Salinity alleviates zinc toxicity in the saltmarsh zinc-accumulator Juncus acutus

1 Salinity alleviates zinc toxicity in the saltmarsh zinc-accumulator Juncus acutus 1 Enrique Mateos-Naranjoa,*; Jesús Alberto Pérez-Romeroa; Susana Redondo-Gómeza; 2 Jennifer Mesa-Marína; Eloy Manuel Castellanosb; Anthony John Davyc 3 4 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 cCentre for Ecology, Evolution and Conservation, School of Biological Sciences, 9 University of East Anglia, Norwich Research Park, Norwich NR4 7TJ, UK 10 11 *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 2 ABTRACT 19 20 The potential importance of Juncus acutus for remediation of Zn-contaminated 21 lands has been recognized, because of its Zn tolerance and capacity to accumulate Zn. 22 Since it is also a halophyte, the extent to which salinity influences its Zn tolerance 23 requires investigation. A factorial greenhouse experiment was designed to assess the 24 effect of NaCl supply (0 and 85 mM NaCl) on the growth, photosynthetic physiology 25 and tissue ions concentrations of plants exposed to 0, 30 and 100 mM Zn. Our results 26 indicated that NaCl supplementation alleviated the effects of Zn toxicity on growth, as 27 Zn at 100 mM reduced relative growth rate (RGR) by 60% in the absence of NaCl but 28 by only 34% in plants treated also with NaCl. This effect was linked to a reduction in 29 Zn tissue concentrations, as well as to overall protective effects on various stages in the 30 photosynthetic pathway. Thus, at 85 mM NaCl plants were able to maintain higher net 31 photosynthesis (AN) than in the absence of added NaCl, although there were no 32 differences in stomatal conductance (gs). This contributed to preserving the trade-off 33 between CO2 acquisition and water loss, as indicated by higher intrinsic water use 34 efficiency (iWUE). Hence, AN differences were ascribed to limitation in the RuBisCO 35 carboxylation, manifested as higher intercellular CO2 concentration (Ci), together with 36 dysfunction of PSII photochemistry (in term of light harvest and energy excess 37 dissipation), as indicated by higher chronic photoinhibition percentages and variations 38 in the photosynthetic pigment profiles in presence of Zn under non-saline conditions. 39 40 Keywords: Chlorophyll fluorescence; Gas exchange; Halophyte; Photoinhibition; 41 Salinity; Zn-stress. 42 3 1. Introduction 43 Juncus acutus L., is a caespitose, halophytic rush, with a sub-cosmopolitan 44 distribution, that inhabits coastal marshes and dune slacks encompassing a wide range 45 of salinity (Fernández-Carvajal, 1982). Together with various other Juncus species, it 46 has been proposed as a bio-tool for wetland restoration projects around the world 47 (Sparks et al., 2013; Marques et al., 2011). In particular, it has potential for the 48 remediation of metal pollution, since it shows great tolerance to excess metals and the 49 capacity to accumulate large amounts of them in its tissues without serious symptoms of 50 toxicity (Mateos-Naranjo et al., 2014; Santos et al., 2014; Christofilopoulos et al., 51 2016). Medas et al. (2017) have recently suggested that J. acutus is able to optimize its 52 response to metal pollution by tuning different biomineralization mechanisms with the 53 minerals and geochemical conditions of the site. Previous studies of metal accumulation 54 and its effects on the performance J. acutus have focused on zinc (Mateos-Naranjo et 55 al., 2014; Santos et al., 2014; Christofilopoulos et al., 2016; Medas et al., 2017), 56 although recently interactions of Zn with Cr, Ni and Cd have also been assessed 57 (Christofilopoulos et al., 2016). 58 Zinc is an essential element for plant metabolism (Kabata-Pendias and Pendias, 59 2001). However, its excess can lead to various phytotoxicity effects on plant 60 metabolism (Chaney, 1993), and specifically on halophytic species (Liu et at., 2016). 