Agricultural and Food Science, Vol. 17 (2008): 165-176 A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 17 (2008): 177–190. 177 © Agricultural and Food Science Manuscript received February 2007 The effects of selenate and sulphate supply on the accumulation and volatilization of Se by cabbage, kohlrabi and alfalfa plants grown hydroponically Roghieh Hajiboland Plant Science Department, University of Tabriz, 51666 Tabriz, Iran, Excellent Center of Biodiversity, University of Tabriz, 51666 Tabriz, Iran, e-mail: ehsan@tabrizu.ac.ir Leila Amjad Plant Science Department, University of Tabriz, 51666 Tabriz, Iran The effect of Selenium (Se) supplementation at five levels of 0 (control), 5, 10, 15, 20 µM in plants supplied with one of four concentrations of sulphur (S) including 0.05, 0.25, 0.5 and 1.0 mM was investigated in two varieties of Brassica oleracea (cabbage and kohlrabi) and alfalfa (Medicago sativa L.) in a hydroponic experiment. In severely S deficient plants (0.05 mM), Se acted as a toxic element, alfalfa was the most susceptible plant that died at this treatment. However, in plants supplied with near adequate (0.5 mM) or adequate (1.0 mM) S, Se acted as a growth promoting element. The most pronounced stimulation of growth was observed in cabbage and the lowest in alfalfa. Increasing S concentration in the medium, reduced Se uptake and transport. In contrast, S uptake and transport increased in response to Se addition. Se volatiliza- tion was higher in alfalfa than cabbage and kohlrabi when expressed on unit shoot dry weight or leaf area basis, but not when expressed per plant. Results suggested that Se supplementation of plants supplied with adequate S, not only had beneficial effects on plants growth but also can have application in enrichment of livestock fodder and human food. Key-words: Alfalfa, Brassica oleraceae, Cabbage, Kohlrabi, Medicago sativa, Selenium supplementation, Sulphur, Transport, Uptake. A G R I C U L T U R A L A N D F O O D S C I E N C E Hajiboland, R. & Amjad, L. Accumulation and volatilization of selenium in two Brassica varieties 178 A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 17 (2008): 177–190. 179 Introduction Selenium (Se) is one of the most widely distributed elements in the earth. A considerable variability exists for its soil concentration from location to location (Mayland et al. 1989). Se has valencies of –II (selenide, Se2-), 0, IV (selenite, SeO3 2-) and VI (selenate, SeO4 2-). From both soils and nutrient solutions plants take up selenate in strong prefer- ence to selenite (Marschner 1995). On the other hand, selenate is the predominant Se species in well aerated soils. Selenite is adsorbed to soil particles to a much greater extent than selenate, therefore selenate is much more readily taken up by plant roots than selenite (Terry et al. 2000). A close chemical and physical similarity exists between Se and sulphur (S). Uptake of sulphate by plant roots is mediated by a high affinity per- mease and selenate competes with sulphate for the binding sites of this permease (White et al. 2004, Sors et al 2005). Therefore, selenate uptake can be strongly decreased by high sulphate supply and the presence of elevated SO4 -2 concentrations has been shown to drastically reduce the uptake of Se for many plants including wheat (Grieve et al., 1999) and several varieties of Brassica (Kopsell and Randle 1999, Suarez et al. 2003). Uptake and accumulation of Se by plants is determined by Se form and concentration and the presence of competing ions (Zayed et al. 1998). Additionally, plant species differ very much in Se uptake and accumulation in the shoots and also in their capacity to tolerate high Se concentrations in the rooting medium or in the shoot tissue or in both. In this regard, plants can be classified into Se-accumulators and non-accumulators, and those between as Se-indicators (Marschner 1995) or secondary Se accumulators (Terry et al. 2000). Se volatilization is the process by which in- organic Se is converted to volatile forms (Zayed et al. 1998). Dimethylselenides the predominant form of volatile Se, is 500 to 700 times less toxic than inorganic forms of Se (Wilber 1980). Thus it is possible to use plant volatilization as one dis- persal pathway for excessive Se concentrations in soils with naturally elevated concentrations of Se, such as those derived from some sedimentary rock or those impacted by anthropogenic inputs. Although Se is not known to be essential for higher plants, it is an essential trace element in mammalian nutrition (Ellis and Salt 2003). Health benefits associated with Se intake include role in reducing viral infection, improving immune function, slowing the aging process and reducing cardiovascular disease. Se status in a population correlates highly with the Se content of locally produced crops (Combs 1989). In areas of the world with low Se in the soil, Se fertilization is practiced to avoid Se deficiencies in animals and humans (Gissel-Nielsen et al. 