Study of the response of wheat to U and Cd combined stress and their uptake and

Abstract. Water culture experiment was adopted to investigate the effects of U and Cd combined stress at different concentrations on the uptake and accumulation of U and Cd and physiological and biochemical characteristics in wheat seedlings. The experiment results showed that the U and Cd uptake by wheat seedlings was higher in roots than in aboveground parts when the total concentration of U and Cd was 5 mg/kg or 20 mg/kg， and the transfer factors （TFs） of U and Cd in wheat seedlings were above 1 when the total concentration was 100 mg/kg. But low concentration of U and Cd were rarely accumulated in wheat seedlings， indicating that wheat seedlings have good tolerance to U and Cd combined stress at low concentration. The experiment results also showed that with the increase in the total concentration of U and Cd， the root activity of wheat seedlings first increased and then decreased， and it exhibited a significant increase when the concentration was 20 mg/kg. Also， with the increase in concentration， the chlorophyll content of wheat seedlings first increased and then decreased， while the SOD activity first decreased and then increased.

Key words： Wheat Uranium Cadmium Uptake Accumulation Physiological response CLC X591

Introduction

Thanks to the development of nuclear industry and extensive application of nuclear technology， as well as activities in other fields， such as industry， agriculture， energy， military， transportation， medical treatment and public health， radionuclide pollution has become one of the important environmental problems difficult to solve nowadays. Radial generated in the process of radionuclide disintegration is the source of such pollution. As the “death” of short-life nuclide happens， primarily such long-life fission products and nuclear materials as 3H， 137Cs， 90 Sr， 239Pu and U form long-term pollution [1]. After these radionuclides enter water and soil， they not only severely damage human beings’ physical health through food chain[2]， but also form big obstacles in eliminating these nuclides， especially the elimination of large-area and low-amount radionuclides in soil， which is far more difficult. In addition， due to mining， metal smelting， and agricultural application of industrial sewage and sludge， a large amount of poisonous and detrimental heavy metal elements enter soil system， which will not only reduce the number of microorganism and soil enzyme activity， but also restrain degradation of organic pollutants in soil， as well as respiratory metabolism， ammonification and nitrification of soil. At present， the area of agricultural acreage being polluted by such heavy metals as arsenic， cadmium， chromium and lead in China is approximately twenty million hectares， which equals one fifth of the total agricultural acreage， and the amount of grain output reduces over ten million tons due to heavy metal pollution every year in China. The amount of agricultural products with excessive Cd is 1.46×109kg[3] every year. Such combined pollution of nuclides and heavy metals directly or indirectly brings hidden dangers to the health of both people and livestock， and also has severe adverse influence on ecological environment. Wheat is a kind of gramineous plants planted in a wide range all over the world and also a kind of food crop whose total output ranks the second in the world. Therefore， whether wheat will gradually transport many elements detrimental to human health from root to grain is extremely important. This experiment takes wheat as material， simulates soil pollution through short-term water culture experiment and studies the features of wheat’s absorption and enrichment of U and Cd， as well as its physiological and biochemical reaction under such stress， thus offering certain scientific proof for the safety of plant growth in contaminative medium and its available values.

1. Materials and methods

1.1 Experimental materials

The plant tested was wheat which come from The Seeds of Mian Yang Corporation.

The Uranium was applied with the from of UO2（NO3）2·6H2O（AR， The chemical regament purchasing dump of China pharma ceutical company in Beijing）， The Cadmium was applied with the from of Cd（NO3）2·4H20（AR， The Kelon chemical reagent factory of Chengdu）.

1.2 Experiment methods

1.2.1 Plant culture

This experiment adopts water culture in Life and Science Laboratory Building in Southwest University of Science and Technology， Mianyang， Sichuan. Sow wheat seeds respectively in soil with pearlite， and after the seedlings grow five to six pieces of leafs， transfer them to a 2L plastic bucket with Hoagland nutrient solution. This bucket is covered by a cystosepiment with holes on it， set one seedling to each hole， ten to each bucket， four treatments in total， and change nutrient solution each three days. The whole culture lasts one month.

