Does the polluted environment modify responses to metal pollution? A case study of two Cistus species and the excess of copper and lead

1 Does the habitat modify responses to metal pollution? A case study of two Cistus species and the excess of copper and lead. S. Rossini Oliva1*, M.D. Mingorance†, E.O. Leidi2, A.J. Fernández3 Abstract Populations of Cistus ladanifer L. and C. salvifolius L. from an abandoned mine area in Riotinto (SW Spain) and unpolluted control sites were examined to test tolerance to copper and lead. Metal tolerance was tested in hydroponic experiments with different Cu (1, 25 and 100 µM Cu) and Pb (0, 100 µM Pb) concentrations. A germination test was also carried out to investigate possible differences between populations. Additionally some physiological and chemical parameters were analyzed in plants from Cu experiment. The results revealed different responses to high Cu and Pb between both species as well as between populations. The populations of C. salvifolius from the contaminated site performed better in the medium with 25 µM Cu than the population from the uncontaminated site. However, no differences in Cu tolerance were found for the populations of C. ladanifer. Interestingly, the opposite result was found for Pb indicating that C. ladanifer has an adaptative tolerance to Pb. Copper contents were higher in roots than in shoots. Germination percentage in both species was greater in seeds obtained in uncontaminated sites and C. ladanifer had a lower germination percentage than C. salvifolius. The same pattern was observed for biomass production. The population of C. salvifolius from contaminated site exhibited a constitutive proline contents. Keywords: metal-tolerance, habitat, population, mining 2 Introduction Metal tolerance is the ability of a species to survive in soils where their metal (loid)s contents are toxic for most other species (Macnair et al., 2000). It can be the result of evolution by natural variation (Ernst, 1992) and differences may be found between populations from contaminated and no contaminated soils. In the same taxonomic groups differences in metal tolerance may appear among populations of the same species (Ernst, 2006). Metal tolerance can be a constitutive or an induced phenomenon (adaptative) as a result of a long period of exposure (Meharg, 1994), but then usually becomes a heritable trait (Verkleij and Schat, 1989). The constitutive metal tolerance was defined as a trait present in all members of populations of a species growing either on contaminated or uncontaminated soils (Meharg, 1994; Boyd and Martens, 1998). Sometimes, the plant species also evolve a multi-tolerance to different metal (loid)s. Mine soils are enriched by potentially toxic elements (PTE) and are usually low in nutrient contents and low soil pH creating hostile environment for plant establishment. Under such conditions plants survival depends on their abilities to modify physiological processes to the limitations imposed by the environment. Plants need to adapt the metabolism to tolerate PTE and by several homeostatic mechanisms maintain the right concentrations of essential elements in cellular compartments to minimize the damage induced by PTE (Clemens, 2001; Ernst, 2006). In metal enriched soils thrives a local flora of metal-tolerant species, which are typically endemic in the area, with more-resistant plant ecotypes developed as a consequence of a selection promoted by the high PTE levels (Reeves and Baker, 1998). Metalliferous sites are extremely important to biodiversity (Whiting et al., 2004). Metal resistance can be reached by two different ways, avoidance and tolerance (Levitt, 1980). Avoidance is defined as an organism ability to prevent excessive metal build up into its body while tolerance results from the ability to cope with excessively accumulated PTE 3 within its body. A metal-tolerant plant species is able to grow, because it can manage the high internal metal concentration using different mechanisms, including biochemical and/or physiological processes. Exudation of organic molecules by roots is considered one of the most important strategies to tolerate high metal concentrations. These organic compounds can chelate metals and facilitate their transport in distinct cellular compartments without inflicting damage (Viehweger, 2014). Root organic acids exudation has been studied due to their potential to stimulate microbial