INTRODUCTION

Copper (Cu) is an essential plant micronutrient, but it can be phytotoxic in concentrations even slightly above the level of sufficiency [1]. Plant species and genotypes differ greatly in their ability to withstand damage induced by Cu exposure. The values of the effective Cu concentrations that lead to 25 to 50% (EC25 or EC50, respectively) degradation in biological performance of sensitive versus tolerant species and genotypes [2] may differ by up to two orders of magnitude.

Copper damage is associated with reactive oxygen species, where it may manifest as unspecific injuries to cellular membrane [3, 4]. Enzyme problems and metallic cation replacement in metalloproteins have also been reported [1, 5]. Furthermore, excessive copper concentrations affect nutrient uptake in sensible species [3, 4]. However, the effects of copper on nutrient uptake in tolerant species have not been studied equally well.

Acacia caven (Mol.) Mol. (Fabaceae) is a typical native species in the central part of Chile that has Mediterranean-type climate [6]. This species is also known as Vachellia caven (Molina) Seigler & Ebinger. In particular, it can be found in abandoned mine tailings in north-central Chile [7]. It has been shown to have constitutive tolerance to Cu stress [6]. On the other hand, Helianthus annuus L. (Asteraceae), the common sunflower, exhibits high Cu sensitivity [8]. Both species have large germplasm, making them suitable for hydroponic experiments.

The main objective of the study was to determine the effect of rising Cu exposure on nutrient uptake rates and root/shoot nutrient contents in two species with distinct copper tolerance: A. caven and H. annuus.

MATERIALS AND METHODS

Hydroponic experiment. A hydroponic experiment was conducted in order to evaluate plant Cu and nutrient uptake. The seeds of certain Chilean native woody species need to be pretreated with acids to achieve germination [9]. Hence, Acacia caven (Mol.) Mol. seeds (Til Til ecotype) were pretreated in concentrated sulfuric acid (90%) for 2 hours and then rinsed with deionized water. A commercial Helianthus annuus L. hybrid variety was chosen for the experiment. Sunflower seeds were disinfected with 70% ethanol for 30 seconds and then rinsed with deionized water. Seeds of both species were germinated on A-6 grade perlite until the radicle reached 10 to 15 mm in length, which occurred at 4 and 3 days for A. caven and H. annuus, respectively.

After germination, one hundred uniform seedlings were harvested as initial growing seedlings (hence forth referred to as the first harvest) and another thirty were transplanted onto polystyrene sheets and then placed over 2.5 L containers with 1/5 strength Hoagland solution. Preference was given to the diluted Hoagland solution because of the plants’ tendency for a stronger Cu response in lower ionic strength solutions, and also to avoid Cu-phosphate precipitation [10]. The composition of the nutrient solution was as follows: 0.5 mM KNO3, 0.5 mM Ca(NO3)2, 0.2 mM MgSO4, 0.1 mM K2HPO4, 0.02 mM Fe-EDDHA 6%, 10 μM H3BO3, 2 μM MnCl2, 0.2 μM ZnSO4 and 0.1 μM MoO3. No buffer was added to the nutrient solution.

Copper treatments were applied upon transplantation at concentrations of 0, 2.0, 3.9, 7.9, and 15.7 μM. Copper was applied from a concentrated Cu stock solution of 18.7 mM CuSO4, stirred and left to equilibrate for 1 hour. Solution pH was in the range of 6.6–6.7, with no statistical differences between the treatments. Copper treatments continued until plant harvest (henceforth referred to as the second harvest). Nutrient solutions were continuously aerated and replaced on a daily basis. Each treatment was replicated three times. All experiments were performed in a controlled climate chamber (light/dark regime: 12/12 h at 22 ± 3°C, relative humidity 46% and light intensity of 27 μmol/(m2 s).

In pre-test, secondary roots appeared on 5th and 4th day after transplant in A. caven and H. annuus, respectively. In order to obtain the maximum principal root elongation, the seedlings were harvested one day before the emergence of secondary roots, which occurred 4 and 3 days after transplant for A. caven and H. annuus, respectively. Seedlings were rinsed with deionized water, following the protocol of other studies on the sample preparation of plant material for the quantitative analysis of metals [11–13]. Subsequently, root length and fresh weight of shoots were recorded. Biomass was dried in a forced-air oven at 45°C for seven days, and then the dry matter was weighed. Dried plant tissues were ground to powder and digested with HNO3–HF–H2O2 in a microwave oven (Milestone 1200; Milestone Microwave System, United States). Cu, Ca, Mg, Fe, and Zn values were established using ICP-MS (Perkin Elmer ELAN 6100).

