Copper and nickel co-treatment alters metal uptake and stress parameters of Salix purpurea × viminalis

Journal of Plant Physiology 216 (2017) 125–134

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Copper and nickel co-treatment alters metal uptake and stress parameters of Salix purpurea × viminalis

MARK

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Kinga Drzewieckaa, Mirosław Mleczeka, , Monika Gąseckaa, Zuzanna Magdziaka, Anna Budkab, Tamara Chadzinikolauc, Zygmunt Kaczmarekd, Piotr Golińskia a

Department of Chemistry, Poznań University of Life Sciences, Wojska Polskiego 75, 60-625 Poznań, Poland Department of Mathematical and Statistical Methods, Poznań University of Life Sciences, Wojska Polskiego 28, 60-637 Poznań, Poland c Department of Plant Physiology, Poznań University of Life Sciences, Wołyńska 35, 60-625 Poznań, Poland d Institute of Plant Genetics, Polish Academy of Science, Strzeszyńska 34, 60-679 Poznań, Poland b

A R T I C L E I N F O

A B S T R A C T

Keywords: Cu/Ni interaction Glutathione Heavy metal stress Phenolics Salicylic acid Salix

Simultaneous treatment of Salix purpurea × viminalis with copper (Cu2+) and nickel (Ni2+) altered metal phytoextraction rates in favor of leaves. Still, metal translocation patters remained unaffected (roots ≈ rods > > leaves ≥ shoots), reaching ∼20 and 14.5 mg kg−1 dry weight in roots for Cu and Ni, respectively. Biometric parameters revealed overall growth inhibition correlated with Cu content in leaves, thus proving its negative effect on photosynthesis. Metal toxicity was strongly affirmed in the case of roots (∼90% loss of root biomass at 3 mM), rather than in the above-ground organs. Plant treatment accelerated the accumulation of soluble carbohydrates, phenolics including salicylic acid and glutathione in Salix leaves. However, significant differences in plant reactions to the applied metals were noted. Metal accumulation in leaves was correlated with soluble sugars and elevated glutathione, and also with total phenolics content, in the case of Cu and Ni, respectively. Glutathione synthesis was induced by both metals, and correlated with salicylic acid in leaves of Nitreated plants.

1. Introduction The concentration of toxic non-functional metals and metalloids, e.g. cadmium (Cd), lead (Pb), mercury (Hg), and arsenic (As), in terrestrial as well as aquatic ecosystems has increased manifolds over the past decades. Other heavy metals such as copper (Cu), nickel (Ni) and zinc (Zn) are essential micronutrients, but at higher concentrations they are also highly toxic and cause retardation of plant growth due to water and nutrient imbalance, decrease of photosynthetic activity and cell constituents’ oxidation, and − as a consequence − negatively influence the condition of entire ecosystems (Nagajyoti et al., 2010). Plants are susceptible to heavy metal toxicity and respond to avoid detrimental effects in a variety of ways. Their resistance to toxic metals is based on multiple mechanisms and has been separated into two reaction modes, i.e. avoidance and tolerance (detoxification and non-specific resistance) (Dalvi and Bhalerao, 2013; Ahmad et al., 2007; Ernst, 2006; Zenk, 1996). Avoidance involves down-regulation of transporters’ activity, change of metal bioavailability in the rhizosphere by exudation of protons or organic acids, and symbiosis with mycorrhizal fungi in order to alter the metal speciation and to restrict the transfer of the metal into

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the plant. However, the uptake by roots has to meet a plant’s demand for the primary metabolic processes and sufficient defense function. Consequently, the only option for survival on metal-polluted soil is the adaptation phenomenon, meaning the evolution of tolerance mechanisms which include cell wall precipitation (binding with pectins), downregulation of transporters via the plasma membrane, rapid complexation with organic acids and peptides, i.e. low molecular weight proteins such as metallothioneins (MTs) and peptide ligands − the phytochelatins (PCs), storage as metal complexes and incorporation into crystals in vacuoles (Emamverdian et al., 2015; Hall, 2002). Resulting metal accumulation within plant tissues and organs combines the metal demand of plant metabolism and the impact of the external supply with its consequences for the storage in roots and for the translocation process from roots to shoots. As a consequence, tolerance of a plant to toxic elements – including non-specific induction of the antioxidant system in the apoplast and cell interior, as well as the biosynthesis of signaling compounds, phytohormones and other regulatory molecules in a controlled response to metal stress – is crucial for plant survival on metal polluted soil. However, simultaneous occurrence of metal ions in soil/ medium alters plant nutrition, metal accumulation rates and also

Corresponding author. E-mail address: [email protected] (M. Mleczek).

http://dx.doi.org/10.1016/j.jplph.2017.04.020 Received 24 November 2016; Received in revised form 10 April 2017; Accepted 11 April 2017 Available online 01 June 2017 0176-1617/ © 2017 Elsevier GmbH. All rights reserved.

Journal of Plant Physiology 216 (2017) 125–134

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the size of the root system to obtain a uniform group of plants and transferred into the Knop’s medium (0.5 L) containing Cu (NO3)2 × 6H2O and Ni(NO3)2 × 6H2O salts each at 0 (control), 0.5, 1, 1.5, 2, 2.5, 3 mM stabilized with steamed ultrapure quartz sand (1.24 kg per pot; pH 7.11, SiO2 content > 97%, moisture content 0.054%) in hydroponic pots (one plant per pot, four plants per treatment). The 14-day treatment was performed in a phytotron under controlled conditions (air temperature 22 ± 1 °C, relative humidity 79 ± 1%), equipped with a fluorescent lamp (MASTER TL-D Secura 58W/830 1SL) providing a radiation (photon) flux of 235 μE s−1 m−2 (μmol s−1 m−2) at the top of the plant with a 16/8 photoperiod. Plants were watered with distilled water to maintain a constant medium level during treatment.