61 The photosynthetic apparatus (i.e. Calvin cycle and photosystem functionality) is 62 especially sensitive to this ion excess (Van Assche and Clijsters, 1986). Despite such 63 potentially deleterious effects, J. acutus is regarded as Zn-hypertolerant, a feature 64 attributable to a series of physiological and biochemical adaptations. In particular, 65 Mateos-Naranjo et al. (2014) showed that carbon assimilation and the efficiency of PSII 66 were not affected by high concentrations of Zn in the culture solution. Furthermore, 67 4 Santos et al. (2014) found that maintenance of the functionality of its photosynthetic 68 apparatus was linked with its ability to overcome oxidative damage produced by excess 69 Zn uptake, through the modulation of its antioxidant enzymatic machinery and efficient 70 dissipation of the cellular redox potential consequent on Zn incorporation into 71 chlorophyll molecules. These studies however, did not take account of the potential 72 interaction of Zn with other important factors characteristic of marshes ecosystems, 73 particularly salinity. It has been demonstrated that the accumulation of sodium in 74 another halophyte, Spartina densiflora, can mitigate its responses to Zn-induced stress 75 (Redondo-Gómez et al., 2011). Hence knowledge of the extent to which salinity might 76 modulate the physiological responses of J. acutus to excess Zn is necessary for a 77 realistic assessment of its metal toxicity thresholds and its potential for the remediation 78 of zinc-polluted saltmarshes. 79 This study employed a factorial experiment which aimed to: (1) investigate the 80 influence of NaCl on the growth responses of J. acutus plants exposed to different Zn 81 concentrations; (2) determine the extent to which this influence could be accounted for 82 by impacts on its photosynthetic apparatus, both in terms of carbon assimilation and 83 efficiency of light-energy use, and (3) assess the nutrient and Zn accumulation patterns 84 consequent on the joint effects of treatment with elevated NaCl and Zn. 85 86 2. Material and Methods 87 2.1. Plant material 88 Seeds of Juncus acutus were collected in December 2013 from different 89 individuals (n = 20) randomly selected from a well-established population in Doñana 90 National Park (Huelva, SW Spain). The seeds were transported to the laboratory for 91 5 germination in a germination chamber (ASL Aparatos Científicos M- 92004, Madrid, 92 Spain) under the following conditions: photoperiod, 16/8 h light/darkness; temperature, 93 24/15ºC; photon flux rat (400–700 nm), 35 µmol m-2 s-1. Germinated seedlings were 94 immediately transferred to individual plastic pots (12 cm in depth, 0.5 L total volume) 95 filled with perlite and placed in a glasshouse (University of Seville, Greenhouse 96 Service) at controlled temperature of 25±3 ºC, and a relative humidity of 40-60%, with 97 natural day light (maximum quantum flux rate of 1000 µmol m-2 s-1). Pots were irrigated 98 with nutrient solution (Hoagland and Arnon, 1938) before the onset of the experimental 99 treatments. 100 101 2.2. Zn and NaCl experimental stress treatments 102 In June 2014, pots containing the J. acutus plants were randomly assigned to 103 three Zn treatments (concentrations of 0, 30 and 100 mM) in factorial combination with 104 two NaCl concentrations (0 and 85 mM) for 40 days. Zn and NaCl concentrations were 105 established by combining Hoagland´s solution with appropriate amounts of 106 ZnSO4·7H2O and NaCl, respectively. Thus, at the beginning of the experiment, the pots 107 were placed in plastic trays containing appropriate solutions to a depth of 1 cm (10 108 replicate pots per stress treatment combination). In order to avoid changes of Zn and 109 NaCl concentration caused by water evaporation from the nutrient solution, levels in the 110 trays were monitored continuously throughout the experimental and topped up to the 111 marked level with Hoagland’s solution (without additional ZnSO4·7H2O or NaCl). 