1.2.2 Experiment design

After one month of culture， adopt combined stress of UO2（NO3）2·6H2O and Cd（NO3）2·4H20 to deal with wheat seedling separately and conduct adsorption test. The detailed methods are： take Hoagland nutrient solution as the basis， and add UO2（NO3）2·6H2O and Cd（NO3）2·4H20 combined conditioning fluid （Table 1） separately， change nutrient solution culturing wheat seedling to UO2（NO3）2·6H2O and Cd（NO3）2·4H20 conditioning fluid. Repeat three times with each ten seedlings. After seven days of treatment， use a part of treated materials to test U and Cd accumulated content of wheat； and the other part to test root activity， chlorophyll content and superoxide dismutase by taking the second piece of plant respectively.

Table 1 Concentration of U and Cd

Note：CK is the control. Each treatment consisted of three replications. mg/kg represents the weight of elements per kilogram nutrient solution.

1.3 Test methods

1.3.1 Test of U and Cd content

After taking out wheat seedling and washing with deionized water， separate root and aboveground part， deactivate enzymes under 105℃ for 30mins， and dry to constant weight under 75℃ in oven， weight their dry weights， grind， and nitrify with microwave digestion system. Finally， test U content of root and aboveground parts of the plant respectively， repeat three times with each sample.

1.3.2 Test of physiological and biochemical indexes

Adopt TTC method to test root activity[4]； at 652nm， adopt 95% ethanol colorimetric method to test absorbance and calculate total chlorophyll content[4]； adopt nitroblue tetrazolium （NBT） to test superoxide dismutase （SOD）[5].

1.4 Data analysis

SigmaPlot11.0， Microsoft Excel 2003（U.S.， Microsoft）， DPS3.1 Software（China） were used for data analysis and all of the testing data repeated three times.

2.Results

2.1 Characteristics of the U and Cd uptake by wheat seedlings

With the increase in concentration， the U uptake by wheat seedlings first decreased and then increased. At the concentrations of 5 mg/kg and 20 mg/kg， the U uptake in roots was higher than that in aboveground parts， and at the concentration of 100 mg/kg， the U content in aboveground parts and roots was the highest， reaching 77.21 μg/g and 76.49 μg/g， respectively. This fact suggests that the U uptake by wheat seedlings was promoted at high concentration， and U is mostly stored in roots. With the increase in concentration， the Cd uptake increased in aboveground parts but decreased in roots， indicating that wheat seedlings have certain tolerance to low concentration of Cd， and the Cd transfer from roots to aboveground parts is elevated as the concentration increases. The comparison showed that Cd uptake was higher than U uptake in wheat seedling roots， and at the concentration of 5 mg/kg， the Cd uptake in roots was the highest， reaching 101.50 μg/g， 1.81 times U uptake. That might be due to the competition between U and Cd in uptake by roots and transfer from roots to aboveground parts.

Table 2 The element content of Wheat and Pea in the different concentration U and Cd treatment

Note： Results represents means±SD. Different small letters represents significant difference at p=0.05 level. Different capital letters represents significant difference at p=0.01 level.

2.2 Characteristics of U and Cd accumulation in wheat seedlings under combined stress at different concentrations

Bioaccumulation factor （BF） is the ratio of element content in the aboveground part of a plant to that in soil. It indicates the difficulty level for element transfer in the soil-plant system and can be used as an indicator for evaluating the capability of plants to transfer U and Cd to aboveground parts. The higher the BF， the higher the mass fraction of U and Cd in aboveground parts and the uptake and transfer capability of plants. Table 3 shows that the BF of U in wheat seedlings remained below 1， first decreasing and then increasing； at the concentration of 20 mg/kg， the BF was the lowest， reaching 0.127， and at the concentration of 100 mg/kg， it was the highest， reaching 0.772， 1.27 times that at the concentration of 5 mg/kg. The fact suggests that the U uptake by wheat seedlings was promoted when the U concentration is high. The BF of Cd changed oppositely， first increasing and then decreasing； at the concentration of 20 mg/kg， the BF was 1.226， while at the concentrations of 5 mg/kg and 100 mg/kg， the corresponding BFs were 43.05% and 52.96%， respectively， of the highest value， indicating that wheat seedlings are more capable of accumulating Cd.

