Tolerance and accumulation of copper in the salt-marsh shrub Halimione portulacoides

1 Tolerance and accumulation of copper in the salt-marsh shrub Halimione portulacoides J. Cambrollé*, J.M. Mancilla-Leytón, S. Muñoz-Vallés, T. Luque and M.E. Figueroa Departamento de Biología Vegetal y Ecología, Facultad de Biología, Universidad de Sevilla, Apartado 1095, 41080 - Sevilla, España *Corresponding author. Postal address: Jesús Cambrollé Silva, Dpto. Biología Vegetal y Ecología, Facultad de Biología, Universidad de Sevilla, Av. Reina Mercedes 6, 41012 Seville, Spain. Tel.: +34-95-4557165; fax: +34-95-4615780. E-mail address: [email protected] 2 Abbreviations: A, net photosynthetic rate; Chl a, chlorophyll a; Chl b, chlorophyll b; Ci, intercellular CO2 concentration; Cx+c, carotenoids; F0, minimal fluorescence level in the dark-adapted state; Fm, maximal fluorescence level in the dark-adapted state; Fs, steady state fluorescence yield; Fv, variable fluorescence level in the dark-adapted state; Fv/Fm, maximum quantum efficiency of PSII photochemistry; ΦPSII, quantum efficiency of PSII; Gs, stomatal conductance; NPQ, non-photochemical quenching; RGR, relative growth rate. 3 Abstract: The present study evaluated the tolerance and accumulation potential of the saltmarsh shrub Halimione portulacoides under moderate and high external Cu levels. A greenhouse experiment was conducted in order to investigate the effects of a range of external Cu concentrations (0 to 60 mmol l-1) on growth and photosynthetic performance by measuring gas exchange, chlorophyll fluorescence parameters and photosynthetic pigments. We also determined total copper, nitrogen, phosphorus and sulphur concentrations in the plant tissues. Halimione portulacoides survived with external Cu concentrations of up to 35 mmol Cu l-1, although the excess of metal resulted in a biomass reduction of 48%. The effects of Cu on growth were linked to a drastic reduction in net photosynthesis. However, H. portulacoides tolerated Cu levels of up to 15 mmol Cu l-1 without suffering adverse physiological effects. Our results indicate that this species could play an important role in the restoration of Cucontaminated soils. Keywords: Copper; Halimione portulacoides; photosynthesis; phytoremediation. 4 Introduction Mining, smelting, land applications of sewage sludge and the use of certain fungicides, among other human activities, have lead to the widespread contamination of soil with copper (Hong-yun et al., 2005). Although Cu is an essential trace element and is involved in a wide range of biochemical and physiological processes such as photosynthesis and respiration (Barón et al., 1995; Pilon et al., 2006), it is potentially toxic to most plants at high concentrations (Dewez et al., 2005). An excess of Cu may alter membrane permeability, protein synthesis, photosynthetic and respiratory processes, enzyme activities, and chromatin structure (Fernandes and Henriques, 1991; Madejón et al., 2009; Van Assche and Clijsters, 1990). Despite this, certain plant species have evolved tolerance to heavy metals and remain unaffected under exposure to extremely high concentrations of Cu (Ernst et al., 2000). Estuarine salt marshes are often highly contaminated with metals, due to human and industrial activities occurring in the estuaries and adjacent areas (Sousa et al., 2008). Furthermore, salt-marsh plants are known to tolerate and accumulate high contents of heavy metals (e.g. Matthews et al., 2005). Halimione portulacoides (L.) Aellen is a halophytic shrub frequently found on sandy and muddy sea-shores and salt marshes around the coasts of Europe, North Africa and South-West Asia. The species is frequently the physiognomic dominant on well-drained and upper marshes, often fringing channels and pools that are flooded at high tide (Chapman, 1950). In several estuaries of the Iberian Peninsula, H. portulacoides grows in sediments featuring extremely high concentrations of metals. In the joint estuary of the Tinto and Odiel rivers (SW Spain), which is one of the world´s most polluted areas in terms of heavy metals, H. portulacoides can be found growing in sediments that contain 300-3000 ppm 5 Cu (Cambrollé et al., 2008; Nelson and Lamothe, 1993; Sáinz and Ruiz, 2006). Moreover, in the Tagus estuary salt marshes (Lisbon, Portugal), this species has demonstrated its ability to tolerate and sequestrate high levels of Cu (Caçador et al. 2000). Recent studies have explored the phytoremediation potential of H. portulacoides under different factors, such as the presence of organic pollutants (Duarte et al., 2007; Almeida et al., 2009a and b); however, certain important aspects, such as the tolerance