % of total Se.Selenate was the major inorganic Se compound in the two tested crops,and selenite was seldom detected since it was adsorbed on samples during extraction.Pakchoi had stronger ability to accumulate Se compared with wheat.Despite a relatively low organic transformation rate,the concentrations of all organic Se compounds,especially methylselenocysteine,in pakchoi were higher than those in wheat.Thus pakchoi is better serving as Se-enriched crop.Among different Se application treatments,the valence of exogenous Se had effects on the distribution and transformation of Se in plants.The soil-applied selenate absorbed by roots could not be organically transformed in time,most of which was transported upward and still stored as selenate.A larger proportion of absorbed Se was transformed into organic compounds and retained in roots when it was applied as selenite in soil.But regarding to the content instead of concentration Se was still mainly distributed in the aboveground part,in which case the form of Se during the transportation needs to be further explored.As for foliar application,Se absorbed by plants seldom transported downwards,and its organic transformation rate was lower than soil application.3.The distribution of Se in subcellular fractions of pakchoi and wheat leaves followed the decend order as: soluble fractions such as vacuole > organelle > cell wall.Vacuole compartmentation played an important role in reducing Se toxicity to the vital area for cell metabolism.Cell wall could not block Se out of plant cell.The speciation of Se varied in different subcellular fractions: selenate was the dominate Se species in soluble fraction,while Se in organelle was in organic form represented by selenomethionine.The Se speciation in the cell wall was undetectable because of its low concentration.The subcellular distribution of Se observed in this study explained the differences among different Se application treatments to a large degree: in the selenate-applied treatment,most of the absorbed Se was stored in vacuole without organic transformation while the absorbed Se in the selenite-applied group was more likely to be transformed into organic form and entered organelle,thus plants could tolerate and accumulate a higher concentration of Se in selenate-applied group.The higher concentration of Se applied,the larger proportion of Se entering vacuole as inorganic Se.The stronger ability of pakchoi to accumulate Se was also accounted for by the larger proportion of Se transported and stored in vacuole.In conclusion,Se-enriched pakchoi and wheat have both inorganic Se represented byselenate and organic Se represented by selenomethionine.Application method,valence of exogenous Se and plant species have significant influence on the content,distribution and speciation of Se in crops,which should be comprehensively considered in the production of Se-enriched crops.