Zinc tolerance and accumulation in the halophytic species Juncus acutus

1 Zinc tolerance and accumulation in the halophytic species Juncus acutus 1 2 Enrique Mateos-Naranjo a*, Eloy M. Castellanosb, Alfonso Perez-Martin 4 c 3 aDepartamento de Biología Vegetal y Ecología, Facultad de Biología, Universidad de 5 Sevilla, 1095, 41080, Sevilla, Spain 6 bDepartamento de Biología Ambiental y Salud Pública, Facultad de Ciencias 7 Experimentales, Universidad de Huelva, 21071, Huelva, Spain 8 c 11 Instituto de Recursos Naturales y Agrobiología de Sevilla (IRNAS), CSIC, Reina 9 Mercedes 10, 41012, Sevilla, Spain 10 *Corresponding author: 12 Enrique Mateos-Naranjo 13 Dpto. Biología Vegetal y Ecología, Facultad de Biología, Universidad de Sevilla, Av 14 Reina Mercedes s/n, 41012 Sevilla, Spain. 15 E-mail: [email protected] 16 Tel.: +34-95-4557495; fax: +34-95-4615780 17 18 19 20 21 22 23 2 ABSTRACT 24 25 The research on species with capacity to tolerate and accumulate zinc is of 26 paramount importance for phytoremediation purposes. An experiment was designed to 27 investigate the effect of Zn from 0 to 100 mmol l-1 on the growth, photosynthetic 28 apparatus and nutrient uptake of the halophytic species Juncus acutus. Gas exchange, 29 chlorophyll fluorescence and photosynthetic pigments concentration were measured. 30 We also determined total zinc, magnesium, potassium, phosphorus and sodium 31 concentrations, as well as C/N ratio. J. acutus showed high tolerance to Zn-induced 32 stress, since all plants survived and none of them showed any toxicity symptoms, such 33 as chlorosis, necrosis or growth reduction at concentrations up to 100 mmol l-1 Zn. The 34 integrity and functionality of the photosynthetic apparatus were unaffected even at zinc 35 concentrations greater than 500 mg Kg-1 on tillers. Likewise, nutrient absorption was 36 relatively unaffected. Zn tolerance was associated with the capacity to accumulate Zn in 37 roots (with values up to 2500 mg Kg-1 41 ) and largely avoid its transport to tillers. These 38 characteristics, along with its ability to establish in a wide variety of ecosystems, render 39 this species a useful phytostabilizer for revegetation of Zn-contaminated lands. 40 42 43 Keywords: Growth response; metal toxicity; nutrient absorption; photosynthesis; Zn-44 stress; photoinhibition. 45 46 3 1. Introduction 47 48 Environmental pollution by heavy metals is a serious problem worldwide, 49 increasing in parallel with the development of human technology. Government, the 50 industry and the public now recognize the potential dangers that metals pose to human 51 health (Duruibe et al., 2007) through the food chain and the health of terrestrial and 52 aquatic communities and ecosystems (Kabata-Pendias and Pendias, 2001). The danger 53 of toxic metals is aggravated by their immutable nature and indefinite persistence in the 54 environment (Garbisu and Alkorta, 2001; Aycicek et al., 2008). Among heavy metals, 55 Zn is considered the main industrial pollutant of both terrestrial and aquatic 56 environments (Barak and Helmke, 1993) and has the greatest mobility and 57 bioavailability of all elements (Morillo et al., 2004). Although Zn is an essential 58 microelement with many roles in plant metabolism (Kabata-Pendias and Pendias, 2001), 59 its excess can lead to toxic effects in plants (Chaney, 1993), with specific effects on the 60 Calvin cycle and photosystem activity (Van Assche and Clijsters, 1986). 61 Many remediation strategies have been considered to counter the detrimental 62 effects of Zn excess, including physical, chemical and biological methods that 63 immobilize or remove metals from the environment (Marques et al., 2011). 