Zinc tolerance and accumulation in the salt-marsh shrub Halimione portulacoides

1 Zinc tolerance and accumulation in the salt-marsh shrub 1 Halimione portulacoides 2 3 J. Cambrollé*, J.M. Mancilla-Leytón, S. Muñoz-Vallés, T. Luque & M.E. Figueroa 4 5 Departamento de Biología Vegetal y Ecología, Facultad de Biología, Universidad de 6 Sevilla, Apartado 1095, 41080 - Sevilla, España 7 8 *Corresponding author. 9 Postal address: Jesús Cambrollé Silva, Dpto. Biología Vegetal y Ecología, Facultad de 10 Biología, Universidad de Sevilla, Av. Reina Mercedes 6, 41012 Seville, Spain. 11 Tel.: +34-95-4557165; fax: +34-95-4615780. E-mail address: [email protected] 12 13 14 15 16 17 18 19 20 21 22 23 24 25 2 Abbreviations: A, net photosynthetic rate; Chl a, chlorophyll a; Chl b, chlorophyll b; 26 Ci, intercellular CO2 concentration; Cx+c, carotenoids; F0, minimal fluorescence level 27 in the dark-adapted state; Fm, maximal fluorescence level in the dark-adapted state; Fs, 28 steady state fluorescence yield; Fv, variable fluorescence level in the dark-adapted state; 29 Fv/Fm, maximum quantum efficiency of PSII photochemistry; ΦPSII, quantum 30 efficiency of PSII; Gs, stomatal conductance; NPQ, non-photochemical quenching; 31 RGR, relative growth rate; SLA, specific leaf area. 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 3 Abstract: 51 52 The halophytic shrub Halimione portulacoides is known to be capable of growth 53 in soils containing extremely high concentrations of Zn. This study evaluated in detail 54 the tolerance and accumulation potential of H. portulacoides under moderate and high 55 external Zn levels. A greenhouse experiment was conducted in order to investigate the 56 effects of a range of Zn concentrations (0 to 130 mmol l-1) on growth and photosynthetic 57 performance by measuring relative growth rate, total leaf area, specific leaf area, gas 58 exchange, chlorophyll fluorescence parameters and photosynthetic pigment 59 concentrations. We also determined the total zinc, nitrogen, phosphorus, calcium, 60 magnesium, sodium, potassium, iron and copper concentrations in the plant tissues. 61 H. portulacoides demonstrated hypertolerance to Zn stress, since it survived with leaf 62 concentrations of up to 2300 mg Zn kg-1 dry mass, when treated with 130 mmol Zn l-1. 63 Zinc concentrations greater than 70 mmol l-1 in the nutrient solution negatively affected 64 plant growth, in all probability due to the recorded decline in net photosynthesis rate. 65 Our results indicate that the Zn-induced decline in the photosynthetic function of H. 66 portulacoides may be attributed to the adverse effect of the high concentration of the 67 metal on photosynthetic electron transport. Growth parameters were virtually unaffected 68 by leaf tissue concentrations as high as 1500 mg Zn kg-1 dry mass, demonstrating the 69 strong capability of H. portulacoides to protect itself against toxic Zn concentrations. 70 The results of our study indicate that this salt-marsh shrub may represent a valuable tool 71 in the restoration of Zn-polluted areas. 72 73 74 75 4 Keywords: Halimione portulacoides; photosynthesis; phytoremediation; Zinc. 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 5 1. Introduction 101 102 Due to their toxicity and capacity for bioaccumulation, pollution by heavy 103 metals is a serious problem. Zinc is a metal characterized by its high mobility and 104 bioavailability (Morillo et al., 2004) and is considered to be a major industrial pollutant 105 of the terrestrial and aquatic environment (Barak and Helmke, 1993). Although Zn is an 106 essential microelement involved in numerous physiological processes (Rengel, 1999), 107 elevated concentrations of this metal in soils can lead to toxicity symptoms in most 108 plants: At high concentrations, Zn causes leaf chlorosis, nutrient imbalances and 109 inhibition of photosynthesis at various stages, resulting in impairment of plant growth 110 (Chaney, 1993; Kabata-Pendias and Pendias, 2001; Van Assche and Clijsters, 1986). 