TF is the ratio of element content in the above-ground part of a plant to that in root. It can be used as an indicator for evaluating the capability of plants to transfer U and Cd from roots to aboveground parts. The higher the TF， the greater the capability of plants to transfer elements from roots to aboveground parts. Table 3 shows that the TFs of U and Cd gradually rose with the increase in U and Cd concentration. The TFs of U and Cd were 1.009 and 1.894， respectively， when the U and Cd concentration was 100mg/kg， but were 0.027-0.669 when the U and Cd concentration was 5 mg/kg and 20 mg/kg， indicating that wheat seedlings are more capable of transferring U and Cd from roots to aboveground parts under combined stress of U and Cd at high concentration.

Table3. The BF and TF of U and Cd in wheat

2.3 Effects of U and Cd combined stress on the root activity of wheat seedlings

Figure 1 shows that with the increase in the total concentration of U and Cd， the root activity of wheat seedlings first increased and then decreased， reaching the peak value of 7.241 mg/（g？h） at the concentration of 20 mg/kg. The root activity of wheat seedlings under combined stress was significantly different from that of controls； there was no significant difference in root activity between the wheat seedlings treated at total concentrations of 5 mg/kg and 100 mg/kg， but significant difference was found between the above wheat seedlings and those treated at the total concentration of 20 mg/kg. When the U and Cd concentration was 20 mg/kg， the root activity of wheat seedlings increased sharply to 2.25 times that of the controls. This fact suggests that U and Cd combined stress at a certain concentration can help the growth of wheat seedling roots； the roots maintain high root activity and good uptake and transfer functions when exposed to lower concentration of U and Cd， but are damaged and show decreased activity as the concentration increases.

2.4 Effects of U and Cd combined stress on the chlorophyll content of wheat seedlings

Figure 2 shows that with the increase in the total concentration of U and Cd， the chlorophyll content of wheat seedlings first increased and then decreased. The chlorophyll content of the wheat seedlings treated at the total concentration of 5 mg/kg was significantly different from that of controls， reaching the peak value of 871.82 mg/kg， 1.08 times that of the controls； no significant difference was found between those treated at the total concentration of 20 mg/kg and controls； the chlorophyll content of the wheat seedlings treated at the total concentration of 100 mg/kg was significantly different from that of controls， 76.39% of the latter. This fact suggests that U and Cd combined stress at low concentration inhibits the decomposition of chlorophyll in wheat seedlings.

2.5 Effects of U and Cd combined stress on the SOD activity of wheat seedlings

Figure 3 shows that with the increase in the total concentration of U and Cd， the SOD activity of wheat seedlings first decreased and then increased. The SOD activity of wheat seedlings under combined stress was significantly different from that of controls； there were significant differences in SOD activity among the wheat seedlings treated at various concentrations. The wheat seedlings showed the lowest SOD activity， 57.25% of that of controls， when treated with 5 mg/kg of U and Cd， and showed the highest SOD activity， 1.58 times that of controls， when treated with 100 mg/kg of U and Cd. This fact suggests that U and Cd combined stress at high concentration can help eliminate superoxide radicals， reduce lipid peroxidation and membrane damage， and enhance reactive oxygen defense in wheat seedlings.

