The Accumulation And Species Of Selenium In Flue-cured Tobacco And The Mechanisms Of Seleniumalleviating Arsenic Toxicity

Selenium(Se) is an essential microelement for human beings and animals. The beneficial or toxic effect of Se on human being and animals was not only dose-dependent, but also related to the chemical form and the bioavailability of Se. At present there were a lot of reports on the uptake of Se by plant and its effects on plant growth and quality and relieving heavy metal toxicity. However, few reports had investigateded the species of Se in plants under the condition of different exogenous Se level. The relationship between the effects of Se on plant growth and stress-resistant abilities and the selenium species change in plant is still unclear. In this paper,the effects of exogenous Se on plant growth, the accumulation and species of Se,and the mechanism of Se alleviating arsenic(As) toxicity to flue cured tobacco were studied by some pot and hydroponics culture experiments, aiming to provide scientific basis for the exploitation of selenoprotein and the application of Se in agriculture safely.The main results were as follows:1. Anion-exchange chromatography(Hamilton, PRP-X100) with hydride-generation atomic fluorescence spectrometry(HG-AFS) is commonly used for the analysis selenium(Se) speciation in the biological samples. The results showed that the best mobile phase was 60Mm(NH4)2HPO4 at Ph 6.0, where the separation of Se species, including selenite [Se(IV)], ivelenite [Se(VI)], selenocystine(Se Cys) and selenomethionine(Se Met), was achieved within 10 minutes with reasonable resolution. In addition, the recovery rates of Se Cys, Se(IV), Se Met and Se(VI) in the Se-enriched samples were 95.72±1.60%, 106.89±2.20%, 93.28±2.82% and 91.38±1.27%, respectively.2. The effects of different levels Se(0-22.2 mg Se kg-1) on the growth, quality and mineral nutrition accumulation of flue-cured tobacco(FCT) were studied through a pot experiment. The results showed that Se stimulated the plant growth at low levels(≤4.4 mg/kg), but inhibited at high dosages(≥11.1mg/kg). The values of nicotine, protein and reducing sugar content were in the optimum range, and the contents of mineral nutrition in roots, stems and leaves of FCT reached the maximum value at 4.4 mg/kg of Se treatment. Se at low levels(≤4.4 mg/kg) improved the quality of FCT by enhancing the uptake on mineral nutrition, especially for N、K、Ca、Mg and Mn remarkably. However, high Se levels(≥11.1 mg/kg) depressed the absorption of mineral nutrition, especially for N、P and K, and ultimately depressed the quality of FCT.3. A potexperiment was conducted to investigate the effects of different Se levels(0-22.2 mg/kg Se) on the plant growth and antioxidant system as well as Se uptake and species in FCT. It showed that the leaf area and number, the biomass and the chlorophyll content of FCT reached the maximum value at 4.4 mg/kg of Se treatment. Se at low levels(≤ 4.4 mg/kg) elevated the capability of antioxidant stress and reduced the malondialdehyde(MDA) content. However, high Se levels(3 11.1 mg/kg) depressed the capability of antioxidant stress and raised the MDA content. The Se content in di fferent parts of flue-cured tobacco significantly increased with the growth of Se level(P<0.05=. According to the detection of different Se speciation, only selenocysteine(Se Cys) was detectable at 2.2 mg/kg treatment; Se Cys, selenite [Se(IV)]and velenite [Se(VI)] were detected in flue-cured tobacco leaves at Se treatment(≥ 4.4 mg/kg), which accounted for 4.6%-10%, 9%-18.7% and 71%-86% respectively; Se Cys, selenomethionine(Se Met) and Se(IV) were detected in root, and organic selenium(66%-84%) was the main Se species at Se≤ 11.1 mg/kg treatment; four Se species [Se Cys, Se Met, Se(IV) and Se(VI)] were detected in flue-cured tobacco roots, and the main Se species was inorganic Se(60%) at 22.2 mg/kg treatment. That is to say, the percentage of organic Se species(Se Cys and Se