64 Phytoremediation has recently gained importance on account of its cost-effective, long-65 term applicability and because it is an ecofriendly, promising clean-up solution for a 66 wide variety of contaminated sites (Weis and Weis, 2004). This methodology depends 67 on the use of plants to act upon the contaminants, by extracting, degrading or 68 immobilizing them (Marques et al., 2011). The research on species which can be useful 69 in metal phytoremediation has become a major issue (Zhang et al., 2010) and these 70 4 species should be chosen on the basis of their capacity to tolerate and accumulate 71 particular contaminants (Marques et al., 2011). 72 There exists a wide variation in sensitivity to metal exposure. However, exists a 73 lack of knowledge about metal toxicity thresholds for native plant species (Ross and 74 Kaye, 1994) and for species used to restore sites contaminated by heavy metals, such as 75 salt marshes. Species of genus Juncus have been employed in wetland restoration 76 projects around the world (Sparks et al., 2013; Marques et al., 2011), but the 77 information on the tolerance and accumulation patterns of heavy metals in these species 78 is really scarce. The present study is focused on the species Juncus acutus L., a 79 halophytic densely caespitose plant with subcosmopolitan distribution that is common 80 in Spanish coastal marsh communities and can be found growing in sediments 81 containing 100–4800 ppm Zn in several estuaries of the Iberian Peninsula (Sáinz and 82 Ruiz, 2006). Moreover, this species has a wide ecological range, tolerating soils with 83 high levels of sulphates and chlorides (Fernández-Carvajal, 1982) and soils with a sandy 84 texture and hydric stress during the dry summer season. Our hypothesis is that all these 85 circumstances highlight the potential of J. acutus to be used for metal remediation in 86 polluted areas. However, no studies have analyzed its growth and physiological 87 responses to zinc excess. 88 The aim of this study was to evaluate the tolerance of J. acutus to elevated 89 concentration of zinc in relation of its survival, growth and photosynthetic response, and 90 quantify the capacity of this species for accumulating this element. 91 92 2. Materials and Methods 93 94 5 2.1. Plant material 95 96 Seeds of J. acutus were collected in December 2011 from the natural marshes of 97 Doñana National Park (37º 15´ N - 6º 58´W; SW Spain) and stored at 4ºC (in darkness) 98 for three months. After that, seeds were placed into a germinator for a month (ASL 99 Aparatos Científicos M-92004, Madrid, Spain) and subjected to an alternating diurnal 100 regime of 16 h of light (photon flux rate, 400-700 nm, 35 μmol m-2 s-1) at 25ºC and 8 h 101 of darkness at 12ºC. Seedlings were then planted in individual plastic pots (11 cm of 102 diameter) filled with perlite and placed in a glasshouse with controlled temperature of 103 21-25ºC, 40-60% relative humidity and natural daylight (minimum and maximum light 104 flux: 250 and 1000 μmol m-2 s-1 108 respectively). Pots were carefully irrigated with 20% 105 Hoagland's solution (Hoagland and Arnon, 1938) as necessary. All the pots received the 106 same irrigation. 107 2.2. Stress treatments 109 110 In October 2012, after five months of seedling culture, the pots (with between 5 111 and 6 tillers) were randomly allocated still inside the glasshouse to five Zn treatments 112 (six pots per tray, one tray per Zn treatment): 0, 10, 30, 60 and 100 mmol l-1 Zn. The 113 treatment with 0 mmol l-1 Zn was considered the control treatment. Zinc treatments 114 were established by combining 20% Hoagland´s solution and ZnSO4·7H2O of the 115 appropriate concentration. The control, 0 mmol l-1 Zn treatment, had exactly 0.002 116 mmol l-1 Zn, as Hoagland´s solution contains a small amount of Zn as an essential trace 117 nutrient. Zn concentrations were chosen to cover variations recorded by Sáinz and Ruiz, 118 (2006) in the salt marshes of the joint estuary of the Tinto and Odiel Rivers. 