toxicity thresholds due to antagonistic and synergistic interactions between essential and toxic elements (Kováčik et al., 2012a; Guala et al., 2010; Li et al., 2009). This aspect is of particular importance in biological methods employing trees and bushes for phytoextraction or phytostabilization of metals, performed at multi-metal polluted sites (tailings of non-ferrous mines of metallic sulfide and lateritic ores, the areas surrounding metal smelters and factories of electroplating industry, etc. where both Cu and Ni are present at high concentrations) (Komanicka et al., 2013; Friesl et al., 2006; Pulford and Watson, 2003). The natural tolerance and easy adaptation of selected plant species to toxic metals has lately focused the interest of plant ecologists, physiologists and biologists and remains the subject of an ongoing debate. The understanding of the genetic and molecular basis of this phenomenon and the identification of metabolites involved in metal tolerance and detoxification processes are of major importance. To date, numerous secondary metabolites, mainly phenolic compounds, free amino acids (proline) and low-molecular-weight peptides (glutathione), have been postulated to possess a dual function and influence both the antioxidant state of the cell and metal detoxification/storage mechanisms (Kováčik et al., 2012b; Korzeniowska et al., 2011; Handique and Handique, 2015; Mijovilovich et al., 2009; Clemens, 2006). Hybrid willow (Salix purpurea × viminalis) was chosen for the present study due to a relatively high tolerance to metals, proven in hydroponical as well as in field studies at metal contaminated sites (Goliński et al., 2015; Tlustoš et al., 2007). The most intensively studied species (Salix viminalis L.) has been recognized as an increasingly useful plant for biomass production as well as environment restoration and reclamation of disturbed landscapes (phytoremediation) (Różanowski et al., 2012; Stolarski et al., 2008; Kuzovkina and Quigley, 2005). In addition, the species has been recognized as accumulator/high accumulator of Cu, Zn, chromium (Cr) and cadmium (Cd), displaying an extraordinary tolerance to heavy metals together with long-term accumulation, fast growth and high biomass yield (Łukaszewicz et al., 2009). In the present study, S. purpurea × viminalis plants were subjected to simultaneous Cu2+ and Ni2+ treatment in a hydroponic system to evaluate the synergistic/antagonistic effects of metals’ co-existence on their toxicity and uptake. Therefore, we performed a complex comparative analysis of metal phytoextraction and the level of biometric parameters and metabolites (physiological biomarkers of plant response to metal-induced stress) following previous assessment for single metal treatments (Drzewiecka et al., 2012; Gąsecka et al., 2012). The aim of the study was to achieve a more comprehensive understanding of plant tolerance to toxic metals with diverse function, toxicity thresholds and competitive properties (such as Cu2+ and Ni2+), and to define the relations between selected primary and secondary metabolites of signaling, and antioxidant and chelating properties (soluble carbohydrates, phenolics and glutathione) in this phenomenon.

2.2. Biometric analysis The mean length of shoots, adventitious roots and leaves, and total (cumulative) leaf area (TLA) were evaluated at the beginning and at the end of the experiment to assess the inhibitory effect of metal treatment on Salix growth. The TLA was evaluated with a DOCUPEN RC 800 portable scanner with ABBYY FineReader 12.0 Sprint and Adobe Photoshop CS6 software. After termination of the experiment, total root biomass was measured by weight. 2.3. Chemical analysis Cu and Ni accumulation in Salix roots, rods, shoots and leaves, the contents of soluble carbohydrates (glucose, fructose and sucrose), total phenolic content (TPC), free, bound and total salicylic acid (SA, SAG and TSA, respectively) and thiols (glutathione – GSH and phytochelatins – PCs), in leaves were analyzed according to the methods previously described in detail by Drzewiecka et al. (2012) and Gąsecka et al. (2012). 2.4. Statistical analysis Statistical analysis was performed with Statistica 10 software provided by StatSoft and the open-source R software environment for statistical computing and graphics. The results were presented in the form of charts and tables as mean values (n = 4) ± 95% confidence intervals or standard deviation, respectively. The consistency of the normal distribution of all investigated parameters (variables) was assessed using the Shapiro-Wilk test. In the case of metal accumulation, a Box-Cox transformation was used to normalize the data. Afterwards, a two-way ANOVA for single and fixed effects of “Cu + Ni addition level” and “Salix purpurea × viminalis organ” was performed and a significant fixed effect was further analyzed with a post-hoc Tukey’s HSD test. The values of biometric parameters and physiological biomarkers of Cu2+ and Ni2+ co-toxicity were analyzed simultaneously with a multivariate analysis of variance (MANOVA) to test the differences between plants treated with different concentrations of both metals simultaneously. For MANOVA analysis, four independent tests were performed, including Wilks’ lambda (a commonly used test) and a Pillai’s trace (a conservative one) followed by one-way ANOVAs with “Cu + Ni addition level” as a single factor. Empirical p-values of one-way ANOVAs were presented within the tables. Significant ANOVAs were followed by a post-hoc Tukey’s HSD test to assess the significance of differences between Cu + Ni levels for each parameter (at α = 0.05) separately and the results were shown within the tables as upper case superscripts. A regression analysis was performed to determine the relationships between metal accumulation, parameters of morphological and physiological reaction of Salix to treatment (dependent variables) and Cu + Ni addition level (independent variable). Analysis of significance for linear regressions was performed with the F-test at α = 0.05, and only significant regressions were presented.

2. Materials and methods 2.1. Plant material and experimental design One-year-old cuttings of Salix purpurea × viminalis hybrid (previously evaluated as Salix viminalis L. cv. ‘Cannabina') collected from a three-year-old rootstock without foliage were used in the experiment (Drzewiecka et al., 2014; Mleczek et al., 2013). Plant material was obtained from Salix spp. collection maintained by the Faculty of Forestry of Poznań University of Life Sciences. To induce rooting, standardized Salix rods (approx. 25 cm long, 15 mm in diameter) were incubated for two weeks in complete Knop’s medium (10 mL of 10% Ca (NO3)2, 2.5 mL of 10% KNO3, 1.2 mL of 10% KCl, 10 mL of 2.5% KH2PO4, 5 mL of 5% MgSO4, 0.25 mL of 0.25% FeCl3 and microelements (μM): 10 NaFeEDTA, 6.25 H3BO3, 0.5 ZnSO4, 0.025 CuSO4, 0.125 Na2MoO4, 1.25 KJ, 0.5 MnCl2 and 0.025 CoCl2 diluted to 1 L with acidified water, pH 5.42). Afterwards, rods were selected according to 126