growth, mobilize low soluble nutrients (e.g., P, Fe, Zn) and produce detoxification of some PTE (Jones and Darrah, 1994; Neumann and Romheld, 1999; Dakora and Phillips, 2002). Other organic compounds, like amino acids, peptides, phytochelatins (PC), and polyamines increased after upon exposure to metals (Sharma and Dietz., 2006). Meir et al. (2012) found differences in root exudation among metallophytes and agricultural plants under Cu treatments. Cistus ladanifer and C. salvifolius are species which grow in soils highly contaminated by As, Cu y Cu indicating that they might hold multi-tolerance to different contaminants. Tolerant plant species have developed greater ability to resist the extreme conditions when compared with members of the same species from non-contaminated sites (Abreu and Magalães, 2009). The hypothesis of this work was to use populations from polluted and unpolluted soils for testing if they hold different mechanisms to tolerate the excess of Cu y Pb. Those species from polluted mine soils might have adapted progressively to high metal availability. If metal tolerance is going to be found in the species from non-contaminated soils, it might indicate constitutive tolerance. This approach might reveal the existence of an acquired resistance or a constitutive tolerance in Cistus which might be useful when planning revegetation of contaminated areas. Therefore, the aim of this study was to 4 determine under controlled conditions (hydropony) the tolerance to Cu and Pb in two native pseudometallophytes collected from polluted or uncontaminated soils. Lead tolerance was studied in terms of biomass production and change of biochemical markers (leaf pigments, amino acids, organic acids). Copper tolerance also included and the analysis of plant chemical composition at the end of the experiment. Material and Methods Plant selection Seed selection was based on previous field observations and was aimed at selecting metaltolerant plants that grew well in the Riotinto mine area. Two shrubs were selected: Cistus salvifolius L. and Cistus ladanifer L. Cistus salvifolius is a very common species in the area and can be found in both polluted and unpolluted soils. They also grow in mining areas of Portugal where the Cu concentration reaches 1267 mg/kg and Pb 7413 mg/kg. The species was considered suitable for phytostabilization of mining wastes in areas with semiarid climate (Abreu et al., 2012). Cistus ladanifer is a pioneer species and its adaptability to widely varying environmental conditions is based on their mechanisms for water and nutrient conservation (Herrera, 1984; Núñez-Olivera et al., 1993, 1996; Talavera et al., 1993). It is able to grow in mining areas with soils polluted by polymetallic contaminations (Santos et al., 2014). Both species can colonize the mining waste only when the pedogenetic process has started and both species are the main components of the matorral shrubland (jarales) in this area. A previous study with the mining soils of Riotinto where C. ladanifer is found reported Cu and Pb concentrations of 192 mg/kg and 331 mg/kg Pb, significantly greater than the European soil baseline (Rossini-Oliva et al., 2016). Copper levels are also high in soils from São Domingo area (South Portugal) where both species may be found (Santos et al., 2014). Seeds of C. ladanifer and C. salvifolius 5 were collected in two different sites. The polluted site was a mining area close to Tinto River (UTM: 29S 715829/4177713) where the vegetation extends along the river margin forming a band in the vicinity to the water course formed by Erica andevalensis, E. australis, Cistus ladanifer, Nerium oleander, Ulex eryocladus, Elychrisum stoechas, Phagnalon saxatile, Cistus populifolius, Cistus monspeliensis and Cistus salvifolius. The uncontaminated area was selected about 30 km away the Riotinto district (Venta del Cruce, UTM 29S 0751275/41247643), without any mining influence where vegetation is jarales (shrub association composed mainly by Cistus species). Seed germination tests Seeds from each population were treated at 100 ºC during 5 minutes since exposure to the heat cracks the testa (Aronne and Mazzoleni, 1989) making germination possible. Seed were placed in Petri dishes with two layers of filter paper moistened with distilled water and kept in a growth chamber (20°C) to test seed germination. Each test was performed with four replicates of 50 seeds