Heavy metal accumulation and tolerance of energy grass (Elymus elongatus subsp. ponticus cv. Szarvasi-1) grown in hydroponic culture

Heavy metal accumulation and tolerance of energy grass (Elymus elongatus 1 subsp. ponticus cv. Szarvasi-1) grown in hydroponic culture 2 Gyula Siposa, Ádám Soltib, Viktória Czechb, Ildikó Vashegyic, Brigitta Tóthd, Edit Csehb, 3 Ferenc Fodorb* 4 5 aSzent István University - Agricultural Research and Development Institute, Bikazug, Szarvas, H-5540, Hungary 6 bDepartment of Plant Physiology and Molecular Plant Biology, Eötvös University, Budapest, Pázmány P. lane 7 1/C, Budapest, H-1117, Hungary 8 c Department of Plant Molecular Biology, Agricultural Institute, Centre for Agricultural Research, HAS, 9 Brunszvik street 2. Martonvásár, H-2462, Hungary 10 dDepartment of Agricultural Botany and Crop Physiology, Institute of Crop Sciences, Centre for Agricultural 11 and Applied Economic Sciences, University of Debrecen, Böszörményi street 138. Debrecen, H-4032 Hungary, 12 13 14 15 * corresponding author 16 Ferenc Fodor 17 e-mail: [email protected] 18 phone/fax: +36 1381 2164 19 20 21 Abstract 22 23 Phytoremediation is a plant based, cost effective technology to detoxify or stabilize 24 contaminated soils. Fast growing, high biomass, perennial plants may be used not only in 25 phytoremediation but also in energy production. Szarvasi-1 energy grass (Elymus elongatus 26 subsp. ponticus cv. Szarvasi-1), a good candidate for this combined application, was grown in 27 nutrient solution in order to assess its Cd, Cu, Ni, Pb and Zn accumulation and tolerance. Its 28 shoot metal accumulation showed the order Pb<Ni<Cu~Cd<Zn. In parallel with this, Pb and 29 Ni had no or very little influence on the growth, dry matter content, chlorophyll concentration 30 and transpiration of the plants. Cu and Cd treatment resulted in significant decreases in all 31 these parameters that can be attributed to Fe plaque formation in the roots suggested by 32 markedly increased Fe and Cu accumulation. This came together with decreased shoot and 33 root Mn concentrations in both treatments while shoot Cu and Zn concentrations decreased 34 under Cd and Cu exposure, respectively. Zn treatment had no effect or even slightly 35 stimulated the plants. This may be due to a slight stimulation of Fe translocation and a very 36 efficient detoxification mechanism. Based on the average 300 mg kg-1 (dry mass) Zn 37 concentration which is 0.03% of the shoot dry mass the variety is suggested to be classified as 38 Zn accumulator. 39 40 Key words: heavy metal accumulation; iron plaque; phytoremediation; Szarvasi-1 energy 41 grass; tall wheatgrass; zinc accumulator 42 43 1. Introduction 44 45 Heavy metal contamination in soils is a worldwide environmental problem. The 46 contamination may be originated from natural and anthropogenic sources, the latter being 47 much more significant. Anthropogenic contamination may occur due to mining, industrial 48 activities, traffic, inadequate use of (phosphate) fertilisers in agriculture and amendment with 49 sewage sludge [1]. In Hungary, a recent environmental disaster underlines the significance of 50 the problem: an industrial accident at a caustic waste reservoir chain of the Ajkai Timföldgyár 51 alumina plant in Ajka, western Hungary in October 2010 flooded about 40 square kilometres 52 and two localities with alkaline wastes of metal containing sludge called red mud. 53 Heavy metals, naturally present or deposited in various concentrations, have different 54 solubility and mobility in the soil but may be mobilised and accumulated by plants [2]. This 55 means a major threat for heavy metal uptake by crop plants but also provide a possibility to 56 remove the metals from the soils by specific plant species. Phytoremediation techniques based 57 on naturally metal accumulating plants (accumulator plants) or chelate-assisted metal 58 mobilization and uptake, i.e. phytoextraction [3,4], may raise another problem of the fate of 59 harvested plant material. Fast growing, high biomass, perennial plants developed or 60 genetically designed for energy production may provide a feasible and cost-effective solution 61 [5]. 