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Furthermore, multivariate linear regressions displaying relationships between the parameters of physiological reaction of S. purpurea × viminalis to Cu + Ni treatment were tested for their significance at α = 0.05. The Akaike information criterion (AIC) was employed to select the best model using the backwards elimination method. Only significant models were presented, and the adjusted coefficient of determination (R2*) was calculated to compare the significance of regressions at different degrees of freedom. To discuss the obtained results, the data were analyzed against the single metal treatments. A contrast analysis employing the least significance difference (LSD) test was performed to assess the synergistic or antagonistic effects between Cu and Ni at α = 0.01 for each addition level separately. Furthermore, the principal component analysis (PCA) of investigated physiological parameters of Salix response to single and simultaneous metal treatments was employed to determine the structure and rules of relations between variables. In order to interpret the existing relationships, orthogonal transformation to a new set of noncorrelated variables (components) was performed.

Table 2 Analysis of significance for linear regressions displaying relationships between metal accumulation, biometric and physiological parameters of Salix purpurea × viminalis response to simultaneous Cu2+ and Ni2+ treatment (dependent variables) and metal concentration in cultivation medium (independent variable) at α = 0.05 (R2–coefficient of determination, p − empirical level of significance). Only significant regressions (p ≤ 0.05) are presented in the table. Dependent variables (Y)

Linear regression analysis (metal addition level as an independent variable X) R2

p

Cu content in Salix organs roots 0.9571 < 0.001 rods 0.9871 < 0.001 shoots 0.9854 < 0.001 leaves 0.9442 < 0.001 Ni content in Salix organs roots 0.9428 < 0.001 rods 0.9573 < 0.001 shoots 0.9264 < 0.001 leaves 0.9757 < 0.001 Biometric parameters root biomass 0.6179 < 0.05 root length 0.7456 < 0.001 shoot length 0.7980 < 0.001 total leaves area 0.8239 < 0.001 leaf length 0.7041 < 0.001 Soluble carbohydrates content in Salix leaves glucose 0.6614 < 0.05 fructose 0.6570 < 0.05 sucrose 0.7439 < 0.001 Phenolics content in Salix leaves free salicylic acid 0.6065 < 0.05 total phenolics 0.5999 < 0.05 Thiol content in Salix leaves glutathione 0.6487 < 0.05

3. Theory/calculation Plant responses to elevated concentration of metals and metalloids vary according to the conditions of metals’ co-existence in the environment. Thus, synergistic and antagonistic effects are observed including metal uptake, translocation rates and induction of plant resistance. The knowledge on the interactions between certain metals − commonly occurring at multi-metal contaminated sites − still remains unclear. Furthermore, metal-induced accumulation of secondary metabolites results from disturbances in soluble carbohydrates metabolism caused by the metal ions. Among these, salicylic acid and glutathione together play a complex role in metal detoxification and regulate the antioxidant state of the cell.

Regression equation

Y = 6.32 × X − 1.37 Y = 4.29 × X + 1.54 Y = 0.93 × X + 0.23 Y = 1.36 × X + 0.80 Y = 4.52 × X − 1.06 Y = 4.38 × X + 0.40 Y = 1.03 × X + 0.32 Y = 1.18 × X + 0.10 Y = −2.45 × X + 7.17 Y = −2.03 × X + 8.15 Y = −2.01 × X + 8.52 Y = −24.10 × X + 184.07 Y = −0.90 × X + 5.26 Y = 80.07 × X − 2.80 Y = 31.36 × X + 43.36 Y = 3.20 × X + 1.01 Y = 4.37 × X + 1.16 Y = 16.18 × X + 8.10 Y = 57.26 × X + 110.95

and 0.62, for shoots length, total leaf area and root biomass, respectively) (Table 2). The accumulation of Cu and Ni differed significantly among Salix organs and decreased in the order roots ≈ rods > > leaves ≥ shoots, reaching each time the highest value at 3 mM treatment (Table S1, Figs. 1A and B). At lower concentrations (up to 1.5 mM for Cu, and 2.5 mM for Ni), metal phytoextraction was higher in wooden rods than in adventitious roots. Cu content in roots reached ∼20 μg g−1 dry weight (DW) at 3 mM, and was significantly higher when compared to Ni (∼14.5 μg g−1 DW), while phytoextraction in rods remained at a comparable level for both metals (∼14 μg g−1 DW). Furthermore, both metals were hardly translocated to the photosynthetic organs. Ni accumulation reached ∼3.6 mg g−1 DW in leaves and was slightly lower in shoots, while Cu content in leaves differed significantly from shoots, reaching ∼4.5 and 2.7 μg g−1 DW, respectively (Figs. 1A and B). A linear regression analysis revealed, that metal phytoextraction was

4. Results Analysis of biometric parameters revealed overall growth inhibition along with an increase of Cu2+ and Ni2+ available for willow plants (Table 1). Significant decreases of their values in relation to control plants were noted for all metal-treated plants with the exception of the total leaf surface at 0.5 mM. The negative impact of metals was strongly affirmed in the case of root system, with nearly 90% loss of root biomass at 3 mM, rather than for the above-ground organs (shoots and leaves). The average leaf length was reduced to 46%, while the photosynthetic area was reduced only to 59%, indicating stronger inhibition of leaf elongation caused by metal treatment. All the investigated parameters of plant growth were significantly (p ≤ 0.05) correlated with Cu + Ni level in the medium. However, stronger negative correlations were observed in the case of shoots and leaves (size of the above-ground organs) than for root system parameters (R2 = 0.82, 0.80

Table 1 The effect of Cu + Ni addition level on biomass parameters of Salix purpurea × viminalis and their change in relation to control plants. Cu + Ni addition [mM]

Leaf length (LL)

[cm] 0 0.5 1 1.5 2 2.5 3 p-value

Total leaf area (TLA)