119 6 At the beginning of the experiment, 1 l of appropriate solution was placed in each 120 tray (Hoagland al 20% + ZnSO4.7H2O) to a depth of 1cm. During the experiment, the 121 levels of trays were monitored and topped up to the marked level with 20% Hoagland´s 122 solution (without additional ZnSO4.7H2O) to limit the change of Zn concentration 123 caused by water evaporation from the nutrient solution. Also, the entire solution 124 (including ZnSO4.7H2 126 O) was changed every three days. 125 2.3. Growth analysis 127 128 At the beginning and the end of the experiment (after 50 days of treatment) three 129 and five entire plants, respectively, from each treatment were dried at 80ºC for 48 h and 130 weighed. Also, before and after the Zn treatment, the number and height of all fully 131 developed tillers were measured. 132 The relative growth rate (RGR) in ash-free dry mass of whole plants was 133 calculated using the formula: 134 135 RGR = (ln Bf – ln Bi) · D-1 (g g-1 day-1 137 ) 136 where Bf = final dry mass, Bi 141 = initial dry mass (an average of the three plants 138 from each treatment dried at the beginning of the experiment) and D = duration of 139 experiment (days). 140 2.4. Gas exchange 142 143 Measurements were taken on random, fully developed photosynthetic tillers (n = 144 7 10, two measurements per plant) using an infrared gas analyser in an open system (LI-145 6400, Li-Cor Inc., Lincoln, NE, USA) after 50 days of treatment. Maximum net 146 photosynthetic rate (A), intercellular CO2 concentration (Ci) and stomatal conductance 147 to CO2 (Gs) were determined at CO2 concentration of 400 µmol CO2 mol-1 air, 148 temperature of 25-30ºC, 42.4 ± 0.4% relative humidity and a photon flux density of 149 1000 µmol m-2 s-1 once a steady-state was reached. A, Ci and Gs were calculated using 150 standard formulas of Von Caemmerer and Farquhar (1981). Photosynthetic area was 151 approximated as the area of a cylinder. Intrinsic water use efficiency (WUEi) was 152 calculated as the ratio between A and Gs 154 . 153 2.5. Tiller water content 155 156 Tiller water content (TWC) was calculated after 50 days of treatment as (n = 5, 157 one measurement per plant): 158 159 TWC = (FW – DW)/FW × 100 160 161 where FW is the fresh mass of the tillers and DW is the dry mass after oven-162 drying at 80ºC for 48 h. 163 164 2.6. Photosynthetic pigments 165 166 At the end of the experimental period, photosynthetic pigments in fully 167 developed, photosynthetic tillers (n=5) from each treatment were extracted using 0.05 g 168 of fresh material in 10 ml of 80% aqueous acetone. After filtering, 1 ml of the 169 8 suspension was diluted with a further 2 ml of acetone and chlorophyll a (Chl a), 170 chlorophyll b (Chl b) and carotenoid (Cx+c) contents were determined with a Hitachi 171 U-2001 spectrophotometer (Hitachi Ltd., Japan) using three wavelengths (663.2, 646.8 172 and 470.0 nm). Concentrations of pigments (µg gfwt-1 175 ) were obtained through 173 calculation following Lichtenthaler (1987). 