3. Discussion and conclusion

The short-term water culture experiment showed that the wheat seedlings had the lowest U uptake in roots and aboveground parts when the total concentration of U and Cd was 20 mg/kg and the highest U uptake in roots and aboveground parts when the total concentration was 100mg/kg； when the U concentration was 5 mg/kg， the U uptake in roots was 18.45 times that in aboveground parts. That suggests that under U and Cd combined stress， U is mostly stored in roots and rarely transferred to aboveground parts. However， Cd accumulation in the aboveground parts and roots of wheat seedlings acted oppositely. With the increase in the total concentration of U and Cd， the Cd content increased in aboveground parts but decreased in roots， indicating that the higher the total concentration， the greater the capability of wheat seedlings to transfer Cd to aboveground parts. When the total concentration was 20 mg/kg， only the BF of Cd was higher than 1， while others were within 0.127-0.772； when the total concentration was 100 mg/kg， the TF of U and Cd were higher than 1. That suggests that under combined stress， Cd is accumulated more than U in wheat seedlings， and U and Cd can be promoted to enter the root cells， pass through the Casparian strips， enter the xylem， combine with the abundant organic acids and amino acids in the xylem， and be transferred to the aboveground parts when their total concentration is increased. In general， the higher the BF and TF， the higher the concentration of elements accumulated in the aboveground parts of plants， and the three indices meet certain criteria in hyperaccumulators. Baker， et al. provided the reference values for Cd hyperaccumulator， i.e. the Cd content in plant leaves or aboveground parts （dry weight） is above 100 mg/kg， the heavy metal content in aboveground parts is higher than that in soil （BF >1）， and the heavy metal content in aboveground parts is higher than that in roots （TF >1） [6]. The results of this experiment showed that wheat is not a U and Cd hyperaccumulator， and wheat seedlings has certain tolerance to relatively low concentration of U and Cd. Thus， it can be concluded that the elements absorbed in wheat roots can hardly be transferred to wheat grains， and large-scale cultivation of wheat can be considered in the soil with mild contamination. The mature wheat has the contaminating elements fixed in roots （and stems and leaves）， so we can consider wheat as an alternative species for rhizosphere fixation of contaminating elements， which is of great significance for reuse of soil and production of food crops.

Root is the main organ for absorbing water and nutrients in plants， and the root growth directly reflects the growth of plants[7]. The experiment results showed that with the increase in the total concentration of U and Cd， the root activity of wheat seedlings first increased and then decreased. The root activity of wheat seedlings was lower than that of controls when treated with 5 mg/kg of U and Cd， increased significantly to the value higher than that of controls when treated at the total concentration of 20 mg/kg， and then decreased as the concentration increased. Patra et al.[8] explained that this phenomenon was due to the active “stimulation effect” of low-concentration heavy metal on plants， but this effect was restricted by concentration.

Chlorophyll is a main pigment for plant photosynthesis. The experiment results showed that U and C combined stress at low concentration （5 mg/kg） did not cause significant damage to the chlorophyll in leaves of wheat seedling， but with the increase in U and C concentration， the chlorophyll content of wheat seedlings decreased significantly. That is almost in accordance with the results of the study by Wei Xueling et al. [9] on the physiological effects of Cd stress on the chlorophyll content of wheat ‘Xihan No. 2’. As shown by the study， the decrease in chlorophyll content is a common phenomenon of plant being poisoned by heavy metals. The possible reason is that heavy metals can inhibit the activity of chlorophyllide reductase and affect the synthesis of amino-γ-levulinic acid [10].

SOD is the first line to defend plant cells from free radicals. The experiment results showed that with the increase in the total concentration of U and Cd， the SOD activity of wheat seedlings first decreased and then increased. The treated wheat seedlings had lower SOD activity than controls only when the total concentration was 5 mg/kg. The reason for improved tolerance of SOD to heavy metals might be that U and Cd combined stress increases O2- and H2O2， the substrates of SOD. Our results are in accordance with the results of the study by Zhang Lihong et al. [11] on the effects of Cd stress on the SOD activity of wheat， indicating that wheat has certain adaptation and resistance to U and Cd.

Acknowledgements

This research was supported by Professor Dan Wang. The instruments employed in this research from the Defense Key Discipline Laboratory of the Nuclear Waste Environmental Security and the center of forecasting and analysis of Southwest University of Science and Technology.

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