% of control

a

6.35 ± 0.15 4.23b ± 0.14 3.77c ± 0.12 3.38de ± 0.07 3.51cd ± 0.04 3.16ef ± 0.05 2.95f ± 0.06 < 0.001

[cm2]

% of control a

67 59 53 55 50 46

194.37 ± 12.72 172.90ab ± 7.50 151.43bc ± 7.33 137.93cd ± 3.71 128.50cd ± 8.76 136.50cd ± 12.77 113.80d ± 5.52 < 0.001

Shoot lenght (SL)

Root lenght (RL)

[cm]

[cm]

% of control

a

89 80 71 66 70 59

9.59 ± 0.14 8.19b ± 0.07 5.12c ± 0.10 3.86f ± 0.12 4.51d ± 0.12 4.29e ± 0.07 3.01g ± 0.06 < 0.001

Roots biomass (RB)

% of control a

81 53 40 47 45 31

10.27 ± 0.17 6.35b ± 0.12 4.85c ± 0.14 3.69e ± 0.19 4.21d ± 0.18 3.42e ± 0.10 2.98f ± 0.12 < 0.001

[g]

% of control a

62 47 36 41 33 29

10.87 ± 0.15 3.75b ± 0.12 2.90c ± 0.13 2.15d ± 0.07 1.86d ± 0.12 1.83d ± 0.11 1.06e ± 0.14 < 0.001

34 27 20 17 17 10

Mean values (n = 4) ± standard deviations. Identical superscripts denote no significant (p > 0.05) differences between Cu + Ni additions for each parameter (within a column) according to a post-hoc Tukey’s HDS test.

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highest accumulation, over 10-times higher than in untreated plants, was noted for glucose and sucrose at 3 and 2.5 mM, respectively. Lower values were observed for fructose, being nearly half of the glucose content at the highest metal concentration. At lower metal levels (0.5–2 mM), the contents of glucose, fructose and sucrose gradually increased, but at 3 mM, a switch between glucose and sucrose accumulation was observed in favor of glucose accompanied by a relative reduction of sucrose content. The accumulation of soluble carbohydrates was significantly (p ≤ 0.05) correlated with the metal addition level, and the strongest relationship was observed in the case of sucrose content (R2 = 0.66 and 0.75 for glucose, fructose and sucrose, respectively) (Table 2). Total phenolic content in Salix leaves ranged from ∼9 to 79 mg GAE g−1 DW (for control and 3 mM treatment, respectively) (Table 4). However, at 2.5 mM a relative drop of their content was observed followed by a conspicuous increase at 3 mM. Among phenolic compounds biosynthesized in Salix leaves, salicylic acid and its glucoside were particularly assayed as biomarkers of metal-induced oxidative stress. Simultaneous treatment of willow plants with Cu2+ and Ni2+ led to an evident increase of salicylic acid accumulation compared to untreated controls, i.e. from ∼2.6 to 60 μg g−1 DW for total (free and glucose bound) salicylic acid in control and 1.5 mM-treated plants, respectively. The accumulation of free salicylic acid reached the highest value at 2.5 mM and was nearly 19 times higher than for control plants (∼17.2 μg g−1 DW) (Table 4). However, a concentration of the metabolite did not strictly follow the increase of metal concentration in the cultivation medium, showing a significant drop at 2 and 3 mM, both for free and glycoside forms of the metabolite. Nevertheless, the overall accumulation of phenolic compounds as well as the content of free salicylic acid in Salix leaves were significantly (p ≤ 0.05) correlated with Cu + Ni concentration in the medium at R2 = 0.66 and 0.61, respectively (Table 2). The presence of thiol compounds in Salix leaves as a response to metal treatment was also determined. The results demonstrated that phytochelatin synthesis was not induced in control or Cu + Ni-treated plants, while the accumulation of their precursor (glutathione) was strongly enhanced by metal treatment and correlated with metal addition level (R2 = 0.65, p ≤ 0.05) (Table 1). The highest GSH content in leaves was 218 μg g−1 DW (∼340% of the control) and was observed at 2.5 mM treatment. After exceeding this value, a significant drop to ∼260% at 3 mM was observed (Table 5). Multivariate regression analysis (Table 6) revealed a significant linear correlation between metal uptake and the accumulation of soluble carbohydrates (glucose, fructose and sucrose) in all Salix organs for both investigated metals. In the case of photosynthetic tissue, Cu accumulation was also correlated with elevated glutathione, and Ni with total phenolic and glutathione contents. An inverse linear dependence on leaf Cu load was observed for all investigated biometric parameters of metal-treated Salix, while Ni accumulation linearly

Fig 1. Copper (A) and nickel (B) content in Salix purpurea × viminalis organs – mean values (n = 4) ± 95% confidence intervals according to two-way ANOVA for “Metal addition level × organ” fixed effect (α = 0.05).

strongly dependent on its concentration in Knop’s medium in the case of each willow organ (R2 = 0.9264–0.9871) (Table 2). However, exponential dependence may also be evaluated for roots, indicating the perpetuation of metal sorption/accumulation by the root system with increasing Cu2+ and Ni2+ levels in the solution (Figs. 1A and B). The MANOVA analysis revealed considerable differentiation of plants as an effect of increasing Cu+2 and Ni+2 concentrations in medium on investigated physiological parameters of Salix plants (both Wilks’ lambda and Pillai’s trace were significant at p ≤ 0.001) (Table S2). This confirmed the hypothesis of a close relationship between metal-induced stress indicators selected for the study, and allowed us to perform one-way ANOVAs for each parameter separately. Glucose, fructose and sucrose contents in willow leaves were significantly higher for all Cu + Ni-treated plants in relation to control ones (Table 3). The

Table 3 The effect of Cu + Ni addition level on soluble carbohydrates (glucose, fructose and sucrose) contents in Salix purpurea × viminalis leaves and their change in relation to control plants. Cu + Ni addition [mM]

Glucose (G)

[mg g−1 DW] 0 0.5 1 1.5 2 2.5 3 p-value

Fructose (F)