characteristics or accumulation patterns of the species in response to rising soil metal concentrations, remain unknown. Knowledge of the physiological impact of elevated levels of metals on this species is essential in order to understand its phytotoxicity limits and, ultimately, to evaluate the potential of the species as a phytoremediation tool. The aim of the present study was to evaluate the tolerance and accumulation potential of H. portulacoides under exposure to moderate and high Cu levels. The specific objectives were: (1) to determine the Cu phytotoxicity thresholds of the study species by analyzing the growth of plants in a range of external Cu concentrations, from 0 to 60 mmol l-1 Cu; (2) to ascertain the extent to which Cu determines plant performance, in terms of influence on the photosynthetic apparatus (PSII chemistry), gas exchange characteristics and photosynthetic pigments; and (3) to examine the possible relationship between the effects of Cu on growth and concentrations of N, P and S within the plant tissues. 6 Materials and Methods Plant material and copper treatments Seeds of Halimione portulacoides were collected in the salt marshes of “La Mata-Torrevieja” (Alicante, SE Spain). The collected seeds were subsequently germinated in perlite moistened with distilled water, and maintained at 25 ºC for 30 days. The resulting seedlings were sown in individual plastic pots (diameter 11 cm) filled with perlite, and placed in a glasshouse with minimum-maximum temperatures of 21-25ºC, 40-60% relative humidity and natural daylight (minimum and maximum light flux: 200 and 1000 μmol m-2 s-1, respectively). Pots were carefully irrigated with 20% Hoagland's solution (Hoagland and Arnon, 1938) as required. When seedlings were between 20 and 25 cm in height (after 4 months of growth), the pots were allocated to six different Cu concentration treatments: 0, 2, 9, 15, 35 and 60 mmol l-1 Cu, applied in shallow trays within the same glasshouse (ten pots per tray, one tray per Cu treatment). Cu treatments were prepared by mixing 20% Hoagland's solution with CuSO4·5H2O of the appropriate concentration. The control, 0 mmol l-1 Cu treatment, in fact contained 0.0005 mmol l-1 of Cu, since Hoagland’s solution contains a small amount of Cu as an essential trace nutrient. These Cu concentrations were chosen in order to reflect the range of levels found by several authors in studies of the salt-marshes of different metal polluted estuaries (e.g. Cambrollé et al., 2008; Nelson and Lamothe, 1993; Caçador et al., 2000), as well as in previous experiments to determine the phytotoxicity thresholds of H. portulacoides. At the beginning of the experiment, a 3 L volume of the appropriate solution was placed in each of the trays to a marked depth of 1 cm. Throughout the experiment, 7 solution levels in the trays were monitored and topped up to the marked level with 20% Hoagland's solution, (with no additional CuSO4·5H2O) in order to limit the change in Cu concentration due to evaporation of the water in the nutrient solution. In addition, the entire solution (including CuSO4·5H2O) was changed on a weekly basis. Growth From each treatment, four complete plants (roots and shoots) were harvested at the beginning, and the remaining six at the end of the experiment (i.e. following 45 days of treatment). These plants were dried at 80ºC for 48 h and then weighed. The relative growth rate (RGR) of whole plants was calculated using the formula: RGR = (ln Bf – ln Bi) · D-1 (g g-1day-1) where Bf = final dry mass, Bi = initial dry mass (average of the four plants from each treatment dried at the beginning of the experiment) and D = duration of experiment (days). Leaf area was determined from the projected area by scanning and digitalising the leaves (Epson V30, Seiko Epson Corp., Nagano, Japan), and using appropriate software (MideBMP v. 4.2.; Ordiales-Plaza, 2000) for processing and analysis. Mineral analysis At the end of the experimental period, leaf and root samples were dried at 80ºC for 48 h and ground, in accordance with the protocols of Redondo-Gómez et al. (2007). 