Data analysis. Relative root elongation was chosen as the endpoint for Cu toxicity assessment due to the simplicity and accuracy of this method [14]. RRE was calculated using the following formula: \(RR{{E}_{i}} = {{100\,\,{{\Re }_{i}}} \mathord{\left/ {\vphantom {{100\,\,{{\Re }_{i}}} {{{\Re }_{{{\text{max}}}}}}}} \right. \kern-0em} {{{\Re }_{{{\text{max}}}}}}},\) \(RR{{E}_{i}} = {{100\,\,{{\Re }_{i}}} \mathord{\left/ {\vphantom {{100\,\,{{\Re }_{i}}} {{{\Re }_{{{\text{max}}}}}}}} \right. \kern-0em} {{{\Re }_{{{\text{max}}}}}}},\) where RREi is relative root elongation in treatment i, REi is root elongation in treatment i and REmax is the maximum root elongation in all treatments. Since root elongation was not normally distributed (P = 0.0001), we used the 75% percentile of the relative root elongation values in our assessment.

Subsequently, we calculated the effective concentrations of Cu in the nutrient solution that inhibited root elongation by 25% (EC25), along with EC25 for foliar Cu concentrations. In order to determine EC25, we used the Hill equation of ExcelMR macro REGTOX version 7.0.6.

The net influx (In) is the net amount of nutrient taken up per unit of root surface area per unit of time. To calculate In, dry-mater nutrient concentration was measured, and root surface was calculated in each harvest. Given that young plants exhibit exponential root growth rates, the average nutrient influx (In) was calculated using the Williams’ formula [15]:

$${{I}_{{\text{n}}}} = \frac{{\left( {{{U}_{2}} - {{U}_{1}}} \right)}}{{\left( {A{{R}_{2}} - A{{R}_{1}}} \right)}}\ln ,$$

where In is the net influx in pmol/cm2 s, U is the entire plant nutrient content per plant in pmol, RA is root surface area per plant in cm2, t is the time between two harvests in seconds, and where subscripts 1 and 2 signify the first and second harvest, respectively.

The specific root length (RLS) is the ratio between root length (RL) and root fresh weight (Rfw). It was established for each species by weighing three samples of approximately 0.2 g of fresh root and directly measuring the length (in cm) of the sample. Root surface was calculated on the basis of root fresh weight assuming a cylindrical shape [16], using the following formula:

$$RA = 2\pi \sqrt {\frac{1}{{\pi \rho R{{L}_{{\text{s}}}}}}} R{{L}_{{\text{S}}}}{{R}_{{{\text{fw}}}}},$$

where RA is root surface in cm2, Rfw is root fresh weight in g, RLS is the specific root length in cm/g and ρ is root density in g/cm, which was assumed at 1 [17]. Partition coefficient (PC) was calculated as the ratio between cation concentration in shoots and cation concentration in roots. In the discussion that follows, the results are presented as the element concentrations in the shoots or roots (expressed in mg/kg) or the element content in the shoots or roots (expressed in μg/plant). In both cases, dry plant weight was used for calculations.

Results were analyzed statistically using the Infostat program with one-way ANOVA. The mean values of the treatments were compared using the Tukey test (α = 0.05).

RESULTS AND DISCUSSION

Among all plant responses, the root length was found to be the most sensitive endpoint (Table 1). Based on this endpoint, A. caven and H. annuus differ greatly in their tolerance to Cu (Table 2). Cu concentration in the solution associated with the calculated EC25 was found to be six times higher in A. caven (15 μM) than in H. annuus (2.3 μM). When effective concentrations were instead based on the measured root Cu concentrations, the difference observed was found to be ten times greater (1044 and 98 mg/kg for A. caven and H. annuus, respectively). The calculated EC25 in this study places A. caven in the first quartile of the most tolerant species and H. annuus in the second quartile, very close to the median [2]. The EC25 value of 7.85 μM of Cu is normally used as the limit between tolerant and non-tolerant species [14], which places A. caven among tolerant species and H. annuus among non-tolerant ones.

Table 1. Fresh and dry weight (g per seedling) and 75th percentile of root length (mm) of Acacia caven and Helianthus annuus cultivated in Hoagland solution spiked with raising copper concentrations.
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Table 2. Effective concentration for 25% reduction in root elongation (EC25)
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The increase of copper in the tissues of both species occurred mainly in the roots (Fig. 1). This phenomenon is known as soil-root barrier [18]. Given its high EC25 value and Cu accumulation in roots, A. caven can be classified as a non-hyperaccumulating tolerant species.