% of control

g

29.42 ± 2.42 45.86f ± 1.46 62.20e ± 2.05 112.04c,d ± 5.16 124.67b,c ± 4.49 102.29d ± 3.83 344.62a ± 6.75 < 0.001

[mg g−1 DW]

Sucrose (S)

% of control

g

156 211 381 424 348 1171

34.50 ± 3.57 72.72f ± 2.53 85.23c,d ± 3.83 95.67b ± 2.57 89.64b,c ± 3.51 81.55d ± 2.63 173.50a ± 4.37 < 0.001

[mg g−1 DW]

% of control

e

211 247 277 260 236 503

1.11 ± 0.07 2.47d ± 0.37 2.42d ± 0.15 6.39c ± 1.09 8.59b ± 1.08 12.19a ± 1.05 7.52b,c ± 1.19 < 0.001

223 218 576 774 1098 677

Mean values (n = 4) ± standard deviations. Identical superscripts denote no significant (p > 0.05) differences between Cu + Ni additions for each parameter (within a column) according to a post-hoc Tukey’s HDS test.

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Table 4 The effect of Cu + Ni addition level on total phenolics and salicylic acid contents in Salix purpurea × viminalis leaves and their change in relation to control plants. Cu + Ni addition [mM]

Total phenolics (TPC)

[mg GAE g−1 DW] 0 0.5 1 1.5 2 2.5 3 p-value

Free salicylic acid (SA)

[μg g−1 DW]

% of control

e

9.39 ± 0.25 17.81d ± 2.06 26.98c ± 2.90 34.63b ± 3.26 36.96b ± 2.25 22.05c ± 1.89 78.73a ± 2.63 < 0.001

Total salicylic acid (TSA)

% of control

e

0.92 ± 0.15 3.22d ± 1.15 2.69d ± 1.29 12.78b ± 1.69 6.35c ± 1.13 17.24a ± 2.04 10.72b ± 1.47 < 0.001

190 287 369 394 235 838

[μg g−1 DW]

% of control

f

350 292 1389 690 1874 1165

2.59 ± 0.55 26.54d ± 3.25 10.41e ± 1.12 60.00a ± 3.74 7.78e ± 1.79 50.17b ± 2.43 35.69c ± 2.81 < 0.001

1024 402 2317 300 1937 1378

Mean values (n = 4) ± standard deviations. Identical superscripts denote no significant (p > 0.05) differences between Cu + Ni concentrations for each parameter (within a column) according to a post-hoc Tukey’s HDS test.

level (0.5 mM), partly resulting from Ni-caused restriction of Cu accumulation in shoots (Table 7). Similarly, Theriault and Nkongolo (2016) found no significant additive effects of combined metal treatment (Ni + Cu) on metal toxicity to white birch (Betula papyrifera) applied at concentrations equivalent to the levels found in the soils of the Greater Sudbury Region (Canada) due to smelting emissions.

Table 5 The effect of Cu + Ni addition level on glutathione contents in Salix purpurea × viminalis leaves and their change in relation to control plants. Cu + Ni addition [mM]

Glutathione (GSH)

[μg g−1 DW] 0 0.5 1 1.5 2 2.5 3 p-value

% of control

5.2. Metal uptake

d

85.17 ± 4.33 152.15c,d ± 7.14 173.69b,c ± 15.21 190.66b,c ± 11.77 235.82b ± 36.69 321.87a ± 34.74 218.53b,c ± 32.95 p < 0.05

179 204 224 277 378 257

In the present study, Cu2+ and Ni2+ applied simultaneously to S. purpurea × viminalis plants slightly stimulated their transport to leaves and diminished their phytoextraction in roots comparing to single metal treatments (Table 7). Still, metal partitioning within willow organs confirmed previous results on low translocation rates of both metals in Salix (Ali et al., 2003). According to Korzeniowska et al. (2011), both Cu and Ni were strongly accumulated in roots, but Ni translocation to leaves may achieve equal levels as the content in the root system (depending on the metal concentration). In the tested willow, Ni2+ triggered restriction of Cu uptake in roots at concentrations up to 1.5 mM, and in shoots for all treatments accompanied by an increase of Cu uptake into rods and leaves. Cu2+ coexistence resulted in inhibition of Ni uptake by roots and slightly altered its translocation in favor of shoots and leaves (Table 7). A similar effect was previously observed in barley (Körner et al., 1987) and mulberry seedlings (Lou et al., 1991). According to those studies, Cu2+ was strongly competitive with Ni2+ and inhibited its accumulation in roots. Furthermore, an increase of Ni2+ in soil decreased Cu content in caryopses of Triticum aestivum (Pandolfini et al., 1992). However, the study of Ghasemi et al. (2009) revealed that Ni2+ elevated Cu accumulation in roots and shoots of Ni hyperaccumulator Alyssum inflatum, causing the increase of plant sensitivity to copper as a result of Cu-caused exclusion of iron (Fe). Still, this synergism was not observed in the case of a non-accumulator species of the genus Alyssum (Ghasemi et al., 2014).

Mean values (n = 4) ± standard deviations. Identical superscripts denote no significant (p > 0.05) differences between Cu + Ni additions for each parameter (within a column) according to a post-hoc Tukey’s HDS test.

influenced only the shoot length. The induction of phenolic compounds was strongly correlated with accumulation of single sugars (glucose, fructose), whereas glutathione and salicylic acid contents were mediated by elevated sucrose and particularly correlated with Ni content in Salix leaves.