1984). Consump- tion of traditional dietary vegetables would be a good way to deliver organic Se to a population, and ensure that beneficial effects may be realize over the life span of an individual. Positive ef- fects of Se biofortification of food crops on hu- man health were frequently reported (Broadley et al 2006). Several forage Brassica species are important vegetable crops that could be enriched with Se to supplement the human diet or can be used as nutritional supplements to meet the nu- tritional requirement of livestock (Ekholm 1997, Ellis and Salt 2003). However, a dietary intake one to two orders of magnitude greater than those leading to Se de- ficiency may cause chronic toxicity. Plants rep- resent the primary route by which Se enters ter- restrial food web, therefore factors that influence the Se uptake by plants may be of considerable importance (Ebbs and Weinstein 2001). It was reported that, members of Cruciferae have an exceptionally high requirement for S (Marschner 1995) and simultaneously accumulate relatively large amounts of Se and might contain, and tolerate, several hundred µg Se g-1 shoot dry matter (Bañuelos et al. 1997). Se uptake and its effect on growth of alfalfa as an important feed crop have been studied by many authors (Soltan- pour and Workman 1980, Mikkelsen et al 1987, 1988). It was shown that alfalfa is highly suscepti- ble to Se and toxicity can occur at contents below 100 µg Se g-1 (Shrift 1969). The critical shoot Se concentrations correspond to a 10% reduction in the yield of some crop species including mustard A G R I C U L T U R A L A N D F O O D S C I E N C E Hajiboland, R. & Amjad, L. Accumulation and volatilization of selenium in two Brassica varieties 178 A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 17 (2008): 177–190. 179 and alfalfa grown in the presence of selenate were presented by White (et al 2004). The main objective of this work was study of the effect of S nutritional status on uptake, ac- cumulation and volatilization of Se. The second aim was to evaluate the beneficial effect of Se on growth of plants particularly in dependence of S supply. Important feed and food crops including two varieties of Brassica oleracea (cabbage and kohlrabi) and alfalfa were used in this work, be- cause of their importance in Se input to food chain directly to human and/or via animals. Material and methods Plant materials Two varieties of Brassica oleracea L. including var. Sabauda (Cabbage) and var. Gongyloides (Kohl- rabi) and one local cultivar of alfalfa (Medicago sativa L. cv. Gareh-yondjeh) were used for this study. Seeds were provided by the Agricultural Research Center, Tabriz, Iran. Plant cultivation and treatments Seeds were surface-sterilized using sodium- hypochlorite at 5%, then were germinated in the dark on filter paper (cabbage and kohlrabi) or vermiculite (alfalfa) soaked with saturated CaSO4 solution. Six-day-old young seedlings were pre- cultured for 16 days in 50% nutrient solution either in hydroponic medium (cabbage and kohlrabi) or on vermiculite (alfalfa). Thereafter, plants were transplanted to 2 l dark pots (one plant per pot) and were pre-cultured for another 2 days. After precul- ture, plants were transferred to treatment solutions with full strength nutrient solution (Hoagland and Arnon 1950) containing one of four levels of S including 0.05, 0.25, 0.5,1 mM and five levels of Se including 0, 5, 10, 15 and 20 µM (as sodium selenate, Na2SeO4.10 H2O). Application of lower S treatments than the complete nutrient solution (1.0 mM) was realized by change in the concentration of MgSO4 and addition of equi-molar amounts of MgCl2 to the medium. Nutrient solutions were changed completely each 4 days and pH of nutri- ent solutions was adjusted each day. Plants were grown under controlled environ- mental conditions with a temperature regime of 25°/18°C day/night, 14/10 h light/dark period, a relative humidity of 70/80% and at a photon flux density of about 400 µmol m-2s-1. Plant harvest and analysis After growing for 14 days, plants were harvested. Each plant was divided into leaves and root, leaves were washed with double-distilled water and after determining fresh weight, leaf area was measured using a planometer. Roots were washed with double-distilled water for 5 min and after blotting dry, fresh weight and root length (Tennant 1975) were determined. After drying at 70 °C for 1 day to determine dry weight (DW), samples were digested in per- chloric acid (1:15 w/v) on heating plate for 5-6 h. Thereafter, digested samples were re-suspended in 1% HCl and made to volume by double-dis- tilled water. S analysis was carried out according to tur- bidity method (Gupta 1999). In an aliquot of the sample solution, BaSO4 turbidity was developed in the presence of sodium chloride / hydrochloric acid buffer, ethanol and glycerol by dissolving BaCl2 crystals. The absorbance of the BaSO4 tur- bidity was measured at 420 nm by spectropho- tometer (Specord 200, Analytical Jena, Germany). The content of S was estimated by reference to the standard calibration curve obtained for K2SO4. Se was determined by atomic absorption spec- trometry (Shimadzu, AA 6500) with an automated hydride generation accessory (HVG-1). The hy- dride generator was operated under the condi- tions of 6.5, 1.0 and 1.0 ml min-1 flow rate for sample, NaBH4 (0.6% w/v in 0.5% w/v NaOH) and HCl (10 M) respectively and nitrogen pres- sure of 350 KPa. The AA spectrometer was set at the wavelength of 196 nm (Norheim and Haugen A G R I C U L T U R A L A N D F O O D S C I E N C E Hajiboland, R. & Amjad, L. Accumulation and volatilization of selenium in two Brassica varieties 180 A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 17 (2008): 177–190. 