174 2.7. Measurement of chlorophyll fluorescence 176 177 Chlorophyll fluorescence was measured using a portable modulated fluorimeter 178 (Mini-PAM, Heinz Walz, Germany) after 50 days of treatment, in tillers similar to those 179 used previously. Measurements were made on each plant in the five zinc treatments (n = 180 10, two measurements per plant). Light and dark-adapted fluorescence parameters were 181 measured at dawn (stable 75 µmol m-2 s-1 ambient light) and at midday (1500 µmol m-2 182 s-1 Plants were dark-adapted for 30 minutes using leaf–clips designed for this 185 purpose. The minimal fluorescence level in the dark-adapted state (F ) to investigate whether zinc concentration affected the sensitivity of plants to 183 photoinhibition (Qiu et al., 2003). 184 0) was measured 186 using a modulated pulse (<0.05 µmol m-2 s-1 for 1.8 µs) too small to induce significant 187 physiological changes in the plant (Schreiber et al., 1986). The data stored were an 188 average taken over a 1.6 seconds period. Maximal fluorescence level in this state (Fm) 189 was measured after applying a saturating actinic light pulse of 10000 µmol m-2 s-1 for 190 0.8 s (Bolhàr-Nordenkampf and Öquist, 1993). The value of Fm was recorded as the 191 highest average of two consecutive points. Values of the variable fluorescence (Fv = Fm 192 - F0) and maximum quantum efficiency of PSII photochemistry (Fv/Fm) were 193 9 calculated from F0 and Fm The same tiller section of each plant was used to measure light-adapted 196 parameters. Steady state fluorescence yield (F . This ratio of variable to maximal fluorescence can be used 194 to quantify photoinhibition (Maxwell and Johnson, 2000). 195 s) was recorded under ambient light 197 conditions. A saturating actinic light pulse of 10000 µmol m-2 s-1 for 0.8 s was then used 198 to produce the maximum fluorescence yield (Fm Using fluorescence parameters determined in both lightand dark-adapted states, 201 the following were calculated: quantum efficiency of PSII (Φ ') by temporarily inhibiting PSII 199 photochemistry. 200 PSII = (Fm' – Fs)/ Fm') 202 (Genty et al., 1989); photochemical quenching (qP = (Fm' – Fs) / (Fm' – F0'), where F0' 203 corresponds to open reaction center traps in the light-acclimated state), and non-204 photochemical quenching (NPQ = (Fm – Fm') / Fm 208 '; Schreiber et al., 1986). 205 Photochemical quenching gives an indication of the proportion of PSII reaction centres 206 that are open (Maxwell and Johnson, 2000). 207 2.8. Chemical analyses of plant tissue samples 209 210 In accordance with protocols of Mateos-Naranjo et al. (2008), at the end of the 211 experiment, tiller and root samples were dried at 80ºC for 48 h and ground. Tillers and 212 roots were carefully washed with distilled water before any further analysis. Then, 0.5 g 213 samples from tillers and roots (taken from five plants per treatment) were digested in 214 triplicate with 6 ml HNO3, 0.5 ml HF and 1 ml H2O2. Ca, Mg, K, P, Na and Zn 215 concentrations in tillers and roots were measured by inductively coupled plasma (ICP) 216 spectroscopy (ARL-Fison 3410, USA). Total N and C concentrations were determined 217 for undigested dry samples with an elemental analyzer (Leco CHNS-932, Spain). 218 16 the long-term effects of the highest Zn concentration on the growth rate of J. acutus 368 could be due to the different development of the photosynthetic area rather than to 369 variations in net photosynthetic rate. Hence, similar rates of CO2 assimilation could be 370 more than compensated for by a greater photosynthetic area in low Zn concentration. 