and dishes/pots were inspected over a 42-day period, daily during the first 2 weeks, and three times a week for the rest of the period. Seeds with a 1- mm-long radicle were scored as germinated and removed. Germination was then calculated as a percentage of germinated seeds per each trial. Plant culture in high Cu or Pb concentration. The seeds were sterilized in 0.3% hypochlorite and washed three times with sterile distilled water, placed to germinate in Eppendorf tubes filled with rockwood in a culture chamber with 16 hours of light at 26 ºC and 8 hours of darkness at 20 ºC. When seeds germinated and seedlings reached 2 cm height they were into 10 liters plastic buckets with nutrient solutions (pH 4.0) containing (in mM): NO3− ,4; H2PO4−,1; SO42− , 2.5; K+ 6 , 4; Ca2+, 2; Mg2+, 1. Micronutrients were supplied as prescribed in the Long Ashton nutrient formula (Hewitt, 1966), except Fe which was provided as Fe-EDDHA at 4 mg/l. When seedlings reached 5 cm height, the experiment was started by adding different concentrations of Cu (25 μM, 100 μM) as CuSO4 while the control treatment contained 1 μM Cu. The metal concentrations were based on the results of previous test and field observations. The nutrient solutions were continuously aerated with an aquarium air pump, and renewed every 3 days to maintain a rather constant nutrient supply and metal concentration. The experiment was carried out during 3 weeks (at this time plants in high Cu concentration started to show chlorotic leaves). The strategy used to test Pb tolerance was somewhat different: only two Pb levels were used (0 or 100 μM). The basic nutrient solution described above contained 0 (control) or 100 μM Pb as Pb(NO3)2. To avoid Pb precipitation in the nutrient solution, it was diluted 1/10 of the original strength for all the elements except P which was 1/100. All Cu and Pb treatments had four replicates. Growth was assessed by measuring plant fresh and dry weights at the end of the assay to estimate Cu and Pb tolerance. Analysis of element concentration in Cu treatments Plants from the Cu treatments were harvested and roots were separated from shoots, rinsed with tap water, gently washed twice with distilled water and dried. Samples were oven-dried at 70°C during 48 h and dry biomass was determined. Dried plant material was then milled and digested with a mixture of HNO3 and H2O2 (Mingorance et al., 1993). Element concentration (B, Ca, Cu, Fe, K, Mg, Mn, P, S and Zn) in each plant part (leaves and roots) was determined by inductively coupled plasma atomic emission spectrometry (ICP-AES). Accuracy was checked by analyzing standard reference material (BCR 62- Olive leaves) and the recovery range was from 90 to 92%. 7 Determination of biochemical parameters Leaves and roots from Cu treated plants were frozen in liquid N2 and stored at -70ºC until analysis. Plant material was then homogenized (mortar and pestle) and centrifuged (10,000 rpm, 5 min at 4ºC). Clear supernatants were used for the analysis of amino acids and organic acids. Free amino acids were separated and quantified after derivatization with phenyl-isotiocyanate by reversed-phase high-performance liquid chromatography (Heinrikson and Meredith, 1984) using a Waters HPLC (Pico.Tag free amino acids column). Organic acids were identified and quantified by isocratic high efficiency ion chromatography-conductivity detector, using a Metrohm 940 Professional IC Vario (Metrosep organic acids 250/7.8 column, Pérola et al., 2010). The chlorophylls a and b and carotenoids were extracted from young leaves with 80% methanol and determined according to Lichtenthaler (1987). All determinations were made in triplicate. For Pb treated plants, only roots were used for the analysis of amino acids and organics acids using the same procedure as indicated above. Statistical analyses Multiple Analysis of Variance (MANOVA) was applied in order to check significant differences between samples among variables. In case variable revealed severe deviation from the assumptions of analysis of variance, logarithmic transformation was done. Linear Discriminant analysis (LDA) was applied to obtain a classification model. Linear Discriminant Analysis is a pattern classification method widely used in data processing. The aim is to create a predictive model which can accurately classify future unknown samples. This classification model was proposed with the aim of establishing whether