181 1986). Series of Se standard solutions consisting of 5 different concentrations ranging from 0 to 40 µg l-1 were prepared by heating sodium selenate (Se(VI)) in the presence of 25% (v/v) hydrochlo- ric acid until reduction to selenite (Se(IV)) was complete (Hobbins 1981). Collection and measurement of volatile Se Plants grown for 14 days at adequate S supply (S = 1.0 mM) and supplemented with three levels of Se (0, 10 and 20 µM) as described above, were used for study of Se volatilization. A transparent acrylic plastic (Plexiglas) chamber was built to capture Se volatilized from plants. The chamber was 0.3×0.3 m in horizontal cross section and 0.5 m in height. Plants, which have been treated for 14 days, were placed in the chamber for 24 h under temperature between 25-28 ºC and illumination of 300 µmol m-2 s-1. One gram of activated carbon (Sigma) was placed in the chamber and volatile Se captured on the activated carbon and extracted by washing with water. The concentration of Se in the wash water was analyzed by atomic absorp- tion following reduction and hydride generation (Lewis et al. 1966). S and Se uptake and transport were calculated on the basis of root DW (Marschner, 1995) ac- cording to the following formulae and were ex- pressed as (mg or µg g-1 root DW): Uptake = (Total S (Se) content of shoot and root) / root DW Transport = Content of S (Se) in shoot / root DW. All required chemicals were purchased from Merck and Fluka chemical companies with pro analysis grade or higher. Experiments were carried out in completely randomized block design with 4 replications. One-way ANOVA followed by Tukey test (p < 0.05) for multiple comparisons was used and performed using Sigma Stat 2.03. Results Effect of Se on plants growth and dry matter production Se affected growth differently depending on S supply level. At very low S supply (0.05 mM in cabbage and 0.25 mM in alfalfa), Se inhibited plants growth at all Se concentrations and for all measured growth parameters. In alfalfa, the toxicity effect of Se at very low S supply (0.05 mM) was very high, so that, plants died in the early growth period and did not remain alive until harvest. However, in kohlrabi supplied by very low (0.05 mM) S, in the presence of 5 µM Se rather an increase in shoot and root DW was observed (Table 1). The most sensitive parameter to Se toxicity in cabbage and kohlrabi was leaf area, which reduced 82% and 74% in cabbage (Table 1) and kohlrabi (Table 2) in response to 20 µM Se respectively. Shoot DW was affected by Se toxicity at 45% and 50% under similar Se concentration in cabbage and kohlrabi respectively. A significant growth improvement was ob- served at Se concentration of 5 and 10 µM when plants were supplied with 0.25 and 0.5 mM S, and at Se concentration of 15 and 20 µM when plants were supplied with 0.5 and 1.0 mM S. The highest growth improvement was observed in cabbage with up to 59% increase in shoot DW and 55% in leaf area. The growth promoting effect of Se was much lower pronounced in kohlrabi and particularly in alfalfa than in cabbage. Only in S sufficient plants (1.0 mM) an increase in shoot DW (35%) was ob- served in response to 20 µM Se in kohlrabi and alfalfa. Changes in other growth parameters such as leaf area were rather in tendency than significant (Table 3). S and Se concentration of shoot and roots Shoot and root concentration of S was not drasti- cally affected by Se addition. However, a signifi- A G R I C U L T U R A L A N D F O O D S C I E N C E Hajiboland, R. & Amjad, L. Accumulation and volatilization of selenium in two Brassica varieties 180 A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 17 (2008): 177–190. 