5. Discussion 5.1. Growth parameters The first quantitative symptom of metal toxicity is retardation of plant growth. Ali et al. (2003) reported a similar level of the detrimental effect of Cu2+ and Ni2+ on Salix acmophylla biomass. Both Cu2+ and Ni2+ are mineral micronutrients essential for higher plants. In Arabidopsis copper is bind to 105 and nickel to 3 proteins (Hänsch and Mendel, 2009; Krämer and Clemens, 2005). As a consequence, their function and average basal contents vary significantly among higher plants (∼1–5 μg g−1 DW for Cu and 2–4 ng g−1 DW for Ni), whereas toxicity thresholds have been found to be at a similar level (∼20–30 μg g−1 DW for Cu and 10–50 μg g−1 DW for Ni) (Chen et al., 2009; Seregin and Kozhevnikova, 2006; Yruele, 2005). However, simultaneous contamination of soil with equal concentrations of Cu2+ and Ni2+ significantly depleted the yield of oats comparing to plants cultivated in soil spiked with these metal separately (Wyszkowska et al., 2007). A synergistic effect on growth reduction was also observed for mulberry seedlings treated with both metals simultaneously (Lou et al., 1991). In the present study, adjustment of the negative effect of Cu2+ and Ni2+ on biomass parameters was not observed. In contrast, a deleterious influence of Ni2+ on Cu-induced retardation of shoot length was observed for all metal combinations excluding the lowest addition

5.3. Accumulation of soluble carbohydrates and phenolics A toxic effect of metals on plant biomass may result from metalinduced disturbances in photosynthesis leading to elevated accumulation of soluble sugars. In the case of maize, Ni2+ treatment at 20–500 μM caused enhanced accumulation of carbohydrates in shoots and strongly inhibited roots growth (Baccouch et al., 2001). Changes in sugars content indicating disturbances in carbohydrates metabolism were previously observed in needles of Scot pine seedlings treated both with Ni2+ and Cu2+ (Roitto et al., 2005). Carbohydrate elevation in shoots/leaves may be a result of photosynthesis repression (mainly photosynthesis rate), but also inhibited starch hydrolysis and/or transport of sucrose caused by the elevated metal level in medium (Araya et al., 2010; Parrot et al., 2005). In the present study, simultaneous treatment of Salix plants with Cu2+ and Ni2+ significantly 129

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Table 6 Analysis of significance for multivariate linear regressions displaying relationships between metal accumulation, biometric and physiological parameters of Salix purpurea × viminalis response to simultaneous Cu2+ and Ni2+ treatment (α = 0.05; a – the “Y intercept” i.e., the value of Y when X is zero, b – estimate of regression coefficient, R2* – adjusted coefficient of determination, P – empirical level of significance for the variable, p – empirical level of significance for the model). Only significant regressions are presented in the table (*** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05). Dependent variables (Y)

Independent variables (X)

Multivariate linear regression coefficients and analysis of their significance

Cu content in Salix organs Roots G, S Rods TPC, S, GSH Shoots G, S, GSH Leaves F, S, GSH Ni content in Salix organs Roots G, S Rods G, S, GSH Shoots S, TPC Leaves TPC, S, GSH Biomass parameters RB Cu (leaves) RL Cu (leaves) SL Ni (shoots), Cu (leaves) LL Cu (leaves) TLA Cu (leaves) Soluble carbohydrates content in Salix leaves G TPC, Ni (roots) F TPC, Cu (rods) S GSH, Ni (leaves),SA Phenolics content in Salix leaves TPC G, F SA S TSA S Thiol content in Salix leaves GSH S

A

b

R2*

P

−1.51 −1.39 −0.22 −0.64

0.04, 0.92 0.10, 0.39. 0.02 0.004, 0.09, 0.004 0.02, 0.13, 0.006

0.8988 0.8982 0.9039 0.8945

***, ***, ***, ***,

*** *, * *, * *, *

*** *** *** ***

−1.26 −1.63 0.10 −0.63

0.02, 0.02, 0.18, 0.02,

0.8928 0.8876 0.8170 0.9089

***, ***, ***, ***,

*** *, * *** **, *

*** *** *** ***

8.60 9.22 9.40 5.74 193.78

−1.81 −1.45 −1.02, −0.70 −0.65 −16.17

0.7062 0.8127 0.8729 0.7751 0.7897

*** *** *, * *** ***

*** *** *** *** ***

−23.43 30.47 −1.36

3.35, 5.56 1.37, 1.99 0.02, 0.99, 0.20

0.9031 0.8836 0.8724

***, ** ***, * *, *, *

*** *** ***

−2.56 0.63 11.11

0.10, 0.25 1.22 2.84

0.9216 0.6994 0.2560

***, ** *** *

*** *** *

103.91

15.99

0.7547

***

***

0.72 0.46, 0.02 0.02 0.15, 0.004

p

F– fructose, G − glucose, GSH − glutathione, LL − leaf length, RL − root length, RB − roots biomass, S − sucrose, SA − free salicylic acid, SL − shoot length, TLA − total leaves area, TPC − total phenolics content. Table 7 Significant contrasts according to Fisher’s LSD test of Cu2+ and Ni2+ simultaneous (Cu + Ni) and individual (Cu or Ni) treatment effects on metal accumulation, biometric and physiological parameters of Salix purpurea × viminalis response (only significant interactions according to two-way ANOVA at α = 0.01 were considered). Parameter

LSD0.01

(Cu + Ni)-Cu 0.5

Cu content in Salix organs Roots 0.12 (−) Rods 0.12 (−) Shoots 0.12 (−) Leaves 0.12 (+) Ni content in Salix organs Roots 0.62 Rods 0.62 Shoots 0.62 leaves 0.62 Biometric parameters shoot length 2.19 Soluble carbohydrates content in Salix leaves glucose 2.40 (−) fructose 2.43 (+) sucrose 7.05 (−) Phenolics content in Salix leaves free salicylic acid 1.74 (+) total salicylic acid 7.71 (+) total phenolics 2.21 (+) Thiols content in Salix leaves glutathione 61.63 (+)

(Cu + Ni)-Ni

1

1.5

2

2.5

3

(−) (+) (−) (+)

(−) (+) (−) (+)

(+) (+) (−) (+)

(+) (+) (−) (−)

(+) (+) (−) (+)

0.5

1

1.5

2

2.5

3

(+) (+) (−) (+)

(−) (+) (−) (+)

(−) (−) (+) (+)

(−) (−) (+) (+)

(−) (+) (+) (+)

(−) (−) (+) (+)

(+) (+)

(+) (+)

(+) (+)

(+) (+)

(+) (+)

(+) (+)

(−)

(−)

(−)

(−)

(−)

(−) (+) (−)

(+) (+) (−)

(+) (+) (−)

(+) (+) (−)

(+) (+) (−)

(+)