112 Furthermore, pH of the solution was monitored and adjusted to 6.5 - 7.0. The entire 113 solution (including ZnSO4·7H2O and NaCl) in the trays was renewed weekly and their 114 6 positions were changed randomly every 2 days to avoid effects of environmental 115 heterogeneity inside the glasshouse. 116 After 40 days of exposure to the stress-inducing treatments, measurements of 117 growth, gas exchange, chlorophyll fluorescence, photosynthetic pigment concentrations 118 and tissue ion concentrations were made. 119 120 2.3. Growth measurements 121 Four plants from each treatment were harvested at the beginning of the 122 experiment and a further ten at the end. Plants were divided in roots and shoots and 123 these biomass fractions were oven dried (60ºC for 48 h) and then weighed. In addition, 124 the number of dead tillers was recorded at the end of the experiment. 125 The relative growth rate (RGR) of whole plants was calculated using the formula: 126 127 RGR = (ln Bf – ln Bi) · D-1 (g g-1 day-1) 128 129 where Bf = final dry mass, Bi = initial dry mass (the mean of the four plants from 130 each treatment sampled at the beginning of the experiment) and D = duration of 131 experiment (days). 132 133 2.4. Photosynthetic physiology 134 7 Gas exchange and chlorophyll fluorescence parameters were measured on the 135 same sections of randomly selected, fully developed photosynthetic tillers (n = 10) 136 using an infrared gas analyzer (LI-6400-XT, Li-COR Inc., NE., USA) and a modulated 137 fluorimeter (FMS-2; Hansatech Instruments Ltd., UK), respectively. The following gas 138 exchange parameters were recorded at a light flux density of 1500 µmol photons m-2 s- 139 1, ambient CO2 concentration (Ca) 400 µmol mol-1 air, leaf temperature of 25 °C and 50 140 ± 5 % relative humidity: net photosynthetic rate (AN), stomatal conductance (gs), 141 intercellular CO2 concentration (Ci), and intrinsic water use efficiency (iWUE). The 142 saturation pulse method was used to determine the energy yields of the Photosystem II 143 (PSII) reaction centers: maximum quantum efficiency of PSII photochemistry (Fv/Fm), 144 quantum efficiency of PSII (ΦPSII; Genty et al., 1989) and non-photochemical 145 quenching (NPQ). As described by Schreiber et al. (1986), a 0.8 s saturating actinic 146 light pulse of 15000 µmol m-2 s-1 was given, at dawn (stable, 50 µmol m-2 s-1 ambient 147 light) and midday (1700 µmol photons m-2 s-1), to photosynthetic tillers previously dark- 148 adapted or exposed to light for 30 min. 149 Finally, the total chlorophyll a (Chl a), chlorophyll b (Chl b) and carotenoid 150 (Cx+c) contents of extracts obtained from randomly selected fully developed 151 photosynthetic tillers (n = 5), were determined with a Hitachi U-2001 152 spectrophotometer (Hitachi Ltd., Japan), using three wavelengths (663.2, 646.8 and 153 470.0 nm). For more details, see Mateos-Naranjo et al. (2008). Concentrations of 154 pigments (µg g-1fw) were calculated according to Lichtenthaler (1987). 155 156 2.5. Tissue ion concentrations 157 Tiller and root samples taken from ten plants per treatment were dried at 80ºC for 158 48 h and ground, according to the protocols of Mateos-Naranjo et al. (2011). Then, 159 8 triplicate 0.5 g samples from each specific tissue were digested in 6 ml HNO3, 0.5 ml 160 HF and 1 ml H2O2. Ca, Mg, K, P, Na and Zn concentrations in the digests were 161 measured by inductively coupled plasma (ICP) spectroscopy (ARL-Fison 3410, USA). 