181 S (mM) Se (μM) Shoot DW (mg per plant) Root DW (mg per plant) Root length (cm per plant) Leaf area (cm2 per plant) 0.05 0 175±4 a 18±3 a 185±39 a 50±8 a 5 77±18 c 7±3 b 29±8 b 13±5 b 10 108±25 cb 9±5 b 38±9 b 14±8 b 15 122±24 b 7±3 b 29±7 b 11±5 b 20 96±19 cb 6±2 b 31±8 b 9±3 b 0.25 0 473±30 b 38±8 bc 265±37 b 142±27 b 5 737±63 a 49±4 ab 427±57 a 203±32 a 10 682±59 a 64±14 a 301±32 ab 176±32 ab 15 425±63 b 36±12 bc 159±33 c 79±12 c 20 350±20 b 27±6 c 181±28 c 58±11 c 0.5 0 648±37 b 51±14 a 165±23 c 182±15 ab 5 555±29 c 46±13 a 309±20 a 161±23 ab 10 756±34 a 67±12 a 297±19 b 218±27 a 15 642±50 bc 52±16 a 189±22 c 137±23 b 20 651±51 b 44±14 a 268±19 b 153±27 b 1.0 0 665±57 b 56±3 ab 246±23 c 196±27 b 5 499±29 c 42±4 b 541±38 a 156±21 b 10 684±36 b 38±12 b 270±19 c 193±23 b 15 1056±67 a 80±18 a 262±30 c 304±42 a 20 962±40 a 61±19 ab 354±14 b 273±21 a Table 1. Effect of various concentrations of Se on shoot and root dry weight (DW), root length and leaf area at different nutritional status of S in cabbage (Brassica oleracea L. var. Sabauda). Data are mean±SD. Values in each column with- in each S treatment followed by the same letter are not significantly different (p < 0.05). Table 2. Effect of various concentrations of Se on shoot and root dry weight (DW), root length and leaf area at different nutritional status of S in Kohlrabi (Brassica oleracea L. var. Gongyloides). Data are mean±SD. Values in each column within each S treatment followed by the same letter are not significantly different (p < 0.05). S (mM) Se (μM) Shoot DW (mg per plant) Root DW (mg per plant) Root length (cm per plant) Leaf area (cm2 per plant) 0.05 0 442±29 b 33±9 b 250±10 a 102±32 a 5 582±31 a 54±11 a 76±11 c 83±10 ab 10 272±39 c 17±8 b 81±17 c 31±8 c 15 276±38 c 21±8 b 190±39 a 45±5 c 20 221±21 c 14±7 b 105±5 b 26±3 d 0.25 0 732±37 a 60±14 a 304±23 b 163±28 a 5 724±30 a 76±17 a 537±23 a 174±11 a 10 461±34 c 50±19 a 148±22 c 79±13 b 15 444±39 c 48±12 a 292±16 b 81±15 b 20 651±43 b 48±18 a 313±20 b 140±25 a 0.5 0 641±36 b 56±18 a 471±19 b 149±31 ab 5 729±47 a 65±16 a 275±27 c 215±32 a 10 758±49 a 61±17 a 174±28 d 179±44 a 15 467±40 c 41±13 a 570±38 a 100±27 b 20 569±33 b 46±10 a 258±28 c 128±23 b 1.0 0 599±36 b 50±5 a 322±24 c 147±6 a 5 632±42 b 62±13 a 391±25 ab 186±17 a 10 577±54 b 53±10 a 311±14 c 157±22 a 15 808±53 a 60±13 a 351±29 bc 173±38 a 20 747±39 a 60±12 a 487±35 a 183±24 a A G R I C U L T U R A L A N D F O O D S C I E N C E Hajiboland, R. & Amjad, L. Accumulation and volatilization of selenium in two Brassica varieties 182 A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 17 (2008): 177–190. 183 cant enhancement of S concentration which was reduced again in response to increasing Se levels in the medium was observed. Such an effect tended to be more pronounced in S deficient plants. As expected, accumulation of Se responded strongly to Se addition. In some S/Se treatment combina- tions, however, Se was not accumulated continu- ously in response to increasing Se levels. After the first increase, a significant reduction was observed followed usually by a further increase in Se accu- mulation (Table 4). Effect of Se addition on S uptake and transport S uptake and transport increased in response to Se treatment from 5 to 20 µM. However, at some S and Se treatment combinations, both uptake and transport decreased with continuing increase of Se concentration particularly in cabbage. However, comparing Se treatment of 0 and 20 µM, a significant enhancing effect of Se on S uptake and transport could be demonstrated in all three studied species/ varieties. The proportion of transported S into shoot changed in response to Se supply, so that, this pro- portion was increased in the presence of Se, but the effect of different Se concentrations were similar. However, such an effect of Se on proportion of S transport was not observed in alfalfa (Table 5). Se uptake, transport and volatilization Se uptake increased with increasing Se concentration in the medium. However, in two varieties of Brassica supplied with very low S (0.05 mM), Se uptake did not change in response to Se addition from 5 to 20 S (mM) Se (μM) Shoot DW (mg per plant) Root DW (mg per plant) Root length (cm per plant) Leaf area (cm2 per plant) 0.05 0 120±35 25±6 246±53 18±4 5 died died died died 10 died died died died 15 died died died died 20 died died died died 0.25 0 126±10 a 17±3 a 243±32 a 15±1 a 5 101±9 a 13±5 a 246±30 a 15±3 a 10 127±15 a 14±5 a 254±24 a 18±7 a 15 131±21 a 15±4 a 253±7 a 17±3 a 20 128±15 a 16±3 a 251±22 a 15±3 a 0.5 0 143±21 a 24±2 a 276±53 a 18±2 a 5 104±17 a 14±5 b 223±45 a 13±1 a 10 150±21 a 18±2 ab 203±35 a 20±6 a 15 185±71 a 20±5 a 237±66 a 21±5 a 20 157±44 a 16±2 b 179±38 a 17±2 a 1.0 0 142±9 b 17±1 a 317±32 ab 17±2 a 5 171±25 ab 15±5 a 358±52 a 25±6 a 10 134±20 b 19±5 a 222±46 b 18±8 a 15 178±29 ab 19±2 a 299±77 ab 19±4 a 20 202±14 a 19±2 a 265±70 ab 25±6 a Table 3. Effect of various concentrations of Se on shoot and root dry weight (DW), root length and leaf area at different nutritional status of S in alfalfa (Medicago sativa L. cv. Garehyounjeh). Data are mean±SD. Values in each column with- in each S treatment followed by the same letter are not significantly different (p < 0.05). A G R I C U L T U R A L A N D F O O D S C I E N C E Hajiboland, R. & Amjad, L. Accumulation and volatilization of selenium in two Brassica varieties 182 A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 17 (2008): 177–190. 