the 8 different groups of samples could be discriminated, such the two plant varieties, the two sampling sites or the three copper treatments. Linear discriminant analysis (LDA) was applied to obtain discriminant functions (DFs) as linear combinations of the inputs variables, minimizing the within-class and betweenclass ratio of the sum of squares (Gardiner, 1997). It was previously employed as unsupervised exploratory analysis of the data structure and as a powerful method for classification. The model was obtained by using a forward stepwise analysis, which selects only the most discriminating variables. Sensitivity (SENS) and specificity (SPEC) calculations were carried out. SENS represents the number of objects belonging to a class that are correctly classified to the correct class, and SPEC corresponds to objects not belonging to a certain class and subsequently classified as pertaining to another. Discrimination of the different groups was also graphically demonstrated representing in the plane the two first discriminant functions. A correlation analysis (Pearson) was done between the Cu concentration in nutrient solution and in plants and the other elements and biomass production. To analyze the germination data, for each trial, the final germination percentages were calculated and values were subjected to analysis of t-test to study differences between populations. Data were analyzed using a statistical package (Statsoft package v6.12). Copper translocation factor (TC) was calculated as Cu shoot/Cu root ratio to recognize the preferential partitioning of this element to the aboveground part (TC values > 1). Results and Discussion Germination studies The germination percentage was significantly different between species (Figure 1) and in C. salvifolius was different between populations (p<0.05). Seeds proceeding from a mining area had a significantly lower germination percentage compared with those from 9 uncontaminated site (p=0.006). The mean germination in C. salvifolius from the contaminated area was 70%, high enough to ensure dispersion and establishment in the Riotinto area, where the species is a significant constituent of the vegetation. In addition both species produce abundant seeds, a key factor to ensure population stability under adverse conditions (Sadras, 2007; Huang et al., 2011). The germination percentage in both species from uncontaminated sites was similar and ranged from 89-93%, whilst in those from the contaminated site C. ladanifer had a higher germination percentage than C. salvifolius. Variability in the germination response of seeds among populations is well known (Martin et al., 1995; Schütz and Milberg, 1997; Baskin and Baskin, 2001; Cruz et al., 2003) and Cistus seed populations have been regarded as possessing considerable heterogeneity in germination behavior (Vuillemin and Bulard 1981; Thanos and Georghiou 1988; Pérez García, 1997). Why seeds from mining areas have a lower germination capacity is an interesting question? Toxicants may alter population viability and this might indicate a maternal imprinting as suggested by several authors (Gutterman 1994a,b,c; Kigel 1995; Pérez García; 1997). The ability to produce seeds with different degrees of dormancy may be a mechanism by which the species adapt to new environmental situations while ensuring the survival. Effect of Cu and Pb on plant growth Plants of both species cultivated with 100 μM Cu showed stunted growth with chlorosis in basal leaves, darkened roots and some of them died unable to tolerate this high Cu concentration. In the Cu excess experiment the MANOVA results (Table 1) show that biomass was affected by species, site, treatments, and the interactions species vs. treatments, species vs. site, site vs. treatment and species vs. site vs. treatment (p<0.05). A biomass 16 it was higher in population from uncontaminated soil. The population of C. salvifolius from uncontaminated soils showed higher contents of Ca, Mn and Mg but lower contents of P and K than plants from the contaminated site. This might mean that the population from contaminated soils was more efficient in the accumulation of K and P. In fact, soils from Riotinto and mining areas of Portugal are poor in P (Abreu et al., 2012; Rossini- Oliva et al., 2016). Copper accumulation was higher in plants from uncontaminated sites, suggesting that plants from polluted soils developed better mechanisms to avoid