111 Despite this, certain plant species have evolved heavy metal tolerance and can grow in 112 Zn-contaminated soils, remaining unaffected by the elevated Zn levels (Ernst et al., 113 2000). For example, it is well known that salt-marsh plants can tolerate and accumulate 114 high contents of heavy metals (e.g. Mateos-Naranjo et al., 2008a; Mattheus et al., 2005). 115 Halimione portulacoides (L.) Aellen is a halophytic shrub frequently found on 116 sandy and muddy sea-shores and salt marshes around the coasts of Europe, North Africa 117 and South-West Asia. The species is frequently the physiognomic dominant on well118 drained and upper marshes, often fringing channels and pools that are flooded at high 119 tide (Chapman, 1950). In several estuaries of the Iberian Peninsula, H. portulacoides 120 grows in sediments featuring extremely high concentrations of metals. In the joint 121 estuary of the Tinto and Odiel rivers (SW Spain), one of the world´s most polluted areas 122 in terms of heavy metals, H. portulacoides can be found growing in sediments that 123 contain 100-4800 ppm Zn (Cambrollé et al., 2008; Nelson and Lamothe, 1993; Sáinz 124 and Ruiz, 2006). Moreover, in the Tagus estuary salt marshes (Lisbon, Portugal), this 125 6 species has demonstrated its ability to tolerate and sequestrate high levels of Zn 126 (Caçador et al., 2000). In view of this ability, several recent studies have explored the 127 phytoremediation potential of H. portulacoides (Almeida et al., 2009; Duarte et al., 128 2007; Sousa et al., 2008). To date, the physiological impact of elevated concentrations 129 of metals on this species remains unknown; however, this knowledge is necessary in 130 order to understand its limits of phytotoxicity and, ultimately, its potential for use in the 131 phytoremediation of areas contaminated by metals. 132 The present study was undertaken to evaluate the tolerance and accumulation 133 potential of H. portulacoides under exposure to moderate and high Zn levels. The 134 specific objectives were: (1) to determine the Zn phytotoxicity thresholds of the study 135 species by analyzing the growth of plants in a range of external Zn concentrations, from 136 0 to 130 mmol l-1 Zn; (2) to ascertain the extent to which Zn determines plant 137 performance, in terms of influence on the photosynthetic apparatus (PSII chemistry), 138 gas exchange characteristics and photosynthetic pigments; and (3) to examine the 139 possible relationship between the effects of Zn on growth and the N, P, Ca, Mg, Na, K, 140 Fe and Cu concentrations in plant tissues. 141 142 143 144 145 146 147 148 149 150 7 2. Materials and Methods 151 152 2.1. Plant material and stress treatments 153 154 Seeds of Halimione portulacoides were collected in the salt marshes of “La 155 Mata-Torrevieja” (Alicante, SE Spain). The collected seeds were subsequently 156 germinated in perlite moistened with distilled water, and maintained at 25 ºC for 30 157 days. The resulting seedlings were sown in individual plastic pots (diameter 11 cm) 158 filled with perlite, and placed in a glasshouse with minimum-maximum temperatures of 159 21-25ºC, 40-60% relative humidity and natural daylight (minimum and maximum light 160 flux: 200 and 1000 μmol m-2 s-1, respectively). Pots were carefully irrigated with 20% 161 Hoagland's solution (Hoagland and Arnon, 1938) as required. 162 When seedlings were between 20 and 25 cm in height (after 4 months of 163 growth), the pots were allocated to five different Zn concentration treatments: 0, 10, 30, 164 70 and 130 mmol l-1 Zn, applied in shallow trays within the same glasshouse (ten pots 165 per tray, one tray per Zn treatment). Zn treatments were prepared by mixing 20% 166 Hoagland's solution with ZnSO4·7H2O of the appropriate concentration. The control 167 treatment, 0 mmol l-1 Zn, in fact contained 0.002 mmol l-1 of Zn, since Hoagland’s 168 solution contains a small amount of Zn as an essential trace nutrient. These Zn 169 concentrations were chosen in order to reflect the range of levels found by several 170 authors in studies of the salt-marshes of different metal polluted estuaries of the 171 southern Iberian Peninsula, around 100-5000 mg Zn kg-1 per total soil DW (Cambrollé 172 et al., 2008; Nelson and Lamothe, 1993; Reboreda and Caçador, 2007b; Sáinz and Ruiz, 173 2006), as well as in previous experiments to determine the phytotoxicity thresholds of 174 H. portulacoides. 