(+)

(+)

(+) (+) (+)

(+)

(+) (+) (+)

(+) (+) (+)

(+) (+)

(+) (−) (+)

(−) (+)

(−) (+)

(+) (−) (+)

(+) (−) (+)

(+)

(+)

(+)

(+)

(+)

(+)

(+)

(+)

(+)

(+)

(+)

by glucose and/or sucrose molecules. In yellow lupine, exogenous sucrose was proven to strongly stimulate the expression of genes encoding key enzymes of flavonoid biosynthesis (PAL – phenylalanine ammonia lyase; CHS – chalcone synthase; CHI – chalcone flavanone isomerase; and IFS – isoflavonoid synthase) (Morkunas et al., 2011). Thus, the elevated accumulation of soluble carbohydrates, serving as donors for

enhanced the accumulation of glucose and fructose in leaves, but Cuinduced sucrose elevation was reduced by Ni2+ co-existence in the medium, indicating distinct modes of their toxicity (Table 7). Metalinduced disturbances in source-sink balance may lead to depletion of plant nutrition and biomass accompanied by allocation of excess carbon to phenolics via induction of shikimate and phenylpropanoid pathways 130

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Vicente and Plasencia, 2011). Our previous studies revealed enhanced biosynthesis of the metabolite in plants challenged with both biotic and abiotic stressors (Phytophthora infestatns, Fusarium proliferatum and F. oxysporum, pea aphid, heavy metals and tropospheric ozone) (Arasimowicz-Jelonek et al., 2014; Mai et al., 2014; Drzewiecka et al., 2012; Dobosz et al., 2011; Borowiak et al., 2012). Among studied plants, the highest accumulation of salicylic acid was observed in leaves of Salix viminalis L. treated hydroponically with Ni2+, and metal treatment resulted in a conspicuous increase of its content from ∼2.5 to 80 μg g−1 DW for 0 and 2.5 mM Ni, respectively. Nevertheless, still scare information is available on biosynthesis, transport and action modes of this metabolite in plants challenged with metal-induced oxidative stress. The majority of studies concern the influence of seeds priming or plants’ pretreatment with salicylic acid on metal uptake and tolerance, and relatively limited data are available on the functioning of endogenous SA during metal stress. As previously documented, exogenous SA greatly diminished cadmium accumulation, translocation rate and toxicity to rice (Choudhury and Panda, 2004), pea (Popova et al., 2009) and flax (Belkadhi et al., 2012) plants propagated from SA-primed seeds. However, Kováčik et al. (2009) observed the elevation of nickel bioaccumulation in photosynthetic tissue as a result of simultaneous treatment of chamomile plants with salicylic acid and Ni2+. This may indicate that salicylic acid possesses a dual function − as a plant protectant (signaling and regulatory metabolite), but also as a metal chelator for some ions. Still, potentiometric titrations revealed that Cu2+ was able to form stable chelates with salicylic acid, in contrast to Ni2+, for which low stability chelates were found (Pecci and Foye, 1960). In the present study, simultaneous treatment of Salix purpurea × viminalis with Cu and Ni ions induced enhanced accumulation of salicylic acid in plant leaves, probably as a result of its in situ biosynthesis and phloem transport from roots. The contrast analysis revealed the antagonistic effect of Cu2+ on Ni2+-induced TSA accumulation at each concentration excluding 0.5 mM (the lowest one). However, a synergic effect was observed for both metals on SA – a biologically active form easily transported via phloem to photosynthetic tissue, which indicates the intensification of Cu2+ and Ni2+ toxicity as the effect of their coexistence in the environment (Table 7). Metal-induced accumulation of endogenous SA was previously documented by Pál et al. (2005) in leaves of maize seedlings under cadmium stress, although its content in roots remained at a constant level. Our previous studies also confirmed changes in salicylic acid content in above-ground organs rather than in roots of plants challenged with oxidative stress (also in the case of root treatment/inoculation with Fusarium spp.) (Waśkiewicz et al., 2013). Enhanced accumulation of salicylic acid and its up- and downstream metabolites was also observed across different species of Thlaspi with Ni/Zn hyperaccumulation abilities, and was accompanied by an increase of glutathione content (Freeman et al., 2005). Therefore, the authors presume that high tolerance of Thlaspi to elevated concentrations of these metals is glutathione-mediated and signaled by constitutively ascending levels of salicylic acid. This contrasts with the conclusion of Tao et al. (2013). In the case of Arabidopsis, the expression of nahG in snc1 plants led to SA deficiency and mitigated the Pb- or Cd-induced growth retardation, proving the involvement of the metabolite in metal phytotoxicity (Tao et al., 2013). However, Pál et al. (2002) reported that SA potentially blocks the activity of phytochelatin synthase (PCS) to maintain a sufficient GSH level in the cytosol to act efficiently as an antioxidant rather than a PC precursor. The authors concluded that an increased GSH pool allows plants to withstand the Ni-induced oxidative stress due to its detoxification and antioxidative properties (Freeman et al., 2004). This was valid also in the present study and was corroborated by a significant dependence of glutathione contents on salicylic acid and sucrose, which probably serves as a signaling molecule inducing SA biosynthesis in Ni-treated Salix. Furthermore, our results demonstrated that PC synthesis was not induced in leaves of Cu + Ni-treated S.