183 S (mM) Se (μM) Cabbage Kohlrabi Alfalfa S Se S Se S Se Shoot 0.05 0 9.7±1.4 ab 2±0.4 c 3.7±0.9 b 20±4 e 1.2±0.1 8±2 5 12.5±0.6 a 2148±131 a 2.6±0.6 b 765±144 d --- --- 10 6.4±0.5 b 1129±190 b 4.9±1.7 a 4476±384 b --- --- 15 9.5±5.1 ab 982±109 b 2.6±0.6 b 1503±211 c --- --- 20 5.5±1.9 b 1002±146 b 2.6±0.8 b 9054±627 a --- --- 0.25 0 15.9±4.0 a 17±5 b 5.9±1.2 c 15±1 b 3.0±0.6 c 4±0.6 e 5 14.1±0.7 a 970±246 a 9.2±1.2 b 889±124 a 5.3±0.7 b 643±138 d 10 15.2±1.2 a 96±11 b 9.2±1.7 b 1092±173 a 5.3±0.3 b 1627±312 c 15 18.5±1.0 a 141±52 b 9.9±1.8 b 1003±171 a 10.9±0.4 a 3123±461 b 20 17.7±4.9 a 911±175 a 14.8±0.2 a 957±119 a 11.1±0.7 a 4316±267 a 0.5 0 19.8±4.2 a 34±13 d 11.1±0.5 b 13±2 d 2.4±0.5 e 4±0.7 e 5 25.4±3.9 a 305±72 c 14.6±2.7 ab 113±27 c 4.4±0.3 d 352±32 d 10 19.9±3.7 a 319±40 c 15.3±1.9 a 239±39 b 5.9±1.0 c 979±49 c 15 20.9±6.7 a 709±97 a 15.2±0.8 a 272±77 b 10.1±0.1 a 1551±460 b 20 23.3±0.9 a 549±86 b 19.9±1.4 a 853±70 a 8.7±0.8 b 2209±244 a 1.0 0 17.6±3.2 ab 52±31c 13.8±0.4 b 12±1 d 2.8±0.7 b 24±5 d 5 29.7±0.5 a 182±34 b 13.6±1.8 b 115±18 c 3.8±0.7 b 330±54 b 10 22.2±1.9 ab 211±28 b 17.7±3.4 ab 1565±273 a 3.7±0.7 b 247±52 b 15 14.5±10.5 b 235±33 b 17.5±1.9 ab 1259±232 a 5.2±0.7 a 116±37 c 20 16.4±10.7 ab 446±67 a 21.2±2.4 a 771±14 b 4.9±1.2 a 883±167 a Roots 0.05 0 22.9±2.1 b 10±2 d 13.5±3.6 a 7±1 d 4.6±0.9 30±4 5 53.3±9.9 b 7640±945 a 9.7±2.1 a 1045±77 b --- --- 10 30.9±6.8 b 4952±80 b 28.2±14.2 a 543±293 c --- --- 15 57.5±24.8 b 2885±649 c 19.6±11.2 a 1410±118 a --- --- 20 128.6±37.5 a 6359±771 a 24.8±11.6 a 817±34 b --- --- 0.25 0 9.3±3.7 b 22±8 e 9.4±1.2 a 24±4 c 7.6±0.9 b 20±3 d 5 12.2±1.4 ab 1580±237 d 3.8±0.3 b 671±18 a 7.2±1.9 b 788±114 c 10 9.8±0.2 b 2611±280 c 7.9±2.8 a 290±30 b 9.4±1.3 ab 1284±233 c 15 15.5±3.3 a 4487±526 b 8.4±1.9 a 558±102 a 6.9±0.7 b 4021±224 b 20 9.9±0.7 b 6633±491 a 5.8±1.3 ab 394±42 b 11.9±2.0 a 5760±788 a 0.5 0 11.0±3.5 ab 32±8 d 9.4±1.4 a 30±4 c 11.4±0.2 a 33±5 e 5 14.6±1.8 a 870±191 c 6.7±1.6 a 1120±164 a 7.8±2.3 ab 578±95 d 10 6.9±1.9 b 1884±380 b 4.4±0.4 a 272±63 b 6.8±1.3 b 1111±152 c 15 9.3±3.0 ab 2933±510 a 8.5±7.0 a 896±194 a 6.1±0.9 b 1524±196 b 20 12.3±0.38 ab 2868±405 a 14.4±7.2 a 1080±126 a 9.9±2.8 ab 2044±115 a 1.0 0 10.8±0.4 bc 25±4 c 10.1±1.6 a 27±5 c 14.8±2.7 ab 30±7 c 5 16.4±2.1 a 446±92 b 8.4±1.5 a 83±23 c 8.4±1.1 b 229±58 b 10 11.8±2.8 b 726±161 b 6.0±0.8 a 855±108 a 9.5±3.2 b 434±82 b 15 7.2±2.4 c 1316±204 a 6.1±1.0 a 797±168 a 11.4±2.9 ab 1115±209 a 20 11.2±0.5 bc 1368±191 a 9.1±3.6 a 512±92 b 19.3±6.2 a 895±68 a Table 4. Concentration of S (mg g-1 DW) and Se (μg g-1 DW) in shoot and roots of cabbage (Brassica oleracea L. var. Sabauda), kohlrabi (Brassica oleracea L. var. Gongyloides) and alfalfa (Medicago sativa L. cv. Garehyounjeh) grown at different levels of S and Se in the medium. Data are mean±SD. Values in each column within each S treatment followed by the same letter are not significantly different (p < 0.05). A G R I C U L T U R A L A N D F O O D S C I E N C E Hajiboland, R. & Amjad, L. Accumulation and volatilization of selenium in two Brassica varieties 184 A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 17 (2008): 177–190. 185 µM. With increasing S supply to plants, an obvious reduction in Se uptake was found. However, Se uptake in plants supplied with 0.5 mM (cabbage) or 1.0 mM (kohlrabi) S, was rather increased in comparison with lower S levels. In contrast to cabbage and kohlrabi, a continuous reduction of Se uptake was observed with increasing supply of S in alfalfa (Fig. 1). A great proportion i.e. up to 97% of Se taken up, was transported into shoot. This proportion did not significantly change in response to increasing S supply. In alfalfa plants at the highest Se concen- tration (20 µM), transport of Se reached up to 40 µg g-1 root DW, which was higher than the highest values in two varieties of Brassica (Fig. 2). As expected, Se volatilization increased with increasing Se concentration in the medium. A distinctive metallic odor characteristic of Se was detected in the growth chamber during the experi- ment. The amount of volatilized Se based on leaf area was much higher in alfalfa than two varieties of Brassica. When was expressed per plant, how- ever, three studied varieties/species were similar in Se volatilization (Fig. 3). Discussion Dual effect of Se on growth Depending on S nutritional status of plants, Se exerted either an inhibitory or a stimulatory effect on growth. However, the extent of both toxic and beneficial effect of Se on growth parameters was S (mM) Se (μM) Cabbage Kohlrabi Alfalfa Uptake Transport Uptake Transport Uptake Transport 0.05 0 118±4 a 96±2 ab 64±18 b 50±14 ab 11±1 6±0.4 5 185±16 a 131±6 ab 37±7 b 28±5 b --- --- 10 108±13 a 77±6 b 107±41 a 79±27 a --- --- 15 213±89 a 156±65 a 54±21 b 35±10 b --- --- 20 209±66 a 81±29 b 66±14 b 41±3 b --- --- 0.25 0 209±83 a 199±80 a 82±21 b 73±20 b 26±7 c 18±6 c 5 223±33 a 211±31 a 91±4 b 88±4 b 48±4 b 41±3 b 10 173±11 a 163±10 a 93±14 b 85±11 b 53±4 b 44±3 b 15 233±14 a 217±11 a 100±40 b 91±38 b 108±7 a 101±6 a 20 239±18 a 229±17 a 207±3 a 201±2 a 125±16 a 113±14 a 0.5 0 261±51 b 250±48 b 136±8 