162 2.6. Statistical analysis 163 Statistical tests were performed in the software package Statistica v. 6.0 (Statsoft 164 Inc.). Generalized linear models (GLM) were used to analyze the interactive effects of 165 Zn and NaCl concentrations (as categorical factors) on the growth and physiological 166 parameters (as dependent variables) of J. acutus plants. Multiple comparisons were 167 analyzed by a LSD (post hoc) test. Before statistical analysis Kolmogorov-Smirnov and 168 Brown-Forsythe tests were used to verify the assumptions of normality and 169 homogeneity of variances, respectively. Differences between tiller and root ion 170 concentrations were compared by the Student test (t-test). 171 172 3. Results 173 3.1. Effects of Zn and NaCl on growth 174 There were significant effects of both zinc and salinity on the RGR of Juncus 175 acutus but no significant interactions (Table 1, GLM: salinity, p < 0.05; Zn, p < 0.01). 176 Thus, in non-saline conditions RGR decreased 25% and 60% in plants grown at 30 and 177 100 mM Zn, respectively, compared to control; however, growth was much less 178 affected by Zn in plants exposed to 85 mM NaCl (i.e. 11% and 34% for 30 and 100 mM 179 Zn, respectively; Fig. 1A). Similarly, the percentage of dead tillers increased sharply 180 with Zn concentration (GLM: Zn, p < 0.01), but this increase was less acute in plants 181 grown in saline conditions (GLM: salinity, p = 0.07; Fig. 1B). 182 9 183 3.2. Effects of Zn and NaCl on photosynthetic physiology 184 There were significant effects of salinity and Zn treatments on net photosynthetic 185 rate (AN) after 40 d of treatment (Table 1, GLM: salinity, p < 0.05; Zn, p < 0.01 and 186 salinity x Zn, p < 0.01). Thus AN decreased progressively with increasing Zn 187 concentration in plants grown at both NaCl concentrations. However, plants exposed to 188 saline conditions maintained higher CO2 assimilation rates at both increased 189 concentrations of Zn than their non-saline counterparts (Fig. 2A). Very similar trends 190 were recorded for stomatal conductance (gs) but salinity did not significantly affect the 191 responses to Zn (GLM: salinity x Zn, p = 0.06; Fig. 2B). In contrast, salinity 192 significantly reduced the intercellular CO2 concentration (Ci) (GLM: salinity, p < 0.05), 193 whereas Zn concentration per se did not. However, Ci values were reduced at the high 194 salinity only in the presence of excess (30 or 100 mM) Zn (Fig. 2C). Salinity and Zn 195 had synergistic effects on intrinsic water use efficiency (iWUE; GLM: salinity x Zn, p < 196 0.05). Thus, plants grown under saline conditions had consistently higher iWUE but the 197 difference was only significant at 30 mM Zn (Fig. 2D). 198 Chlorophyll fluorescence parameters were also affected by the combination of Zn 199 and salinity treatments. Fv/Fm values, both at dawn and midday, tended to decrease with 200 increasing Zn concentration in plants grown in non-saline conditions. However, in 201 plants exposed to salinity, this effect was less marked and only evident at the highest Zn 202 concentration treatment (Table 1,GLMMd and Pd: salinity x Zn, p < 0.05; Fig. 3A, B). ΦPSII 203 values at dawn and at midday followed a similar pattern to those of Fv/Fm (GLMMd: 204 salinity x Zn, p < 0.05; Fig. 3C,D), except that the differences in predawn values were 205 minimal. NPQ values at midday increased markedly with Zn concentration, both in the 206 absence and presence of salinity, but this effect was substantially stronger in the absence 207 16 This beneficial effect could be linked with the key role of Na accumulation in plant 358 osmotic adjustment (Shabala et al., 2009). Hence, it is possible that the higher Na 359 concentration in tissues of J. acutus under saline conditions and the reduction in gs in 360 the presence of Zn might help to alleviate any water stress ascribed to Zn toxicity. 