carbon skeletons for secondary metabolites, may affect signaling pathways of primary and secondary metabolism activated in plants under conditions of oxidative stress (Morkunas et al., 2011; Rosa et al., 2009; Morkunas et al., 2005; Roitto et al., 2005). This was confirmed in the investigated Salix by a strong linear correlation between TPC and simple sugar (glucose and fructose) contents in the case of both single (Cu, Ni) and combined (Cu + Ni) treatments. Among numerous secondary metabolites synthesized in plant cells and secreted under metal stress, phenolic compounds are considered essential (Janas et al., 2010; Kováčik et al., 2010; Kováčik et al., 2008; Białońska et al., 2007; Ali et al., 2003). According to Rastgoo et al. (2014), both Cu2+ and Ni2+ were able to induce accumulation of phenolics in Aeluropus littoralis leaves. This is a wide group of biomolecules including flavonoids, phenolic acids, stilbenes, tannins, etc. with one or more aromatic rings in the chemical structure bonded to hydroxyl groups giving them unique antioxidant (mainly cinnamic acid derivatives, e.g. chlorogenic and ferulic acids) and chelating (protocatechuic acid, tannins) properties (Kısa et al., 2016; Ghasemzadeh and Ghasemzadeh, 2011; Michalak, 2006; Gratão et al., 2005; Roitto et al., 2005). Furthermore, phenolic acids are structural components of lignin polymer (e.g. p-coumaric, ferulic and sinapic acids), and enhanced lignification of the plant cell wall under metal stress provides a mechanical barrier limiting metal entry to the cell interior (Finger-Teixeira et al., 2010). The activity of phenolic metabolism enzymes (e.g. SKHD – shikimate dehydrogenase, PAL), and resulting total accumulation of phenolics as well their composition/profile differs significantly in plants from heavily polluted habitats and may serve as indicators in biomonitoring studies in areas with intense anthropopression (Loponen et al., 2001). Our previous study confirmed that willow plants cultivated at a metal contaminated site (Ni-Cu tailings facilities) accumulated relatively higher levels of phenolics (i.e. chlorogenic, trans-cinnamic, ferulic, p-coumaric, vanilic and sinapic acids) and total flavonoids in leaves than control plants (Gąsecka et al., 2017). In the current study, simultaneous treatment of Salix purpurea × viminalis with Cu2+ and Ni2+ led to the enhanced accumulation of phenolic compounds in leaves, and a significant synergic effect of both metals on TPC was observed, proving the augmentation of Cu2+ and Ni2+ cotoxicity (Table 7). However, Roitto et al. (2005) proved an antagonistic effect of Cu2+ on Ni-induced elevation of selected flavonols (quercitin, kaempferol, catechin and astragalin) in needles of Pinus sylvestris L. However, a similar effect was not observed for condensed tannins, probably involved in Ni detoxification, which may ameliorate metal ions through chelation (their content in Cu + Ni-treated plants was comparable as for Ni). This indicates significant variation of the phenolics profile, which depends on metal type and is accompanied by an overall increase of phenolic content in metal-treated plants. 5.4. Salicylic acid and glutathione accumulation In the group of phenolic compounds, salicylic acid is a widely distributed secondary metabolite playing a complex role in plant growth and development (endo- and exogenous SA regulates seed germination, leaf elongation, flowering and thermogenesis, etc.), and in response mechanisms against numerous environmental stressors (Wani et al., 2016; Kovács et al., 2014; Dixon and Paiva, 1995). During pathogen invasion, enhanced SA biosynthesis in hypersensitive response (HR) induces suicidal programmed cell death (PCD) to limit the attack and restrict the infection site. Furthermore, salicylate serves as an intra- and interplant signaling molecule to develop systemic acquired resistance (SAR) with the induction of pathogenesis-related proteins (PRs) in uninfected organs and also in neighbor plants (Raskin, 1992). Salicylic acid’s role in plant resistance to biotic stressors has been studied in depth, but its functioning in response mechanisms against abiotic factors, mainly of anthropogenic origin (such as meal(loid)s, xenobiotics, and tropospheric ozone) is still unclear and remains the subject of an ongoing debate (Berkowitz et al., 2016; Khan et al., 2015; Rivas-San 131

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phenolics and glutathione (serving both as antioxidants and metal chelators) in leaves was induced at a comparable level by both metals applied separately, as well as fructose and glucose contents, and the synergistic effect was observed for simultaneous metal application. However, for extensive studies of reaction mechanisms, the phenolic compounds should be profiled to distinguish the role of particular compounds in metal chelation and free radical scavenging. Regardless to the metal, simple sugars (glucose, fructose) and total phenolics were strongly correlated, indicating a cross-talk between primary and secondary metabolism in metal-stressed plants (signaling compounds and carbon donors) (Fig. 2). Furthermore, sucrose accumulation in willow leaves was strongly induced by Cu2+ rather than Ni2+. In contrast, Ni2+ caused elevated accumulation of salicylic acid, indicating distinct reaction modes of the plant to Cu2+ and Ni2+. Simultaneously, glutathione accumulation was correlated with free and less with total salicylic acid contents in leaves of Ni-treated Salix (Fig. 2), proving the SA-induced biosynthesis of GSH under conditions of metal stress. Acknowledgments This work was supported by the National Science Centre of Poland [grant number 2014/15/B/NZ9/02172]. Appendix A. Supplementary data

Fig. 2. Principal component analysis (PCA) of investigated physiological parameters of Salix purpurea × viminalis response to single (Cu, Ni) and simultaneous (Cu + Ni) metal treatments (F – fructose, G – glucose, GSH – glutathione, S – sucrose, SA – free salicylic acid, SAG – salicylic acid glucoside, TSA – total salicylic acid).

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jplph.2017.04.020.

purpurea × viminalis. Apparently, PCs are not responsible for in Cu and Ni detoxification and tolerance in this Salix clone despite high levels of glutathione probably induced by the elevated salicylic acid. A lack of PC synthesis in metal-treated plants was also reported for the Ni-hyperaccumulating tree Sebertia acuminate (Sagner et al., 1998), the Co hyperaccumulator Crotalaria cobalticola (Oven et al., 2002), Sedum alfredii from a Zn and Pb polluted site (Sun et al., 2005) and various clones of Salix viminalis exhibiting different metal tolerance (Gąsecka et al., 2012; Landberg and Greger, 2004). Resulting GSH elevation was found particularly important in Cu2+ and Ni2+ tolerance of various Thlaspi species (Freeman et al., 2004; Freeman et al., 2005), whereas an Arabidopsis mutant (cad2) with a reduced capacity to produce GSH was found to be hypersensitive to Cu2+ (Cobbett et al., 1998). In the present study, similarly as for SA and TPC, both metals acted synergistic on GSH content when applied simultaneously; however, the addition of Ni2+ caused greater intensification of plant reaction to treatment than Cu2+ (Table 7).

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