c 127±6 c 31±7 c 20±7 c 5 324±54 a 309±52 ab 170±17 b 164±15 b 58±4 b 50±1 b 10 230±49 b 223±47 b 195±23 b 190±23 b 47±6 bc 41±5 b 15 268±28 ab 259±25 b 182±17 b 173±10 b 102±2 a 96±1 a 20 359±17 a 346±17 a 261±18 a 246±11 a 100±9 a 90±6 a 1.0 0 221±29 bc 210±298 b 175±9 c 165±8 c 36±5 c 21±2 d 5 373±16 a 357±14 a 147±24 c 138±23 c 38±2 c 29±1 cd 10 415±6 a 403±7 a 199±21 b 193±21 c 40±8 c 30±4 c 15 199±52 c 192±49 c 242±30 ab 236±29 a 74±9 a 62±6 a 20 274±32 b 263±32 b 273±27 a 264±24 b 62±10 b 43±4 b Table 5. Effect of various concentrations of Se on uptake and transport of S (mg g-1 root dry weight, (DW)) at different S nutritional status in cabbage (Brassica oleracea L. var. Sabauda), kohlrabi (Brassica oleracea L. var. Gongyloides) and alfalfa (Medicago sativa L. cv. Garehyounjeh). Data are mean±SD. Values in each column within each S treatment followed by the same letter are not significantly different (p < 0.05). A G R I C U L T U R A L A N D F O O D S C I E N C E Hajiboland, R. & Amjad, L. Accumulation and volatilization of selenium in two Brassica varieties 184 A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 17 (2008): 177–190. 185 0 10 20 30 40 50 60 Se Transport (mg g-1 root DW ) 0.05 mM S 0.25 mM S 0.5 mM S 1.0 mM S Cabbage 0 10 20 30 40 50 60 Se Transport (mg g-1 root DW) Kohlrabi 0 10 20 30 40 50 60 0 5 10 15 20 Se Conce ntration (µM) Se Transport (mg g-1 root DW) Alfalfa 0.05 mM S 0.25 mM S 0.5 mM S 1.0 mM S 0 10 20 30 40 50 60 Se Uptake (mg g -1 root DW) Cabbage 0 10 20 30 40 50 60 0 5 10 15 20 Se Conce ntration (µM) Se Uptake (mg g-1 root DW ) Alfalfa 0 10 20 30 40 50 60 Se Upt ak e (mg g root DW) Kohlrabi-1 Fig 1. Changes in the Se uptake (mg g-1 Root DW) at different nutritional status of S in cabbage (Brassica ol- eracea L. var. Sabauda), kohlrabi (Brassica oleracea L. var. Gongyloides) and alfalfa (Medicago sativa L. cv. Garehyounjeh) treated by different levels of Se. Error bars indicate SD. Fig 2. Changes in the Se transport (mg g-1 Root DW) at different nutritional status of S in cabbage (Brassica ol- eracea L. var. Sabauda), kohlrabi (Brassica oleracea L. var. Gongyloides) and alfalfa (Medicago sativa L. cv. Garehyounjeh) treated by different levels of Se. Error bars indicate SD. 0.0 0.2 0.4 0.6 0.8 1.0 Cabbage Kohlrabi Alfalfa Cabbage Kohlrabi Alfalfa Plant Varie tie s/Spe cie s Plant Varie tie s/Spe cie s Se Volatilization (µg plant-1d -1 ) b 0 20 40 60 80 100 120 140 Se Volatilization (µg m-2 d -1) 0 µM Se 10 µM Se 20 µM Se a Fig 3. Se volatilization in cabbage (Brassica oleracea L. var. Sabauda), kohlrabi (Brassica oleracea L. var. Gongyloides) and alfalfa (Medicago sativa L. cv. Garehyounjeh) treated by different levels of Se (S=1.0 mM) on the basis of leaf area (μg m-2 d-1) and per plant (μg plant -1 d-1). Error bars indicate SD. A G R I C U L T U R A L A N D F O O D S C I E N C E Hajiboland, R. & Amjad, L. Accumulation and volatilization of selenium in two Brassica varieties 186 A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 17 (2008): 177–190. 187 highly dependent on plant species/varieties. For example a significant growth stimulatory effect of 5 µM Se was observed in strongly S deficient kohlrabi (0.05 mM) while alfalfa plants died at the same treatments. The toxic effect of Se to plants results mainly from interferences of Se with S metabolism and replacing S-amino acids by corresponding Se- amino acids and their subsequent incorporation into proteins (Terry et al. 2000). Different tolerance to Se toxicity of Brassica varieties compared with alfalfa in this work is likely the result of differ- ences in the extent of Se-amino acids formation and incorporation into proteins and/or different production of nonproteinogenic methylated Se- amino acids. In addition of Se hyperaccumulators, in some secondary accumulators such as broccoli (Brassica oleracea var. Italica) (Lyi et al. 2005) the accumulation of these non toxic species of Se-amino acids has been suggested to be the basis of Se tolerance (Neuhierl et al. 1999). It could be suggested that studied varieties of B. oleracea in this work possess similar mechanism for preven- tion of Se toxicity. Further studies are necessary for proving of involving biochemical pathways in these varieties. Among growth parameters, root length and leaf area with up to 82% and 83% reduction in cabbage and 74% and 58% reduction in kohlrabi respectively, were the most sensitive parameters to Se toxicity. A high susceptibility of root length and leaf area could be likely explained by a direct effect of Se toxicity on cell division and/or expan- sion of cells. Enhancement of growth at low Se concentra- tions demonstrated in this work was also observed in Arabidopsis (White et al., 2004) and rye grass (Hartikainen et al. 2000). However, the growth improvement in this work was much higher than that reported for other species e.g. 8% increase for shoot DW in ryegrass (Hartikainen et al. 2000). Growth improvement due to Se supplementation, was much higher in