62 Succesful clean up of metals from the soil is based on the efficiency of plants to take 63 up and accumulate them in their roots and translocate them to the shoots whereas tolerance to 64 toxicity influences plant growth: large biomass also provides higher capacity for storage. 65 These processes depend on various transport proteins the presence and function of which 66 should be taken into account when new species are characterised. 67 Szarvasi-1 energy grass (Elymus elongatus subsp. ponticus cv. Szarvasi-1) was bred 68 from a native population of tall wheatgrass in Hungary that was adapted to slightly salty 69 habitats [6,7]. It has a fibrous root system that may reach 3.5 m whereas the shoot may grow 70 to 1.8-2.2 m. In spite of its high biomass yield it is well adapted to drought, flood and frost 71 and does not require special soil conditions but prefers sandy and alkaline soils. As a 72 perennial grass it may live up to 10-15 years. Its industrial uses are well documented but there 73 are only limited data available on its natural element composition or requirement and 74 accumulation as well as tolerance to toxicity [8]. 75 Heavy metals, such as cadmium, copper, lead, mercury, nickel and zinc are major 76 pollutants, particularly in areas with high anthropogenic pressure [9]. Szarvasi-1 energy grass 77 may be potentially applied in renewable energy production combined with phytoextraction or 78 phytostabilization. The aim of the present work was to assess the natural ability of Szarvasi-1 79 energy grass to accumulate or tolerate different heavy metals, Cd, Cu, Ni, Pb, Zn from 80 nutrient solution. Hydroponic culture was chosen for the experiments because it excludes the 81 different adsorption, mobility and retention characteristics of the metals in soil. 82 83 2. Results 84 2.1. Physiological responses to heavy metal treatments 85 86 The control and heavy metal containing nutrient solutions had very similar, slightly acidic pH 87 values which have been increased to slightly alcaline levels during cultivation of the plants 88 (Table 1). The extent of increase was smaller in case of Cd and Cu treatments. 89 Root and shoot growth was not affected, compared to the untreated control by Pb and 90 Zn applied in the nutrient solution in 10 M concentration for a month (Fig. 1). Ni and Cd 91 caused about 20 and 35% inhibition in the root and shoot growth, respectively. Cu had the 92 strongest effect on Szarvasi-1 decreasing the root and shoot dry mass by 90 and 75%, 93 respectively. When Cd and Cu were applied the relative dry matter content of the roots 94 increased with 76 and 138% whereas that of the shoots with 44 and 56%, respectively (Fig. 95 2). 96 The chlorophyll (Chl) concentration of the leaves changed most markedly under Cu 97 treatment that caused about 50% decrease leading to visible symptoms (Fig.3). Cd and Ni 98 caused a smaller but significant decrease compared to the control while the effect of Pb was 99 insignificant. The transpiration, measured as stomatal conductance for water vapour, 100 decreased by 79 and 91% in the plants treated with Cd and Cu, respectively (Fig. 4). Zn 101 increased the Chl concentration and stimulated the transpiration compared to the control, 102 although these changes were not significant. 103 104 2.2. Heavy metal concentration 105 106 Heavy metals applied in the treatments in 10 μM concentration were adsorbed by the roots in 107 different amounts (Fig. 5). Cd and Ni concentrations were very similar and the lowest among 108 the five metals. Zn concentration was twice as large while Cu concentration was almost 4 109 times larger than that of Cd and Ni. Pb was adsorbed in the highest amount (280 mol g-1 DW 110 = 1.35 mg kg-1 DW). Half of the roots of each plant were undertaken a washing procedure 111 (with CaSO4 +Na2EDTA) in order to remove the loosely bound part of the adsorbed metals. 112 The metal concentration was recalculated using the same dry mass data. The results revealed 113 that most of the Cd (79%) and Ni (93%) were not removable. In case of Zn only 41% 114 remained in the roots after washing. However, the root concentrations of the three metals 115 were statistically not different after the washing (26-27 μmol g-1 DW). In case of Cu and Pb 116 most of the adsorbed amount was removed in the washing procedure: only 8 and 1% 117 remained, respectively, resulting in the lowest concentrations. 