371 This response might provide positive feedback, since larger photosynthetic areas would 372 induce higher growth rates which would in turn induce more photosynthetic area, 373 amplifying the difference between plants at different zinc concentrations over time. 374 Thus, in our experiment, the largest differences in RGR were related to variations in the 375 number of tillers, which might be related to differences in photosynthetic area and hence 376 to reduction in light interception. In line with our results, Delperee and Lutts (2008) also 377 found that growth inhibition was not correlated with CO2 assimilation rate for Solanum 378 lycopersicum under cadmium stress conditions. This was explained by the presence of 379 several mechanism of tolerance related with oxidative stress control and the protection 380 of photosystems. It is possible that J. acutus used the same protection system. In this 381 respect, the hypertolerance of J. acutus to Zn stress was also reflected in the integrity 382 and functionality of its photochemical apparatus. Several studies have reported a direct 383 effect of zinc on the photosynthetic electron transport chain (Vaillant et al., 2005; 384 Mateos-Naranjo et al., 2008), which may be associated with a substantial stress 385 response. Our data showed that Fv/Fm values were always lower at midday than at 386 dawn, a fact that indicated that J. acutus experienced some degree of dynamic 387 photoinhibition at the higher light flux. Several authors have defined dynamic 388 photoinhibition as a reversible mechanism controlling the dissipation of excess 389 luminous energy by means of thermal dissipation (NPQ), which is in agreement with 390 the greater NPQ at midday than at dawn in our data. This was supported by the lower 391 ΦPSII at dawn than at midday, a decrease due to the increase in NPQ, which indicates 392 17 that the plants dissipate light as heat, thereby protecting the leaf from light-induced 393 damage (Maxwell and Johnson, 2000). Dawn values of Fv/Fm On the other hand, the reduction in the absorption of essential mineral elements 397 has been described as one of the effects of heavy metals on plants (Chaney, 1993; 398 Kabata-Pendias and Pendias, 2001). In this regard, our mineral nutrient analyses 399 indicated that the presence of zinc in nutrient solution did not generate large nutritional 400 imbalance in J. acutus plants, especially in tillers tissues, although root and tillers Ca 401 and Mg concentrations were lower at the highest zinc level. The interactions Zn-Ca and 402 Zn-Mg have been previously described by several authors (Kabata-Pendias and Pendias, 403 2001). Thus, the reduction in Mg concentration in tillers could be linked with a 404 decrease in chlorophyll content recorded in this experiment, since the most familiar role 405 of Mg in photosynthesis is as the central atom of the chlorophyll molecule (Shaul, 406 2002). Finally, Na concentration for tillers and roots increased with external Zn 407 concentration. Redondo-Gómez et al. (2011) determined that the accumulation of Na in 408 the tissues of Spartina densiflora favored recovery of the photosynthetic apparatus of 409 this species against zinc excess. This result is linked with the high integrity showed by 410 photosynthetic apparatus of J. acutus to Zn stress. 411 were close to optimal 394 values for unstressed plants (approximately 0.84; Bjorkman and Demmig, 1987), this 395 fact revealing no presence of chronic and irreversible photoinhibition. 396 412 5. Conclusions 413 414 J. acutus shows a high tolerance to zinc-induced stress, as proved the fact that all 415 plants were able to survive and did not show any visible Zn toxicity symptoms, such as 416 chlorosis, necrosis or a strong growth inhibition at concentrations up to 100 mmol l-1 417 18 Zn. Likewise, unaffected photosynthesis and efficiency of PSII photochemistry 418 apparatus might indicate that J. acutus is not experiencing metal toxicity, despite the 419 fact that Zn concentrations recorded in its tillers tissues (> 500 mg Kg-1) were greater 420 