cabbage than kohlrabi and al- falfa. The positive growth response of plants to Se added at small concentrations, have been attributed to the antioxidative effect of Se (Hartikainen et al. 2000). Se and S uptake and transport Supply of plants with increasing concentrations of S, reduced Se uptake and transport. Because of chemical similarities, sulphate (SO4 -2) and selenate (SeO4 -2) ions may be in direct competition for the same binding sites in plants during active absorption (White et al. 2004). Another cause of inhibition of selenate uptake by high external sulphate is related to the reduction of selenate activity by sulphate in the external solution as was calculated by Shennan et al. (1990). In contrast to the inhibitory effect of S on Se uptake, rather a significant enhancement of S up- take and proportion of transported S into shoot was observed in Se treated plants. Similar effect of Se on S uptake and accumulation was observed by other authors (Kopsell and Randle 1999, White et al. 2004). It seems likely that an antagonistic effect of Se on S uptake and transport have been reported only in experiments with high SeO4 2-:SO4 2- ratio in the medium e.g. a 1 SeO4 2-:1 SO4 2- ratio by Ferrari and Renosto (1972). In experiments used lower ratio of Se to S e.g. 1:4 and 1:2 in the nutrient solution, S concentration in plants increased with increasing Se concentrations (Kopsell and Randle 1999). The cause of stimulatory effect of selenate on S uptake and transport is not known exactly, but it was sug- gested that rhizosphere selenate promotes sulphate uptake, possibly by preventing the reduction in the abundance and/or activity of sulphate transport- ers by sulphate and/or its metabolism (White et al. 2004). In severely S deficient cabbage and kohlrabi (at 0.05 mM S, remind at this level of S, alfalfa plants were died in the presence of Se as low as 5 µM), Se uptake and transport was very high and did not re- spond to increasing Se concentration in the medium. A response to Se concentration was observed only in plants supplied with 0.25 mM S and higher. It could be attributed to a passive Se influx from medium to root and then transport into shoot. S is one of the important structural components of plasma mem- branes (Marschner 1995) and concentration of sul- folipids was reported to reduce by about two folds in S-starved plants (Nikiforova et al 2005). There- fore, it could be suggested that, sever S deficiency results in loss of membrane integrity and therefore A G R I C U L T U R A L A N D F O O D S C I E N C E Hajiboland, R. & Amjad, L. Accumulation and volatilization of selenium in two Brassica varieties 186 A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 17 (2008): 177–190. 187 passive fluxes of ions. It is well known that, pro- duction of reactive oxygen species and reduction of antioxidant enzymes damages membrane integrity (Elstner 1982). Results of our previous experiment demonstrated that sever S deficiency inhibit activ- ity of ascorbat peroxidae, catalase and superoxide dismutase and increased H2O2 concentration (Haji- boland and Amjad 2007). There are numerous re- ports on the induction of oxidative stress and loss of membrane integrity due to nutrients deficiency (Yu et al. 1998, Candan and Tarhan 2003). An unusual increase in the uptake and trans- port of Se was observed in cabbage (supplied with 0.5 mM S) and kohlrabi (supplied with1.0 mM S) compared with plants treated with rather higher Se and lower S concentrations. These changes are likely the result of the effect of S nutritional status on affinity of S carriers for Se. White et al (2004) suggested that Se and S enter Arabidopsis through multiple transport pathways with contrasting S/Se selectivity, whose activities vary between plants of contrasting nutritional status. For examination of this hypothesis for studied species/varieties in this work a short-term uptake experiment is nec- essary. The translocation of high proportion of Se taken up from root to shoot was observed in this work indicates a low potential of roots for incor- poration of selenate into organic molecules. It was shown that in contrast to selenite, plants transport and accumulate substantial amounts of selenate in leaves (Zayed et al. 1998) likely because it is not readily converted to organic forms of Se in roots (Terry et al. 2000). Se accumulation and volatilization Se accumulated to a great extent in S deficient plants, which was the result of both increased uptake and transport of Se and concentration effect due to S deficiency- induced growth inhibition. S deficient cabbage, kohlrabi and alfalfa plants accumulated up to 1.2, 9.1 and 4.3 mg g-1 DW Se in shoot and 6.6, 1.4 and 5.8 mg g-1 DW Se in root. The maximum values of Se concentration of S sufficient plants were 0.4, 1.6 and 2.2 mg g-1 DW for shoot and 1.4, 0.9 and 2.0 mg g-1 DW for root of cabbage, kohlrabi and alfalfa respectively. Se accumulation in dry matter of leaves in various Brassica varieties treated by 20 µM