175 8 At the beginning of the experiment, a 3 L volume of the appropriate solution was 176 placed in each of the trays to a marked depth of 1 cm. Throughout the experiment, 177 solution levels in the trays were monitored and topped up to the marked level with 20% 178 Hoagland's solution, (with no additional ZnSO4·7H2O) in order to limit the change in 179 Zn concentration due to evaporation of the water in the nutrient solution. In addition, 180 the entire solution (including ZnSO4·7H2O) was changed on a weekly basis. 181 182 2.2. Growth 183 184 From each treatment, four complete plants (roots and shoots) were harvested at 185 the beginning, and the remaining six at the end of the experiment (i.e. following 75 days 186 of treatment). These plants were dried at 80ºC for 48 h and then weighed. 187 The relative growth rate (RGR) of whole plants was calculated using the formula: 188 189 RGR = (ln Bf – ln Bi) · D-1 (g g-1day-1) 190 191 where Bf = final dry mass, Bi = initial dry mass (average of the four plants from each 192 treatment dried at the beginning of the experiment) and D = duration of experiment 193 (days). 194 Leaf area was determined from the projected area by scanning and digitalising 195 the leaves (Epson V30, Seiko Epson Corp., Nagano, Japan), and using appropriate 196 software (MideBMP v. 4.2.; Ordiales-Plaza, 2000) for processing and analysis. Specific 197 leaf area (SLA) was calculated as the ratio of leaf area to leaf dry mass. 198 199 200 9 2.3. Chemical analysis of plant samples 201 202 At the end of the experimental period, leaf and root samples were dried at 80ºC 203 for 48 h and ground, in accordance with the protocols of Redondo-Gómez et al. (2007). 204 Leaves and roots were carefully washed with distilled water prior to further analysis. 205 Then, 0.5 g samples were digested with 6 ml HNO3, 0.5 ml HF and 1 ml H2O2. 206 Measurements of Ca, Cu, Fe, K, Mg, Na, P and Zn were conducted by inductively 207 coupled plasma (ICP) spectroscopy (ARL-Fison 3410, USA). Total N concentration 208 was determined from undigested dry samples using an elemental analyzer (Leco CHNS209 932, Spain). 210 211 2.4. Gas exchange 212 213 Gas exchange measurements were taken from randomly selected, fully 214 expanded leaves (n = 10, one measurement per plant plus four extra measurements 215 taken randomly), following 75 days of treatment, using an infrared gas analyzer in an 216 open system (LI-6400, LI-COR Inc., Neb., USA). Net photosynthetic rate (A), 217 intercellular CO2 concentration (Ci) and stomatal conductance to CO2 (Gs) were 218 determined at an ambient CO2 concentration of 360 µmol mol-1, temperature of 219 20/25ºC, 50 ± 5% relative humidity and a photon flux density of 1000 µmol m-2 s-1. 220 Values of the parameters A, Ci and Gs were calculated using the standard formulae of 221 Von Caemmerer and Farquhar (1981). 222 223 16 (2000 and 2006) found EC50-plant values of between 1.2 and 3.4 mmol l-1 Zn for five 374 reclamation grass species and six restoration forbs. 375 Salt marsh plants generally accumulate different percentages of metals within 376 their belowand above-ground biomass, with a higher percentage of metals found in the 377 roots rather than in the shoots (Fitzgerald et al., 2003; Matthews et al., 2004). In our 378 greenhouse experiment, H. portulacoides tissue concentrations of Zn were higher in the 379 below-ground biomass than in the leaves at external Zn concentrations of 10 and 30 380 mmol l-1; the transfer factor (TF), which is defined as the ratio of the metal 381 concentration in shoots to that in the roots (Sun et al., 2009), was therefore below 1.0 in 382 these Zn treatments. Previous studies showed that the roots of this salt-marsh shrub 383 accumulate more metals than the above-ground biomass (Caçador et al., 2000; 384 Reboreda and Caçador, 2007a; Reboredo, 1991). Sousa et al. (2008) found that this 385 species