than toxicity thresholds recorded for plants. Furthermore, Zn excess did not affect water 421 relations of this species and overall absorption of essential mineral elements. All these 422 results suggest that J. acutus is a hypertolerant species to zinc. Moreover, the capacity 423 of this species to accumulate great amount of Zn in its roots (> 2500 mg Kg-1 430 Zn) could 424 be accounted for by the development of such mechanisms as compartmentation, which 425 could control the ion transport into tillers, thereby improving its tolerance to Zn. 426 Consequently, the hypertolerance to zinc proved by these results, together with its 427 ability to establish in a wide variety of ecosystems, reflect that this species is suitable as 428 a phytostabilizer for revegetation of Zn-contaminated lands. 429 19 Acknowledgements 431 432 We are grateful to Antonio J. Ruiz Rico for revision of the English text of this 433 manuscript, Carmen Carrasco for technical assistance and Antonio Diaz-Espejo for his 434 altruistic help in lending some of the equipment. We also thank the Spanish Science and 435 Technology Ministry for its support (project CTM2008-04453) and Seville University 436 Glasshouse General Services for their collaboration. 437 438 439 20 References 440 441 Aycicek, M., Kaplan, O., Yaman, M., 2008. Effect of cadmium on germination, 442 seedling growth and metal contents of sunflower (Helianthus annuus L.). Asian J. 443 Chem. 20, 2663-2672. 444 Barak, P., Helmke, P.A., 1993. The chemistry of Zinc. In: Robson, A. (Ed.), Zinc in 445 Soils and Plants, Developments in Plants and Soil Sciences. Kluwer Academic 446 Press, New York, pp. 1-13. 447 Björkman, O., Demming, B., 1987. Photon yield of O2 Bolhàr-Nordenkampf, H.R., Öquist, G., 1993. Chlorophyll fluorescence as a tool in 451 photosynthesis research. In: Hall, D.O., Scurlock, J.M.O., Bolhàr-Nordenkampf, 452 H.R., Leegood, R.C., Long, S.P. (Ed.), Photosynthesis and Production in a 453 Changing Environment: a Field and Laboratory Manual. Chapman & Hall, 454 Londres, pp. 193-206. 455 evolution and chlorophyll 448 fluorescence characteristics at 77K among vascular plants of diverse origins. 449 Planta 170, 489-504. 450 Cambrolle, J., Mancilla-Leyton, J.M., Muñoz-Valles, S., Figueroa-Luque, E., Luque, 456 T., Figueroa, M.E., 2013. Evaluation of zinc tolerance and accumulation potential 457 of the coastal shrub Limoniastrum monopetalum (L) Boiss. Environ. Exp. Bot. 85, 458 5057. 459 Chaney, R.L., 1993. Zinc phytotoxicity. In: Robson, A.D. (Ed.), Zinc in Soils and 460 Plants. Kluwer Academic Publishers, Londres, pp. 131-150. 461 Delpérée, C., Lutts, S., 2008. Growth inhibition occurs independently of cell mortality 462 in tomato (Solanum lycopersicum) exposed to high cadmium concentrations. J. 463 Integr. Plant. Biol. 50, 300-310. 464 21 Duruibe, J.O., Ogwuegbu, M.O.C., Egwurugwu, J.N., 2007. Heavy metal pollution and 465 human bio toxic effects. Int. J. Phys. Sci. 2, 112–118. 466 Evangelou, M.W., Daghan, H., Schaeffer, A., 2004. The influence of humic acids on 467 the phytoextraction of cadmium from soil. Chemosphere 57, 207–213. 468 Fernández-Carvajal, M.C., 1982. Revisión del género Juncus L. en la Península Ibérica 469 III. Anales del Jardín Botánico de Madrid, Madrid. 470 Fitzgerald, E.J., Caffrey, J.M., Nesaratnam, S.T., McLoughlin, P., 2003. Copper and 471 lead concentrations in salt marsh plants on the suir Estuary, Ireland. Environ. 472 Pollut. 123, 67-74. 473 Flexas, J., Ribas-Carbo, M., Diaz-Espejo, A., Galmes, J., Medrano, H., 2008. Mesophyll 474 conductance to CO2 Garbisu, C., Alkorta, I., 2001. Phytoextraction: A cost-effective plant-based technology 477 for the removal of metal from the environment. Bioresource Technol. 