selenate in the nutrient solution, was reported to be 0.15-0.5 mg g-1 DW (Souza et al. 2002) which are in the range or slightly lower than observed amounts in S sufficient plants in this work. The average Se concentration of crop plants from non-seleniferous soils varies between about 0.01 and 1.0 mg kg-1 DW (Marschner 1995) e.g. ap- proximately 1000 times lower than the Se amounts in this work. The Se concentration of shoot in soil grown ryegrass treated by 13 µmol kg-1 soil (1 mg kg-1) was reported to be 16 µg g-1 DW (Hartikainen et al. 2000) which was much lower than values observed at similar treatments in this work, e.g. 211-1565 µg g-1 DW. Using soils contaminated with moderate levels of Se, it was shown that some Brassica species (B. juncea and B. napus) could be classified as secondary Se accumulator plants with a typical Se concentration of several hundred mg of Se kg-1 DW in their shoot tissues (Bañuelos et al. 1997). For hydroponically grown plants, critical toxic Se concentration in the shoot was determined at 320-330 µg g-1 DW for ryegrass and white clo- ver (Smith and Watkinson 1984). Other authors reported significantly higher amounts, e.g. up to 840-900 µg g-1 DW for ryegrass and strawberry clover (Hopper and Parker, 1999). Studied plants in this work showed rather a growth promotion at tissue concentrations as high as 446 (cabbage), 771 (kohlrabi) and 883 (alfalfa) µg g-1 DW. Unexpectedly, Se volatilization based on dry matter of leaf (not shown) and leaf area was higher in alfalfa than two varieties of Brassica. It means that, the activity of enzymes involving in the forma- tion of volatilized Se is higher in alfalfa than cab- bage and kohlrabi. However, because of different biomass, the amounts of volatilized Se expressed as µg per plant, were similar among three studied species/varieties. The volatilized Se in some va- rieties of Brassica was reported to be 0.2-0.8 µg g-1DW (Souza et al. 2002) which is in the range of our observed data for cabbage and kohlrabi but much lower than amounts determined for alfalfa. A G R I C U L T U R A L A N D F O O D S C I E N C E Hajiboland, R. & Amjad, L. Accumulation and volatilization of selenium in two Brassica varieties 188 A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 17 (2008): 177–190. 189 Phytovolatilization is an important remediation process because it prevents Se from entering the food chain. Se is volatilized mostly as dimethyl- selenide (Wilber 1980). Se has long been recognized to be an essen- tial micronutrient for animals and human. To prevent Se deficiency, 0.05 to 0.1 µg Se g-1 diet is required, Se toxicosis in animals is the conse- quence when dietary concentrations of Se exceed 5 to 15 µg g-1 (Mayland et al. 1989). These critical concentrations were exceeded by all treatments in this experiment and are consisted with the ability of these plants to accumulate Se, therefore, these plants cannot be used without dilution. The vegetable Brassica have been identified as good delivery system for organic Se in human diets (Kopsell and Randle 1999). Results of this work show that, alfalfa could be considered as one suitable plant species for Se supplementation of livestock fodder. However, adequate S nutrition of plants is necessary in order to reduce the risk of Se toxicity for animals and human. Conclusion Lower susceptibility to toxic concentrations of Se in combination with greater growth stimulation by supplemental Se in two Brassica varieties com- pared with alfalfa reveals a mechanism function- ing only in Brassicaceae. Such mechanism may facilitate an efficient internal detoxification of Se as well as higher efficiency for utilization of this element in growth improvement. Due to the pres- ence of glucosinolates in family Brassicacea, they could be the candidate buffering molecules. These molecules likely trap excess Se particularly in the absence of enough S in plant tissues and exclude Se from protein fractions. Prevention of toxicity may allow plants to recruit low levels of free Se for growth likely via activation of antioxidant defence system, enhancement of reduced cell metabolites or change of lignification and auxin metabolism. References Bañuelos, G.S., Ajwa, H.A., Wu, L., Guo, X., Akohoue, S. & Zambrzuski, S. 1997. 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Toxicology of selenium: a review. Clini- cal Toxicology 17:171–230. Yu, Q., L. Osborne & Rengel, Z. 1998. Micronutrient defi- ciency changes activities of superoxide dismutase and ascorbate peroxidase in tobacco plants. Journal of Plant Nutrition 21:1427–1437. Zayed, A.M., Lytle, C.M. & Terry, N. 1998. Accumulation and volatilization of different chemical species of sele- nium by plants. Planta 206:284–292. A G R I C U L T U R A L A N D F O O D S C I E N C E 190 The effects of selenate and sulphate supply on theaccumulation and volatilization of Se by cabbage,kohlrabi and alfalfa plants grown hydroponically Introduction Material and methods Results Discussion Conclusion References