Cu accumulation: these differences were more evident in the root Cu contents. Similar results were found by Gan et al. (2013) in a copper tolerant species. This result should explain the difference in biomass production between populations. A negative correlation was found in both populations between Cu content in plant and treatments with biomass production (p< 0.05). Similar results were reported in other species for Zn accumulation (Bert et al., 2000; Meerts and Van Isacker (1997). According to Meharg (2005) ecotypes adapted to metal enriched soils take up less potentially toxic element than metal sensitive ecotypes. Colzi et al. (2012) suggested that a change in root cell wall composition observed only in a Cu tolerant population might be one of the factors that guarantee a low apoplastic Cu accumulation and might also limit symplastic Cu contents. Sousa et al. (2008) reported that cell walls retain a highest content of metals compared with other cellular compartments. The increase of root lignification observed in Cu tolerant populations should also be limiting the Cu uptake (Colzi et al., 2015). Copper content was again higher in the roots than in the shoots in both populations and translocation factor was always <1 (Fig.6?) in both populations. Marschner (1995) reported that usually roots are the preferential site of Cu accumulation when Cu in the nutrient solution is high. In most metallophytes Cu tolerance is based on metal exclusion (Ernst et al., 1992), a pattern also found in pseudometallophyte species (Poschenrieder et al., 2001; Rossini- 17 Oliva et al., 2016). Transport processes have been recognized as a central mechanism of metal detoxification and tolerance (Hall, 2002; Hall and Williams, 2003). When a metal ions exceeds a metal-specific threshold, they can turn toxic (Sharma and Dietz, 2006). For both Cistus species, 100 µM Cu might be a threshold value for Cu toxicity. This value depends on the species: Erica andevalensis, a species that also grows in Riotinto mining area tolerates more than 100 µM Cu (Rossini-Oliva et al., 2010). Copper is an essential element for plants although elevated concentrations can turn it toxic and plants possess different mechanisms to cope with high level of potentially toxic elements. The critical toxicity level of copper in leaves is 20 mg kg−1 to 30 mg kg−1 dry matter (Robson and Reuter 1981; Kabata-Pendias 2001). Tables 4-5 show that at 100 µM Cu both species and populations reached higher Cu values in their shoots and this might explain the growth reduction. At 25 µM Cu in solution, shoot Cu contents in C. salvifolius and C. ladanifer was in a similar range to that reported by Abreu et al. (2012) and Santos et al. (2014) for plants growing in the mining area of São Domingo. Like in C. ladanifer, K and P decreased in both roots and shoot of C. salvifolius when Cu increased in the nutrient solutions. A negative correlation (p<0.05) was found between Cu with K, P and Mn in roots and shoot. In non tolerant plants, high levels of Cu might damages root plasma membranes promoting K efflux (Marschner, 1995, Hall, 2002). A damage in root membranes may have led to the above mentioned reduction in root K concentration but C. salvifolius from contaminated sites was able to maintain a significantly higher K concentration (Table 5). There is a relation between shoot growth and P deficiency (Marschner, 1995), this should be one of the reasons of the decrease of growth in high Cu concentration observed in both species at high Cu concentration. In facts correlation analysis revealed a positive correlation between biomass with P and K content in roots and shoot. 18 Effect of Pb on biochemical parameters The presence of Pb in the root medium did not affect the contents of photosynthetic pigments in the leaves (p>0.005). However, high Pb concentration in the nutrient solution produced significant changes in the concentration of some root organic acids and free amino acids (Table 4). The presence of Pb in the root medium led to a significant positive correlation with the root contents of oxalate (r= 0.773, p<0.001), maleate (r=0.881, p<0.001) and citrate (r=0.592, p<0.001) and the amino acids tyrosine (r=0.744, p<0.001), arginine (r=0.647, p<0.001), valine (r=0.551, p<0.001), histidine (r=0.535, p<0.001) and asparagine (r=0.436, p<0.01). It is interesting to note that the significant increase in the concentration of oxalate, maleate, succinate