118 In the shoot, Cd and Cu concentrations were similar while Ni and Pb were 119 significantly lower. Zn concentration was the highest reaching 4.71 µmol g-1 DW (300 mg kg120 1 DW) which is 0.03% of the shoot dry mass. 121 122 2.3. Essential metal concentration 123 124 The concentration of Fe was similar in the roots and shoots of control, Ni, Pb and Zn treated 125 plants (Fig. 6). However great difference was found between the Cd and Cu treated plants. 126 The latter two treatments caused a very high increase in the total Fe concentration of the 127 roots, 140 and 400 % by the Cd and Cu treatment, respectively, and the non-removable 128 fraction was still several times higher than in the control. The shoot concentration was 129 significantly changed (increased) only by Zn. Total Mn concentrations in the roots of Ni and 130 Pb treated plants were the same as in the control (Fig.7). Zn treatment reduced the Mn 131 concentration to one third of the control while Cd and Cu further reduced it to a minimal 132 level. The non-removable fraction of Mn accounted for 33-53% of the total amount. The 133 shoot contained Mn at a similar level in the control, Ni and Pb treatment while Zn slightly 134 reduced it. Cd and Cu decreased the shoot Mn to about half of the control. 135 Zn and Cu concentrations of the plants were compared in Figs. 8 and 9 when they 136 were applied at low (microelement) concentration. Only the roots of Cd and Ni treated plants 137 contained Zn in significantly lower concentration compared to the the control (Fig.8). 138 However, the non-removable fraction was different from the total only in the control. In the 139 shoot, only the Cu treated plants contained Zn at a lower level than the control. Cu 140 concentrations were almost identical to the control in the Ni, Pb and Zn treatment in both 141 roots and shoots while in the Cd treated plants it was 50% higher in the root and 20% lower in 142 the shoot (Fig.9). 143 144 3. Discussion 145 146 3.1. Heavy metal uptake 147 148 The metal content of shoot tissues depends on the uptake and translocation ability of root and 149 vascular tissues. In the hydroponic culture, the root system of Szarvasi-1 energy grass 150 adsorbed highly different amounts of heavy metals at slightly acidic to slightly alkaline pH in 151 the order: Pb>Cu> Zn> CdNi (Fig 5.). This finding is in agreement with previous work on 152 tall wheatgrass [10] and may be explained by different mechanisms. Cu, Zn and Ni are 153 essential transition metals required for normal growth in the order Zn>Cu> Ni and are readily 154 soluble in the applied experimental conditions. Their adsorbance may be driven by active 155 uptake. Cd is a nonessential heavy metal that is present in the nutrient solution in free divalent 156 ionic form [11]. However, after applying a washing procedure (CaSO4 + Na2EDTA solution) 157 in order to remove the portion deposited only to the apoplastic spaces, we found that Cd, Ni 158 and Zn were taken up by the roots in very similar amount, while Cu uptake was smaller. The 159 influx of transition metals is mediated by specific transporter proteins. 160 Zn uptake was first reported to be regulated by ZIP family genes in Arabidopsis 161 thaliana [12,13], however, the exact function of ZIPs is poorly known, yet [14]. In rice, a 162 Strategy II plant in Fe uptake, OsZIP1 and OsZIP3 seems to be important for Zn uptake from 163 soil [15,16]. In barley, Zn-DMA (deoxy mugineic acid – a phytosiderophore released by the 164 plant) is preferred over Zn2+ for uptake through roots [17]. In contrast, rice plants absorb less 165 Zn-DMA compared to Zn2+ [18]. Tall wheatgrass is a close relative to barley, thus DMA166 chelated Zn uptake can be predicted. 167 Cd may enter the root cells using different pathways provided by ZIP family 168 transporters, ZNT1 [19] and IRT1 which latter mediates Fe2+ uptake in non-graminaceous 169 plants [20] but was also found in rice [21]. In rice, Cd2+ uptake into the symplasm was shown 170 to be linked to Ca2+ transport, as accumulation of Cd is inhibited by La3+ and high Ca2+ 171 concentrations [22]. Wheat LCT1 (low-affnity cation transporter) was shown to have a role in 172 both Cd2+ and Ca2+ uptake [23]. In rice, OsNramp5 and OsNramp1 were reported as a root 173 plasma membrane transporter of Mn2+ and Cd2+ [24] and Fe2+ and Cd2+ [25], respectively. 