is able to retain a high quantity of metals in the root cell wall, and stated that 386 compartmentation and detoxification mechanisms are crucial to allow H. portulacoides 387 to tolerate high levels of heavy metals. Our study shows that 388 H. portulacoides could limit Zn transport into the shoots up to an external concentration 389 of 30 mmol l-1, since leaf metal levels became higher than those in roots when the 390 external Zn exceeded this concentration; TF values were therefore higher than 1.0 at 70 391 and 130 mmol l-1 external Zn. Kabata-Pendias and Pendias (2001) indicated that, 392 although roots often contain much more Zn than shoots, at extremely high levels of Zn 393 in soils this metal can be translocated from the roots and accumulated within the shoots. 394 Zinc is relatively active in biochemical processes and is known to be involved in 395 several biological and chemical interactions with several other elements. In addition, the 396 supply of Zn is expected to decrease the uptake of most nutrients (Chaney, 1993; 397 Fageria, 2001). In our experiment, Zn-Cu, Zn-Fe and Zn-Mg interactions were not 398 17 found, despite the fact that such interactions have been widely observed and reported 399 (Kabata-Pendias and Pendias, 2001). However, the presence of Zn did alter the 400 concentrations of the macroelements, N, P, Ca, Na and K. The tendency of N 401 concentration in H. portulacoides roots to increase slightly with increasing external Zn 402 could be due to a higher binding of Zn by proteins and amino acids in root tissues 403 (Olsen, 1972). Sousa et al. (2008), in a field study with H. portulacoides determining 404 metal concentrations in several fractions of plant material, found that a high percentage 405 of root Zn was being stored in the proteic fraction. 406 A zinc-phosphorus interaction has been widely observed and reported in many 407 crop plants (Fageria, 2001) although the specific mechanism of this interaction is not 408 yet known. In our greenhouse experiment, the observed phosphorus distribution 409 between roots and leaves, with respect to increasing external Zn, suggested that the 410 upward translocation of P could be being affected by the excess of the metal. However, 411 despite several studies reporting the antagonistic effect of these elements (Kabata412 Pendias and Pendias, 2001), the P concentrations recorded in tissues of H. portulacoides 413 in this study did not suffer a marked decrease with increasing external Zn. Päivöke 414 (2003) also found a reduction of root P content with increasing Zn supply in seedling 415 and adult plants of Pisum sativum, while shoot concentrations remained unaltered. 416 Moreover, metals can replace Ca ions at essential sites on the cell membranes, and 417 metal-induced changes in membrane properties affect K+ and H+ extrusion, as well as 418 the function of membrane carriers and ion channels (Breckle and Kahle, 1991; El-Enany 419 et al., 2000; Janicka-Russak et al., 2008). All these effects, leading to an increase in 420 non-specific membrane permeability, could be responsible for the reported imbalances 421 in Ca, Na and K concentrations in tissues of H. portulacoides. Several field and 422 18 greenhouse studies have found similarly elevated Na concentrations in plants growing 423 in high metal conditions (e.g. Päivöke, 2002; Steinnes et al., 2000). 424 Zinc toxicity inhibits photosynthesis at various stages and through different 425 mechanisms (Chaney, 1993; Van Assche and Clijsters, 1986). The net photosynthesis 426 rate (A) in H. portulacoides was unaltered when exposed to Zn levels of up to 70 mmol 427 l-1, a value that is considerably higher than those recorded by several authors. For 428 example, Vaillant et al. (2005) reported that the photosynthesis of four Datura species 429 decreased at 2.5 mmol l-1 Zn in the nutrient solution, and Mateos-Naranjo et al. (2008b) 430 found that external Zn concentrations from 10 mmol l-1 caused marked reductions in A 431 in the salt-marsh cordgrass Spartina densiflora. There were no clear effects of Zn on 432 stomatal conductance in our