8 Leaves and roots were carefully washed with distilled water prior to further analysis. Then, 0.5 g samples were digested with 6 ml HNO3, 0.5 ml HF and 1 ml H2O2. Measurements of Cu, P and S were conducted by inductively coupled plasma (ICP) spectroscopy (ARL-Fison 3410, USA). Total N concentration was determined from undigested dry samples using an elemental analyzer (Leco CHNS-932, Spain). Gas exchange Gas exchange measurements were taken from randomly selected, fully expanded leaves (n = 10, one measurement per plant plus four extra measurements taken randomly), following 45 days of treatment, using an infrared gas analyzer in an open system (LI-6400, LI-COR Inc., Neb., USA). Net photosynthetic rate (A), intercellular CO2 concentration (Ci) and stomatal conductance to CO2 (Gs) were determined at an ambient CO2 concentration of 360 µmol mol-1, temperature of 20/25ºC, 50 ± 5% relative humidity and a photon flux density of 1000 µmol m-2 s-1. Values of the parameters A, Ci and Gs were calculated using the standard formulae of Von Caemmerer and Farquhar (1981). Chlorophyll fluorescence Chlorophyll fluorescence was measured in randomly selected, fully developed leaves (n = 12) using a portable modulated fluorimeter (FMS-2, Hansatech Instruments Ltd., England) following 45 days of treatment. Lightand dark-adapted fluorescence parameters were measured at dawn (stable, 50 µmol m-2 s-1 ambient light) and at midday (1600 µmol m-2 s-1) in order to investigate the effect of Cu concentration on the 9 sensitivity of plants to photoinhibition. Plants were dark-adapted for 30 minutes, using purpose designed leaf–clips. The minimal fluorescence level in the dark-adapted state (F0) was measured using a modulated pulse (<0.05 µmol m-2 s-1 for 1.8 µs) which was too small to induce significant physiological changes in the plant. The data recorded represented an average taken over a 1.6 second period. Maximal fluorescence in this state (Fm) was measured after applying a saturating actinic light pulse of 15000 µmol m-2 s-1 for 0.7s. The value of Fm was recorded as the highest average of two consecutive points. Values of variable fluorescence (Fv = Fm - F0) and maximum quantum efficiency of PSII photochemistry (Fv/Fm) were calculated from F0 and Fm. This ratio of variable to maximal fluorescence correlates with the number of functional PSII reaction centres, and dark adapted values of Fv/Fm can be used to quantify photoinhibition (Krivosheeva et al., 1996). The same leaf section of each plant was used to measure light-adapted parameters. Steady state fluorescence yield (Fs) was recorded following adaptation of the plants to ambient light conditions for 30 minutes. A saturating actinic light pulse of 15000 µmol m-2 s-1 for 0.7 s was then used to produce the maximum fluorescence yield (Fm') by temporarily inhibiting PSII photochemistry. Using fluorescence parameters determined in both lightand dark-adapted states, the following were calculated: quantum efficiency of PSII (ΦPSII = (Fm' – Fs)/ Fm'), which measures the proportion of light absorbed by the chlorophyll associated with PSII that is used in photochemistry (Maxwell and Johnson, 2000); and non-photochemical quenching (NPQ = (Fm – Fm') / Fm'; Redondo-Gómez et al., 2006), which is linearly related to heat dissipation (Maxwell and Johnson, 2000). 16 macronutrient (Tyler, 1976). Mateos-Naranjo et al. (2008a) also detected a reduction of leaf and root P content with increasing Cu supply in Spartina densiflora. On the other hand, the increased concentration of S in leaves of H. portulacoides with increasing amounts of external Cu, could be due to an increase in the synthesis of sulphur-rich compounds involved in metal tolerance, such as glucosinolates, metallothioneins and phytochelatins (Ernst et al., 2008; Hassinen et al., 2011). Cambrollé et al. (2011b) also report a rise in S concentration in the roots of Glaucium flavum at elevated Cu levels. Finally, in our study, the excess Cu resulted in reduced plant N concentration, particularly in the leaf tissues at 35 mmol l-1 external Cu. Several studies have shown negligible reductions in the activities of most enzymes related to nitrogen metabolism (such as nitrate reductase) under Cu exposure, demonstrating the impact of Cu on N metabolism (Llorens et al., 2000; Xiong et al., 2006). Nitrogen deficiencies affect all levels of plant function, from metabolism to resource allocation, growth, and development (Stitt and Krapp, 1999; Stitt et al., 2002). Copper plays a significant role in several physiological processes, including photosynthesis, and it is commonly recognized that Cu toxicity induces inhibition of photosynthetic function (Kabata-Pendias and Pendias, 2001). Previous reports have shown that external Cu concentrations above 20 mg l-1 inhibit the photosynthetic process (Baszynski et al., 1982; Bibi and Hussain, 2005; Lidon and Henriques, 1991; Vassilev et al., 2002). However, the gas exchange parameters in H. portulacoides were unaltered on exposure to external Cu levels of up to 15 mmol l-1 (1000 mg Cu l-1). An external Cu concentration of 35 mmol l-1 had a drastic effect on net photosynthesis rate (A) and stomatal conductance (Gs), with no direct relationship between both parameters since there was no reduction in