174 The uptake and translocation of Cu is little known, it was found that P-type heavy 175 metal ATPases (HMAs) are involved [26,27]. Cu+ transport into the cytosol is also mediated 176 by COPT family transporters in A. thaliana [28]. In graminaceous plants, the uptake of Cu 177 (and Zn) may be mediated by the release of phytosiderophores which (is increased under Fe 178 and Zn deficiency and) plays a distinct role in Fe acquisition [29]. ZIP2 and ZIP4 proteins are 179 also suggested to be transporting Cu2+ in Arabidopsis [30]. Cu2+ can form stable NA chelate 180 even under mild acidic conditions which complexes may have a role in the Cu translocation. 181 The uptake of Cu2+-chelates cannot be excluded in Strategy-II plants, either. Gunawardana et 182 al [31] showed that Cu uptake is enhanced by the presence of hystidine in the hydroponic 183 solution in ryegrass (Lolium perenne). 184 The uptake and translocation of Ni is poorly known, too. Ni may enter the cells in a 185 rather unspecific route through plasmalemma CNGCs (cyclic nucleotide gated channels) [32]. 186 Nishida et al. [33] showed that AtIRT1, the primary Fe2+ uptake transporter in the root, 187 mediates Ni accumulation in Arabidopsis thaliana. But there is no evidence for a specific Ni 188 uptake in Strategy-II plants up to now. 189 Taking all these into account, ZIP-family transporters or chelation based strategies 190 (NA and DMA chelation) may be involved in the uptake of Cu, Ni and Zn. Cd uptake may 191 also interfere with that of Fe and Mn. Thus, regular disturbances in the essential transition 192 metal uptake and translocation in heavy metal treated Szarvasi-1 energy grass can be 193 explained as complex interference in these systems. 194 The various uptake mechanisms described above show that probably it is not the way 195 of influx that matters as it does not provide explanation for the higher adsorption and lower 196 uptake of Cu compared to the other transition metals in Szarvasi-1 energy grass. The 197 formation of Fe-plaque in the roots of Cu treated plants may account for the retention of Cu 198 and also Fe on the roots [34]. Such an unspecific mechanism may be predicted in the case of 199 Cd and Pb, too. In Cd treated plants this is underlined by the accumulation of Fe and Cu in the 200 root apoplast. Pb is a nonessential heavy metal that may produce relatively insoluble 201 precipitates with the constituents of the nutrient solution in sulphate and phosphate (or 202 chloride) form on the root surface [35]. Soluble Pb concentration can be increased with 203 complexing agents like EDTA or citrate [8] which serves as the basis for Pb mobilization in 204 polluted soils during “induced phytoextraction” [36,37,38]. Applying the (CaSO4 + 205 Na2EDTA) washing procedure, we found that Pb taken up by the roots was almost negligible. 206 Pb uptake may be mediated either by CNGC [32] or P-type ATPase transporters [39]. 207 However, as most of this metal is removable from the root apoplast its uptake may occur 208 through more unspecific routes, too. The ionic radius of Pb2+ is much larger compared to the 209 other metal ions tested, thus it may be assumed to surge into stelar tissues through internal 210 wounding by lateral root formation [40] or may be taken up by endocytosis [41]. 211 212 3.2. Heavy metal translocation 213 214 5.2. Mass measurements 363 364 The roots of the 10 plants grown in a single pot were separated into two portions. The roots of 365 the first 5 plants were centrifuged between filter papers at 300 g to remove traces of nutrient 366 solution before drying but no other treatment was applied. The other 5 roots were rinsed with 367 0.5 mM CaSO4 solution and then transferred to 200 ml 0.5 mM CaSO4 solution containing 10 368 mM Na2EDTA (pH 4.05) and were shaken for 1.5 h at 125 rpm [11]. After rinsing again with 369 CaSO4 the roots were centrifuged between filter papers at 300 g. The filtered roots were 370 weighed. Dry mass was determined after drying at 80 °C. Final data are extrapolated to one 371 single plant. 