361 362 5. Conclusions 363 We may conclude that the presence of NaCl in the growth medium, at 364 concentrations representative of estuarine environments, considerably reduces the 365 deleterious effects of elevated Zn concentrations on the growth and development of J. 366 acutus. This beneficial effect was largely mediated by the reduction of Zn levels in J. 367 acutus tissues, together with an overall protective effect on its photosynthetic apparatus, 368 manifested as improved carbon harvesting, functionality of the photochemical apparatus 369 (PSII) and photosynthetic pigment concentrations. Furthermore, amelioration by NaCl 370 was linked with the maintenance of a more advantageous water balance. These 371 ecophysiological characteristics would enhance the fitness and competitive ability of J. 372 acutus in zinc-polluted estuaries and saltmarshes, providing a tolerant bio-tool for the 373 management and restoration metal pollution in salinized lands. 374 375 17 Acknowledgements 376 377 This work has been supported by Ministerio de Economía y Competitividad (MINECO 378 Project CGL2016-75550-R cofunded by FEDER). J.A Pérez-Romero thanks Ministerio 379 de Educación, Cultura y Deporte for its personal financial support (FPU014/03987). 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Md (midday), Pd (predawn), T (tiller and R (root). 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 Parameter Na Zn Na x Zn RGR 0.03* 0.00** 0.06 Dead Tillers 0.07 0.00** 0.19 AN 0.02* 0.00** 0.01* gs 0.30 0.00** 0.06 Ci 0.04* 0.05 0.22 iWUE 0.02* 0.00** 0.03* Fv/Fm, Md 0.00* 0.00** 0.02* Fv/Fm, Pd 0.00** 0.00** 0.02* ΦPSII, Md 0.09 0.00** 0.02* ΦPSII, Pd 0.01** 0.00** 0.06 NPQ, Md 0.01** 0.00** 0.10 NPQ, Pd 0.63 0.51 0.62 Chl a 0.00** 0.88 0.00** Chl b 0.06 0.57 0.04* Cx+c 0.02* 0.22 0.04* [Zn]T 0.00** 0.00** 0.00** [Zn]R 0.00** 0.00** 0.00** [Na]T 0.00** 0.00** 0.00** [Na]R 0.00** 0.00** 0.00** [K]T 0.95 0.58 0.47 [K]R 0.00** 0.00** 0.00** [Mg]T 0.00** 0.00** 0.00** [Mg]R 0.00** 0.00** 0.00** [Ca]T 0.00** 0.00** 0.00** [Ca]R 0.00** 0.00** 0.00** [P]T 0.02* 0.00** 0.04* [P]R 0.00** 0.00** 0.01** [Mn]T 0.78 0.00** 0.88 [Mn]R 0.00** 0.00** 0.00** 25 Table 2. Ion concentration in tiller and roots of Juncus acutus treated with a range of Zn concentration in combination with 0 mM and 85 mM NaCl, after 40 days. Values represent mean ± SE, n = 5. Different letters indicate means that are significantly different from each other Treatments Tiller concentration Zn (mM) NaCl (mM) Zn (mg Kg-1) Na (mg g-1) K (mg g-1) Mg (mg g-1) Ca (mg g-1) P (mg g-1) Mn (mg Kg-1) 0 0 32.3 ± 0.5a 0.97 ± 0.1a 29.9 ± 0.1a 3.54 ± 0.3a 5.89 ± 0.1a 2.95 ± 0.2a 35.5 ± 0.6a 30 0 304.6 ± 1.4b 1.84 ± 0.2b 29.4 ± 0.3a 3.19 ± 0.2a 4.97 ± 0.2b 2.39 ± 0.1b 27.3 ± 0.3b 100 0 611.7 ± 0.8c 3.65 ± 0.2c 30.2 ± 0.1a 3.04 ± 0.1a 4.09 ± 0.2c 2.50 ± 0.2b 23.9 ± 0.4c 0 85 36.9 ± 0.6a 7.75 ± 0.5d 28.9 ± 0.5a 3.31 ± 0.3a 4.20 ± 0.2c 2.89 ± 0.5a 32.7 ± 0.2a 30 85 248.5 ± 0.5d 6.96 ± 0.1e 29.7 ± 0.2a 3.06 ± 0.2a 3.85 ± 0.4c 2.82 ± 0.2a 27.3 ± 0.4b 100 85 412.3 ± 1.1e 8.41 ± 0.3d 30.9 ± 0.3a 2.83 ± 0.1b 3.71 ± 0.3c 2.52 ± 0.3b 22.9 ± 0.1c Root concentration 0 0 87.3 ± 0.7a 1.48 ± 0.1a 28.9 ± 0.2a 5.48 ± 0.2a 15.84 ± 0.5a 3.36 ± 0.5ab 39.3 ± 0.2a 30 0 2122.6 ± 1.3b 2.92 ± 0.2b 24.5 ± 0.3b 4.60 ± 0.2a 17.36 ± 0.2a 4.25 ± 0.5a 39.9 ± 0.4a 100 0 2479.0 ± 0.3c 5.78 ± 0.4c 16.8 ± 0.4c 4.07 ± 0.1b 8.54 ± 0.2b 2.39 ± 0.2b 33.5 ± 0.1b 0 85 58.4 ± 1.2d 21.17 ± 0.2d 20.2 ± 0.4b 4.87 ± 0.2a 13.27 ± 0.6a 2.63 ± 0.1b 33.5 ± 0.1b 30 85 1455.4 ± 2.2e 16.14 ± 0.3e 20.5 ± 0.2b 3.62 ± 0.1b 8.67 ± 0.2b 2.71 ± 0.1b 27.5 ± 0.2c 100 85 1969.2 ± 1.1f 13.95 ± 0.4f 17.3 ± 0.3c 3.41 ± 0.2c 4.89 ± 0.1c 2.67 ± 0.1b 26.4 ± 0.2c 32