and citrate in roots exposed to Pb (Table 4) had also a significant effect by site and species only in root oxalate. An increase in root oxalate content (and exudation) occurred when Pb tolerant rice varieties were exposed to 20 µM Pb in the nutrient solution (Yang et al., 2000). In response to 100 µM Pb, oxalate was the main organic acid released by Pinus sylvestris roots either with or without ectomycorrhiza (Johansson et al., 2008). Therefore, the increase in organic acids recorded in Cistus roots exposed to Pb suggests the vacuolar compartmentation of the toxic metal where it might form insoluble salts (Jones, 1998; Nakata, 2003). Among the amino acids and amides, asparagine significantly increased its concentration as well as others amino acids (Table 4) related to metal chelation (histidine, arginine) (Sharma and Dietz, 2006). Other amino acids (phenylalanine, tyrosine) are related to phenylpropanoid metabolism which is induced by general stress conditions (Dixon and Paiva, 1995). Exposure to Pb activates the phenylpropanoid pathway in roots (Pawlak-Sprada et al., 2011) and lignins are involved in the root Pb sequestration where 19 most of Pb in excess is kept (Marmiroli et al., 2005; Cheng et al., 2014; Pourrut et al., 2013). Only in the cases of citrate and oxalate, the effect of site (that is non-polluted vs polluted populations) was significant (Tabla 4). Discriminant analysis for Pb treatment showed that the two species can be separated at SENS = 94%. Fig. ? and equations system 4 (Supplement materials) show variables that differentiate both species: chlorophyll b, histidine, valine, acetate, lactate and succinate confirming data of Table 4. Regarding the study of Pb treatments applied (0 µM or 100 µM) the discrimination obtained between them was also at SENS of 100% (Figure ?) for the variables carotenoids, chlorophyll b, phenylalanine, valine, malonate and malate (see equations system 5, Supplementary material). Carotenoids, chlorophyll b, phenylalanine and malonate were the parameters that weighted to discriminate treatment 100 µM. When we tested if C. ladanifer and C. salvifolius population should be differentiated from control and Riotinto site data showed that C. salvifolius was again able to discriminate both populations mainly by the variables carotenoids, tartrate, lactate and alanine (see equations 6 Supplementary material nd Fig Pb.?), being the C. salvifolius in Riotinto (sR) group the more discriminated of the rest (Fig. Pb.?). Conclusions Results show that Cu and Pb tolerance varies with species and populations. 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Chlorophyll b content (mean values ± 0.95 confidential intervals, n=4) in Cistus ladanifer and C. salviifolius from different sites. Uncontaminated Contaminated C.salvifolius C.ladanifer Species 0,3 0,4 0,5 0,6 0,7 0,8 Chlorophyll b (mg/g) 33 Figure 4 a-b. Proline (a) and asparagine (b) mean content (n=3) in leaf of Cistus salviifolius treated with different Cu concentrations. Cu concentration ( M) 020 40 60 80 100 120 Proline in leaf saps (nmoles ml-1) 0 1000 2000 3000 4000 5000 C.salviifolius Control C. salviifolius RT Cu concentration ( M) 020 40 60 80 100 120 Asparagine in leaf saps (nmoles ml-1) 0 10000 20000 30000 40000 C. salviifolius Control C. salviifolius RT a b 34 35 DF1 DF2 -6 -5 -4 -3 -2 -1 0 1 2 3 4 -6 -5 -4 -3 -2 -1 0 1 2 3 4C. salviifolius C. ladanifer DF1 DF2 -5 -3 -1 1 3 5 -5 -3 -1 1 3 5 1 µM Cu 25 µM Cu 100 µM Cu DF1 DF2 -5 -3 -1 1 3 5 -5 -3 -1 1 3 5C ladanifer in control C salviifolius in control C ladanifer in Riotinto C salviifolius in Riotinto Figure 5 a-c. Distribution in the plane of the DFs of Cistus samples treated with Cu grouped by Cistus species (a), by Cu treatments (b) and by Cistus populations in control (unpolluted) and Riotinto (polluted) sites (c). a b a b c 36 DF1 DF2 -6 -5 -4 -3 -2 -1 0 1 2 3 4 -6 -5 -4 -3 -2 -1 0 1 2 3 4C. salviifolius C. ladanifer DF1 DF2 -6 -5 -4 -3 -2 -1 1 2 3 4 -6 -5 -4 -3 -2 -1 1 2 3 4 0 µM Pb 100 µM Pb DF1 DF2 -6 -5 -4 -3 -2 -1 -0 1 2 3 4 -6 -5 -4 -3 -2 -1 0 1 2 3 4C ladanifer in control C salviifolius in control C ladanifer in Riotinto C salviifolius in Riotinto a b c 37 Figure 6 a-c. Distribution in the plane of the DFs of Cistus samples treated with Pb grouped by Cistus species (a), by Pb treatments (b) and by Cistus populations in control (unpolluted site) and Riotinto (polluted) sites (c). 38 39 40 41