372 373 5.3. Element analysis 374 375 Measurements were made with three parallel samples (each containing 5 dried plants) after 376 acidic digestion. 5-10 ml ccHNO3 was added to each gram of the samples for overnight 377 incubation. Then the samples were pre-digested for 30 min at 60 °C. Finally, 2-3 ml H2O2 (30 378 m/m%) was added for a 90 min boiling at 120 °C. The solutions were filled up to 10-50 ml, 379 homogenised and filtered through MN 640W filter paper. The element content of the filtrate 380 was determined by ICP-MS. Data were converted from ppm to μmol g-1 units in order to 381 ensure better comparison between treatments. 382 383 5.4. Chlorophyll concentration 384 385 The measurements were made with the first fully developed leaves. The Chl concentration 386 was determined photometrically (Shimadzu UV-2101PC) from 80% acetone extracts using 387 the equations of Porra et al. [60]. Each measurement was carried out on three individual 388 plants in each treatment group. 389 390 5.5. Stomatal conductance 391 392 Stomatal conductance was measured with a porometer (DELTA-T Devices Ltd.) on the 393 abaxial epidermis of the middle sections of the youngest, fully developed leaves. 394 Transpiration was calculated as mmol H2O m-2 s-1. Each measurement was carried out three 395 times on three individual plants in each treatment group. 396 397 5.6. Definition of indices 398 399 Translocation factor (TF) and phytoextraction capacity (PC) was defined after Vashegyi et al. 400 [8] with some modifications. Translocation factor of Mei = shoot Mei concentration ( mol g-1) 401 / Mei concentration in the washed roots ( mol g-1). Phytoextraction capacity of Mei = shoot 402 total Mei content (g) * 100 / total amount of Mei supplied to the nutrient solution during the 403 entire growth period (g) 404 405 5.7. 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Kriedemann, Determination of accurate extinction 573 coefficients and simultaneous equations for assaying chlorophyll a and b extracted with four 574 different solvents: verification of concentration of chlorophyll standards by atomic absorption 575 spectroscopy, Biochim. Biophys. Acta 975 (1989) 384–394. 576 577 578 579 580 581 Captions to Figures 582 583 Figure 1 Root and shoot dry mass of 37 day-old Szarvasi-1 energy grass grown in nutrient 584 solutions amended with different metals (Cd, Cu, Ni, Pb, Zn) in 0 or 10 μM concentration. 585 (Data are shown as mean±SD, n=6, significant differences between data are idicated with 586 different letters, P<0.05) 587 588 Figure 2 Dry matter content in the roots and shoots of 37 day-old Szarvasi-1 energy grass 589 grown in nutrient solutions amended with different metals (Cd, Cu, Ni, Pb, Zn) in 0 or 10 μM 590 concentration. (Data are shown as mean±SD, n=6, significant differences between data are 591 idicated with different letters, P<0.05) 592 593 Table 1. pH values of the nutrient solutions amended with different heavy metals (Cd, Cu, Ni, 667 Pb, Zn) in 0 (ctr) or 10 μM concentration at preparation (Day 0) and after 4 days of plant 668 growth (Day 4) in unbuffered, aerated hydroponic culture of one month-old Szarvasi-1 energy 669 grass. (Data are presented as mean±SD, n=6, significant differences between data are idicated 670 with different letters, P<0.05)) 671 672 treatment Day 0 Day 4 673 ctr 4,70 7,66±0,05 a 674 Cd 4,67 6,73±0,01 b 675 Cu 4,70 6,11±0,08 c 676 Ni 4,60 7,42±0,12 d 677 Pb 4,76 7,297±0,20 d 678 Zn 4,78 7,72±0,05 a 679 680 681 682 683 684 685 686 687 688 689 690 Table 2. Translocation factor [TF = shoot Mei concentration (mol g-1) / Mei concentration of 691 the washed roots (mol g-1)] and phytoextraction capacity [PC = shoot total Mei content (g) 692 *100 / Mei supplied to the nutrient solution during the whole growth period (g)] of Mei and 693 Fe in Szarvasi-1 energy grass grown in nutrient solutions amended with different metals 694 (Mei) in 0 (ctr) and 10 μM concentration. (The concentration of Fe was also 10 μM in all 695 treatments and the untreated control.) 696 697 treatment (Mei) TF (Mei) TF (Fe) PC (Mei) PC (Fe) 698 Cd 0,077 0.031 0.071 0.069 699 Cu 0.159 0.009 0.041 0.035 700 Ni 0.020 0.102 0.038 0.129 701 Pb 0.160 0.281 0.024 0.173 702 Zn 0.174 0.188 0.467 0.226 703 Fe (ctr) - 0.265 - 0.159 704