Met in flue-cured tobacco leaves and root) declined, whereas the ratio of inorganic Se species [Se(IV) and Se(VI)] increased with the growth of Se level. The correlation analysis showed that the superoxide dismutase(SOD) activity as well as the glutathione(GSH) and MDA contents are positively correlated with the Se(IV) and Se(VI) contents at P<0.01 and excessive inorganic Se might destruct the reactive oxygen species(ROS) balance and enhance the MDA content.4. An ion depletion hydroponic experiment was conducted to study the effects of different Ph value, accompany ions and inhibitors on the Se(Ⅳ) and Se(Ⅵ) absorption kinetics characteristics of different genotypes of FCT. The results showed that FCT have faster rate on absorbing Se(Ⅵ) source than Se(Ⅳ) source, rsulting in lower Km and Cmin values for Se(Ⅵ) source. FCT var.Yunyan87 had lower Km and Cmin value and stronger affinity to Se than FCT var. K326. The Imax value of two genotypes of FCT rised,while the Km and Cmin value reduced significantly at Ph6. Compared with CK, the Imax value of two genotypes of FCT with addition of accompany ion decreased significantly. Under the condition of Se(Ⅳ) source, the Imax value of PO43- treatment was significantly lower, but Km and Cmin value were significantly higher than those of SO42- treatment. However, the results were inverse at Se(Ⅵ) source condition. Compared with control, addition inhibitors reduced the Imax value significantly. The Imax value of CCCP treatment was significantly lower, but the Km and Cmin values were significantly higher than those of La Cl2 treatment.5. Uptake, speciation distribution, the effects on the flue-cured tobacco growth and antioxidant system of the single and interactive effects of Se at different levels(0-5 mg/L Se) and As at different doses(0-5 mg/L As) were studied through a hydroponic experiment. The results showed that low Se levels(≤1 mg/L) and low As doses(1 mg/L) stimulated the growth of flue-cured tobacco by reducing the malondialdehyde(MDA) content. However, high Se and As levels(5mg/L) inhibited the plant growth and raised the MDA content, and meanwhile the biomass of the aboveground parts and underground parts declined notably. Under high As doses(5mg/L) stress, the optimum level of Se application promoted the growth of flue-cured tobacco by elevating the antioxidant capacity and reducing the malondialdehyde(MDA) content. The antagonistic effect was obvious for Se affecting As uptake with high As levels(5mg/L), similar inhibition of Se uptake by As has been found with high Se doses(5mg/L). The main Se species was organic Se at low Se doses(0.1mg/L), and the proportion of organic Se species increased from 58.2% to 100% and 29.9% to 68.5% in the shoots and roots of flue-cured tobacco with the increasing of As doses. However, the percentage of organic Se at high Se level(5mg/L) reduced from 68.2% to 41.7% and 66.5% to 31.7% in the shoots and roots of flue-cured tobacco with the raise of As levels. According to the detection of different As speciation, As(V)(63.3%-94.4%) was the main As species in the shoots while As(Ⅲ)(36.9%-73.9%) was the major As species in the roots at low As levels(1mg/L). Nevertheless, the main As species were As(Ⅲ)(68.1%-90.1%) with high As levels(5mg/L). When As was maintained at the same value, the proportion of As(Ⅲ) dropped whereas the percentage of As(V) raised gradually with the increase of Se levels.6. With Se-enriched tobacco leaves e as materials, the albumin, globulin, prolamin and glutelin were extracted by continuous extraction method, using the DR assay for hydroxyl radical and pyrogallol assay for superoxide radical anion. The results showed that a good correlation existed between the dose of Se-containing protein and the ability of free radical scavenging. The antioxidation activities of the proteins were albumin>glutelin>globulin>prolamin. There was significant positive correlation between the antioxidant activity and the selenium content in protein.