Fig. 1.
figure 1

Copper concentration (mg/kg) in shoots and roots of Acacia caven and Helianthus annuus cultivated in modified Hoagland solution spiked with rising copper concentrations. Bars indicate standard deviation. 1—Acacia caven (roots), 2—Acacia caven (shoots), 3—Helianthus annuus (roots), 4—Helianthus annuus (shoots).

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Copper uptake kinetics in both species adjust to a straight line as described by Nye [19]. Such line is given by \({{I}_{{\text{n}}}} = \left( {C{\text{|}}L - {{C}_{{L\,{\text{min}}}}}} \right){{I}_{{\text{n}}}} = \left( {C{\text{|}}L - {{C}_{{L\,{\text{min}}}}}} \right)\), where In is net influx (pmol/cm2 s), α is root absorbing power (cm/s), CL is Cu concentration in solution (pmol/cm3) and CL min is CL at In = 0. The difference in α between the species was not statistically significant. Likewise, the difference in CL min between the species was not statistically significant (Table 3). Such behavior was unexpected because species [14] or even genotypes of the same species [20] usually exhibit some differences.

Table 3.   Calculated influx values for Acacia caven and Helianthus annuus cultivated in Hoagland solution spiked with rising copper concentrations
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In this study, rising Cu concentrations in the exposure solution resulted in a reduction in the Ca influx rate in H. annuus, whereas no significant effect was noted for A. caven (Table 4). The same Cu exposure concentrations did not have any impact on the influx rates calculated for Fe, Mg, or Zn.

Table 4. Net influx (pmol/cm2 s) of Ca, Fe, Mg and Zn in seedlings of Acacia caven and Helianthus annuus cultivated in Hoagland solution spiked with rising copper concentrations
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In A. caven, no differences were found between Cu treatments in terms of Ca contents or Ca concentrations in shoots and roots (Table 5). Likewise, in H. annuus, Ca concentrations varied slightly in the roots between Cu treatments, whereas Ca contents remained unchanged in the roots between Cu treatments. On the other hand, there was a considerable reduction in Ca concentrations and Ca contents in the shoots of H. annuus (Table 5). The observed behavior cannot be related to plant tolerance to Cu. In Minuartia hirsuta, Ca concentrations in roots and shoots decreased in tolerant population and increased in sensitive one as a response to the increase of Cu concentration in the nutrient solution [21]. However, in Elsholtzia argyi, Ca concentrations maintained in roots and decreased in shoots in tolerant population, but decreased in both roots and shoots in sensitive population [22]. Thus, more studies should be carried out with other species in order to explain the observed patterns.

Table 5. Calcium concentrations (expressed in mg/kg) and calcium contents (expressed in μg/plant) in roots and shoots of Acacia caven and Helianthus annuus seedlings exposed to rising copper concentrations in the nutrient solution
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In other words, H. annuus was unable to maintain root-to-shoot Ca translocation due to rising concentrations of Cu in the nutrient solution. In the toxic range of Cu in H. annuus (>2.3 µM), the Ca influx dropped with rising concentrations of Cu in the nutrient solution (Fig. 2), indicating that both cations compete for the same binding sites. This behavior is consistent with cations competition described in the biotic ligand model [23–25].

Fig. 2.
figure 2

Relation between In Cu and In Ca of Helianthus annuus cultivated in modified Hoagland solution spiked with rising copper concentrations. Bars indicate standard deviation.

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Basic divalent cations in general and Ca in particular have the potential to ameliorate metal toxicity through ion competition and the decreased electrical potential of the cell membrane [26–28]. In addition, Ca uptake can further protect the plants due to its role in the synthesis and activation of antioxidants that are essential for cell membrane integrity [29, 30].

To conclude, Acacia caven and Helianthus annuus displayed great differences in their tolerance to Cu, the former being a tolerant and the latter being a non-tolerant species. However, rising Cu concentrations in the nutrient solution cause an increase in Cu concentrations mainly in the roots in both species. Furthermore, both species exhibited equal Cu uptake kinetics parameters indicating that density and affinity of Cu binding sites in roots are similar in both species. Thus, our findings suggest that distinct species exhibit different toxicity responses at the same level of binding site saturation. Since we are not aware of any other study discussing this tendency, this adds an aspect of novelty to the present study.

Moreover, Cu had a greater affinity for Ca binding sites on the roots of H. annuus in comparison to A. caven. Further studies are necessary to uncover the tolerance mechanisms of A. caven that protect it from Cu toxicity. Nevertheless, the ability of A. caven to maintain Ca influx in view of rising Cu may have a role in the high tolerance of this species.