experiment, thus indicating that the decline in A may be 433 attributed to non-stomatal limitations. Zinc may decrease the activity of enzymes 434 involved in C fixation (Mysliwa-Kurdziel et al., 2004), and thus the increase of 435 intercellular CO2 concentration (Ci) found in plants exposed to the highest external 436 concentrations of Zn in this study may be explained by modifications in RuBisCO 437 activities. Inhibition of enzyme activity in the presence of heavy metals could be due to 438 substitution of Mg2+ in the active site of RuBisCO subunits by metals ions (Siedlecka 439 and Krupa, 2004). In this study, we recorded a modification in the Zn/Mg concentration 440 ratio, caused by an increase in Zn concentration while Mg remained constant, which 441 could be related to a decrease in RuBP carboxylase. 442 Several studies have reported a direct effect of zinc on the photosynthetic 443 electron transport chain (e.g. Di Baccio et al., 2009; Vaillant et al., 2005), which may be 444 associated with a substantial stress response. In our experiment, maximum quantum 445 efficiency of PSII (Fv/Fm) did show a significant reduction at midday compared to the 446 values recorded at dawn. At midday, the measured reduction in Fv/Fm values indicated 447 19 that H. portulacoides had experienced photoinhibition at the higher light flux. The 448 quantum efficiency of PSII (ΦPSII) also showed this difference between sampling 449 times. ΦPSII decreased as a consequence of the increase in NPQ, indicating that the 450 plants dissipated light as heat, thus protecting the leaf from light-induced damage 451 (Maxwell and Johnson, 2000). Fv/Fm and ΦPSII were significantly affected by Zn 452 concentrations above 70 mmol l-1, suggesting that an excess of zinc enhances the 453 photoinhibition induced by light stress. 454 Photoinhibition is caused by damage to photosynthetic components; the effect 455 can be short-term and reversible (dynamic photoinhibition), or long-term and 456 irreversible (chronic photoinhibition; Werner et al., 2002). Values of dawn Fv/Fm at the 457 highest Zn external concentration remained lower than control parameters for unstressed 458 plants (Björkman and Demmig, 1987), revealing chronic photoinhibition or 459 photodamage. This decline in Fv/Fm was caused by lower values of Fm, which indicated 460 a decrease in the proportion of open reaction centres (Maxwell and Johnson, 2000), 461 possibly due to the recorded decrease in the concentration of chlorophyll. The 462 involvement of trace metals in thylakoyd reactions and Chl synthesis has been 463 documented by several authors (Küpper et al., 1998, 2002; von Wettstein et al., 1995). 464 Several studies have similarly demonstrated reductions in photosynthetic pigment 465 content caused by elevated metal concentrations (e.g. Cambrollé et al., 2011b; 466 Redondo-Gómez et al., 2011). Our results suggest that the Zn-induced decline in the 467 photosynthetic function of H. portulacoides may be attributed to the adverse effect of 468 the excess of this metal on photosynthetic electron transport, as a consequence of either 469 the decrease in chlorophyll synthesis or the increase in its degradation. 470 471 472 20 5. Conclusions 473 474 The photosynthetic function of H. portulacoides was negatively affected by Zn 475 concentrations above 70 mmol l-1 in the nutrient solution. In this manner, and despite the 476 fact that Zn concentrations of between 100 and 500 mg Zn kg-1 DW in mature leaf 477 tissue are generally considered excessive or toxic (Kabata-Pendias and Pendias, 2001), 478 growth parameters were practically unaffected by leaf tissue concentrations as high as 479 1500 mg Zn kg-1 dry mass. Moreover, this species survived with leaf concentrations of 480 up to 2300 mg Zn kg-1 dry mass, when treated with 9000 mg Zn l-1 (130 mmol l-1). This 481 study of the physiological responses of H. portulacoides to experimental Zn treatments 482 ranging from 0 to 130 mmol l-1 (0-9000 ppm) has provided new insights into the heavy 483 metal tolerance of this salt-marsh shrub. 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