77, 229–478 236. 479 : current knowledge and future prospects. Plant Cell Environ. 475 31, 602-621. 476 Genty, B., Briantais, J.M., Baker, N.R., 1989. The relationship between the quantum 480 yield of photosynthetic electron transport and quenching of chlorophyll 481 fluorescence. BBA-Gen Subjects 990, 87-92. 482 Hoagland, D., Arnon, D.I., 1938. The water culture method for growing plants without 483 soil. Cal. Agric. Exp. Sta. Bull. 347, 1-39. 484 Kabata-Pendias, A., Pendias, H., 2001. Trace elements in soils and plants. CRC Press, 485 Boca Ratón, Florida. 486 Lichtenthaler, H.K., 1987. Chlorophylls and carotenoids: pigments of photosynthetic 487 biomembranes. Method Enzymol. 148, 350-382. 488 22 Marques, B., Lillebo, A.L., Pereira, E., Duarte, A.C., 2011. Mercury cycling and 489 sequestration in salt marshes sediments: An ecosystem service provided by Juncus 490 maritimus and Scirpus maritimus. Environ. Pollut. 159, 1869–1876. 491 Mateos-Naranjo, E., Redondo-Gómez, S., Cambrollé, J., Luque, T., Figueroa, M.E., 492 2008. Growth and photosynthetic responses to zinc stress of an invasive 493 cordgrass, Spartina densiflora. Plant Biol. 10, 754–762. 494 Matthews, D.J., Moran, B.M., Otte, M.L., 2004. Zinc tolerance, uptake, accumulation in 495 the wetland plants Eriophorum angustifolium, Juncus effusus and Juncus 496 articulatus. Wetlands 24, 859-869. 497 Maxwell, K., Johnson, G.N., 2000. Chorophyll fluorescencea practical guide. 498 J. Exp. Bot. 51, 659-668. 499 Melis, A., 1999. Photosystem-II damage and repair cycle in chloroplasts: what 500 modulates the rate of photodamage in vivo?. Trends Plant. Sci. 4, 130-135. 501 Morillo, J., Usero, J., Gracia, I., 2004. Heavy metal distribution in marine sediments 502 from the southwest coast of Spain. Chemosphere 55, 431-442. 503 Paschke, M.W., Redente, E.F., Levy, D.B., 2000. Zinc toxicity thresholds for important 504 reclamation grass species of the western United States. Environ. Toxicol. Chem. 505 19, 2751-2756. 506 Paschke, M.W., Perry, L.G., Redente, E.F., 2006. Zinc toxicity thresholds for 507 reclamation forb species. Water Air Soil Poll. 170, 317-330. 508 Perez-Martin, A., Flexas, J., Ribas-Carbó, M., Bota, J., Tomás, M., Infante, J.M., Diaz-509 Espejo, A., 2009. Interactive effects of soil water deficit and air vapour pressure 510 deficit on mesophyll conductance to CO2 in Vitis vinifera and Olea europaea. J. 511 Exp. Bot. 60, 2391–2405. 512 23 Pollard, A.J., Powell, K.D., Harper, F.A., Smith, J.A.C., 2002. The genetic basis of 513 metal hyperaccumulation in plants. Crit. Rev. Plant Sci. 21, 539–566. 514 Qiu, N., Lu, Q., Lu, C., 2003. Photosynthesis, photosystem II efficiency and the 515 xanthophyll cycle in the saltadapted halophyte Atriplex centralasiatica. New 516 Phytol. 159, 479486. 517 Redondo-Gómez, S., Mateos-Naranjo, E., Vecino-Bueno, I., Feldman, S.R., 2011. 518 Accumulation and tolerance characteristics of chromium in a cordgrass Cr 519 hyperaccumulator, Spartina argentinensis. J. Hazard Mater. 185, 862-869. 520 Ross, S.M., Kaye, K.J., 1994. The meaning of metal toxicity in soil-plant systems. In: 521 Ross, S.M., (Ed.), Toxic metals in soil-plant systems. Wiley, New York, pp. 27-522 71. 523 Sáinz, A., Ruiz, F., 2006. Influence of the very polluted inputs of the Tinto-Odiel 524 system on the adjacent littoral sediments of southwestern spain: A statistical 525 approach. Chemosphere 62, 1612-1622. 526 Schreiber, U., Schliw,a W., Bilger, U., 1986. Continuous recording of photochemical 527 and non-photochemical chlorophyll fluorescence quenching with a new type of 528 modulation fluorimeter. Photosynth Res. 10, 51-62. 