intercellular CO2 concentration (Ci). The decrease of stomatal conductance may be related to an alteration in the K:Ca ratio in the guard cells 17 and/or changes in the concentration of abscisic acid, which controls stomatal movement (Marschner, 1999). The marked decline of Gs in the absence of a decrease in Ci, indicates that the reported reduction in photosynthetic activity at 35 mmol l-1 Cu could be partially due to the effects of the metal on the photosynthetic apparatus. It is recognized that an excess of copper has a direct effect on the photosynthetic electron transport chain (Jegershöld et al., 1995; Ouzounidou and Ilias, 2005). In our experiment, maximum quantum efficiency of PSII (Fv/Fm) did show a significant reduction at midday compared to the values recorded at dawn. At midday, the measured reduction in Fv/Fm values indicated that H. portulacoides had experienced photoinhibition at the higher light flux. Quantum efficiency of PSII (ΦPSII) also showed this difference between sampling times. ΦPSII decreased as a consequence of the increase in NPQ, indicating that the plants dissipated light as heat, thereby protecting the leaf from light-induced damage (Maxwell and Johnson, 2000). Fv/Fm and ΦPSII were strongly affected by Cu concentrations above 15 mmol l-1, suggesting that an excess of Cu enhances the photoinhibition induced by light stress. Photoinhibition is caused by damage to photosynthetic components; the effect can be short-term and reversible (dynamic photoinhibition), or long-term and irreversible (chronic photoinhibition; Werner et al., 2002). The midday value of Fv/Fm at the highest external Cu concentration did not recover at dawn, and in fact remained lower than control parameters for unstressed plants (Björkman and Demmig, 1987), revealing chronic photoinhibition or photodamage. Our results suggest that the decrease of net photosynthesis registered at 35 mmol l-1 external Cu may have been caused not only by factors affecting stomatal closure, but also, to a large extent, by damage to the photosynthetic apparatus. The reported decline in Fv/Fm was caused by higher values of F0, which can be attributed to a reduction in the energy transfer from the antennae of the 18 PSII to the reaction centres (Maxwell and Johnson, 2000). Similar results were obtained by Ralph and Burchett (1998) in Halophila ovalis exposed to Cu and Zn. The decline in chlorophyll fluorescence parameters registered in H. portulacoides at the highest external Cu concentration may be related to the notable decrease in the concentration of chlorophyll recorded in this treatment. Copper in excess causes degradation of the chloroplast inner structure and pigment content (Ciscato et al., 1997; Ouzounidou, 1996). Furthermore, nitrogen deficiency has a negative impact on chloroplasts (Lawlor, 2002) and it is known that the biosynthesis of chlorophylls is dependent on the supply of assimilated N (Xiong et al., 2006). In this way, we recorded a dramatic decline in leaf N concentration at 35 mmol l-1 Cu, which probably affected the Chl synthesis. Halimione portulacoides can tolerate external Cu levels of up to 15 mmol l-1 (1000 mg Cu l-1) without suffering adverse physiological effects. This may be partially achieved by the capacity of the roots to retain large quantities of metal, preventing their translocation to photosynthetic tissues. Furthermore, despite the fact that Cu concentrations of between 20 and 100 mg Cu kg-1 DW in leaf tissue are generally considered excessive or toxic (Kabata-Pendias and Pendias, 2001), growth parameters of this species are unaffected by leaf tissue concentrations as high as 80 mg Cu kg-1 DW. Halimione portulacoides is able to survive with external Cu levels of 35 mmol l-1; however, upward translocation from the roots seem to increase markedly at this Cu concentration, reaching toxic levels of Cu in the leaves which causes nutrient imbalances and a severe decline in photosynthetic function. These factors contribute to an overall reduction in carbon gain and, consequently, to a reduction in plant growth. This study provides new insights into the heavy metal tolerance of H. portulacoides, and contributes to the knowledge regarding this species, which may be potentially useful in the restoration of Cu-contaminated areas, since the plant is capable of producing a significant amount of biomass and sequestrating high concentrations of this metal without suffering adverse physiological effects. 19 Acknowledgements We thank the Spanish Ministry of Science and Innovation (project CTM200804453) and the Seville University Glasshouse General Service for their collaboration. 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