529 Shaul, O., 2002. Magnesium transport and function in plants: the tip of the iceberg. 530 Biometals, 15, 309–323. 531 Sparks, E.L., Cebrian, J., Bilber, P.D., Sheehan, K.L., Tobias, C.R., 2013. Cost-532 effectiveness of two small-scale salt marsh restoration designs. Ecol. Eng. 53, 533 250-256. 534 24 Stefani, A., Arduini, I., Onnis, A., 1991. Juncus acutus: germination and initial growth 535 in presence of heavy metals. Ann. Bot. Fenn. 28, 37-43. 536 Tripathi, R.D., Srivastava, S., Mishra, S., Singh, N., Tuli, R., Gupta, D.K., Maathuis, 537 J.M., 2007. Arsenic hazards: Strategies for tolerance and remediation by plants. 538 Trends Biotechnol. 25, 158–165. 539 Vaillant, N., Monnet, F., Hitmi, A., Sallanon, H., Coudret, A., 2005. Comparative study 540 of responses in four Datura species to zinc stress. Chemosphere 59, 1005–1013. 541 Van Assche, F.V., Clijsters, H., 1986. Inhibition of photosynthesis by treatment of 542 Phaseolus vulgaris with toxic concentration of zinc: effects on electron transport 543 and photophosphorylation. Physiol. Plantarum 66, 717-721. 544 Verkleij, J.A., Schat, H., 1990. Mechanism of metal tolerance in higher plants. In: 545 Shaw, A.J. (Ed.), Heavy metal tolerance in plants: evolutionary aspects. CRC 546 Press, Boca Raton, pp. 179-193. 547 Von Caemmerer, S., Farquhar, G.D., 1981. Some relationships between the 548 biochemistry of photosynthesis and the gas exchange of leaves. Planta 153, 377-549 387. 550 Weis, J.S., Weis, P., 2004. Metal uptake, transport and release by wetland plants: 551 implications for phytoremediation and restoration. Environ. Inter. 30, 687-700. 552 Zhang, X.S., Xia, H.P., Li, Z.A., Zhuang, P., Gao, B., 2010. Potential of four forage 553 grasses in remediation of Cd and Zn contaminated soils. Bioresource Technol. 554 101, 2063-2066. 555 556 557 25 Figure captions 558 559 Fig. 1. Growth analysis of Juncus acutus in response to treatment with a range of Zn 560 concentrations for 50 d. Total dry mass (A); relative growth rate, RGR (B); number of 561 tillers (C) and mean height of tillers (D). Values represent mean ± SE, n = 5. Different 562 letters indicate means that are significantly different from each other (Tukey test, P < 563 0.05). 564 Fig. 2. Chlorophyll a, Chl a (A); Chlorophyll b, Chl b (B) and carotenoids, Cx+c (C) 565 concentrations in randomly selected, fully developed photosynthetic tiller of Juncus 566 acutus in response to treatment with a range of Zn concentrations for 50 d.Values 567 represent mean ± SE, n = 5. Different letters indicate means that are significantly 568 different from each other (Tukey test, P < 0.05). 569 Fig. 3. Maximum quantum efficiency of PSII photochemistry, Fv/Fm (A); quantum 570 efficiency of PSII, ΦPSII Fig. 4. Concentration of Zn (A); calcium, Ca (B); sodium, Na (C); potassium, K (D); 576 phosphorus, P (E) and magnesium, Mg (F) in tillers (○) and roots (●) of Juncus acutus 577 in response to treatment with a range of Zn concentrations for 50 d. Values represent 578 mean, n = 5. Different letters indicate means that are significantly different from each 579 other (Tukey test, P < 0.05). 580 (B) and non-photochemical quenching, NPQ (C) at dawn (○) 571 and at midday (●) in randomly selected, fully developed photosynthetic tillers of Juncus 572 acutus in response to treatment with a range of Zn concentrations for 50 d. Values 573 represent mean ± SE, n = 10. Different letters indicate means that are significantly 574 different from each other (Tukey test, P < 0.05). 575 Fig. 5. C/N ratio for tillers (○) and roots (●) of Juncus acutus in response to treatment 581 with a range of Zn concentrations for 50 d. Values represent mean, n = 5. Different 582