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International Journal of Phytoremediation Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/bijp20
Effect of Salinity on Zinc uptake by Brassica juncea a
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Luís A. B. Novo , Emma F. Covelo & Luís González
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Department of Plant Biology and Soil Science , University of Vigo, As Lagoas , Marcosende , Vigo , Spain Accepted author version posted online: 29 Oct 2013.Published online: 06 Jan 2014.
Click for updates To cite this article: Luís A. B. Novo , Emma F. Covelo & Luís González (2014) Effect of Salinity on Zinc uptake by Brassica juncea , International Journal of Phytoremediation, 16:7-8, 704-718, DOI: 10.1080/15226514.2013.856844 To link to this article: http://dx.doi.org/10.1080/15226514.2013.856844
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International Journal of Phytoremediation, 16:704–718, 2014 C Taylor & Francis Group, LLC Copyright ISSN: 1522-6514 print / 1549-7879 online DOI: 10.1080/15226514.2013.856844
EFFECT OF SALINITY ON ZINC UPTAKE BY BRASSICA JUNCEA Lu´ıs A. B. Novo, Emma F. Covelo, and Lu´ıs Gonz´alez
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Department of Plant Biology and Soil Science, University of Vigo, As Lagoas, Marcosende, Vigo, Spain Salinity is a major worldwide problem that affects agricultural soils and limits the reclamation of contaminated sites. Despite the large number of research papers published about salt tolerance in Brassica juncea L., there are very few accounts concerning the influence of salinity on the uptake of trace metals. In this study, B. juncea plants divided through soil sets comprising 0, 900 and 1800 mg Zn kg−1, were treated with solutions containing 0, 60 and 120 mmol L−1 of NaCl, with the purpose of observing the effect of salt on Zn uptake, and some physiological responses throughout the 90 days experiment. Increasing concentrations of NaCl and Zn produced a decline in the ecophysiological and biochemical properties of the plants, with observable synergistic effects on parameters like shoot dry weight, leaf area, or photochemical efficiency. Nevertheless, plants treated with 60 mmol L−1 of NaCl accumulated striking harvestable amounts of Zn per plant that largely exceed those reported for Thlaspi caerulescens. It was concluded that salinity could play an important role on the uptake of Zn by B. juncea. The potential mechanisms behind these results are discussed, as well as the implications for phytoremediation of Zn on saline and non-saline soils. KEY WORDS: salinity, NaCl, Zn, phytoremediation, trace metals, Brassica juncea
INTRODUCTION Salinity is a major problem in the soils throughout the world. More than 800 million hectares of land all over the world are salt affected, which accounts for more than 6% of the planet’s total land area (Munns and Tester 2008). Salinity afflicts large areas of agricultural soils that become substantially or partially unproductive, and there are signs that irrigation systems and type of irrigation water have significantly contributed to converting arable lands in saline lands (Ashraf and McNeilly 2004). According to Ghassemi, Jakeman and Nix (1995), about 20 to 27% of the world’s irrigated lands may be salt affected. However, most of this salt influenced soil has arisen from natural causes, as result of the accumulation of salts over long periods of time in arid and semiarid zones (Munns and Tester 2008). Sites contaminated with industrial waste or wastewater, are often subjected to multiple stresses like trace metals, salinity or drought (Bauddh and Singh 2012), that carry a wide range of deleterious effects to plants. Detrimental consequences of salinity to plants include the impairment of mineral nutrition, growth inhibition, water status imbalance and
Address correspondence to Lu´ıs A. B. Novo, Department of Plant Biology and Soil Science, University of Vigo. As Lagoas, Marcosende, 36310 Vigo, Spain. E-mail: [email protected] 704
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secondary stresses such as oxidative and photosynthetic damage (Ashraf and McNeilly 2004; Munns and Tester 2008; Siddiqui, Mohammad, and Khan 2009; Ahmad et al. 2012). Zinc (Zn) is an essential micronutrient that influences several metabolic processes of plants, but alike other trace metals its phytotoxicity implies growth and development inhibition, leaf chlorosis and oxidative damage to the plants (Nagajyoti, Sreekanth and Lee 2010). Phytoremediation is a useful green and inexpensive technique to mitigate the impact of trace metals in soils, especially if compared to chemical and mechanical strategies that are more intricate, costly and environmental harmful (Baker et al. 1994; Cheng 2003; Mendez and Maier 2008). A large number of species among the Brassica genus have been broadly tested for phytoremediation of trace metals. They have an intrinsic skill to tolerate these trace metals, but also to promote their stabilization in the rhizosphere and even to accumulate them in elevated concentrations in the root and shoot (Kumar et al. 1995; Ebbs and Kochian 1997). Brassica juncea (L.) Czern and Coss is noted for its aptitude to endure, stabilize, extract and accumulate metals such as Cd, Cr, Cu, Ni, Pb or Zn (Ebbs et al. 1997; Ebbs and Kochian 1998; Prasad and Freitas 2003; Bluskov et al. 2005; Meyers et al. 2008; Purakayastha et al. 2008; Dede, Ozdemir, and Dede 2012). In spite of the substantial number of research articles published about salt tolerance in B. juncea, not much has been reported about the effect of NaCl on the uptake of trace metals. The species is known to be moderately salt tolerant (Ashraf and McNeilly 2004; Siddiqui, Mohammad, and Khan 2009), and although experiments carried out to date demonstrate glycophytes cannot rival with halophytes (Zaier et al. 2010; Manousaki and Kalogerakis 2011; Tapia et al. 2011), promising results indicate its potential for the phytoextraction of trace elements like Cd or Pb under salinity stress (Zaier et al. 2010; Bauddh and Singh 2012). The aim of this study is to understand the effect of salinity stress on the uptake of Zn by Brassica juncea, assess the combined impact of Zn and salt on the plant growth, and determine the suitability of this species for phytoremediation of Zn in saline soils.
MATERIAL AND METHODS Plant Material And Experimental Design Healthy B. juncea seeds acquired at Herbiseed (Herbiseed, Berkshire, United Kingdom) were allowed to germinate in trays homogeneously filled with commercial organic substrate (pH 5.6, salinity 1.5 g L−1, 180 mg N L−1, 200 mg P2 O5 L−1, 250 mg K2 O L−1, 150 mg Mg L−1, and 120 mg S L−1) from Gramoflor (G¨artner-substrat, Gramoflor GmbH & Co. KG, Vechta, Germany), under greenhouse conditions: photoperiod of 11:13 h Light:Dark, temperature of 20 ± 2◦ C and 65 ± 5% relative air humidity. Seeds were given 14 days to germinate. Soil humidity was kept constant with deionized water almost to field capacity (Veihmeyer and Hendrickson 1931). Seedlings germinated till two fully expanded leaves, were transferred to 16 cm diameter plastic pots, filled with the same organic substrate (hereinafter referred to as soil) used in the germination process, at the rate of 3 seedlings per pot. After 14 days, plants were thinned to one per pot following uniform criteria. Growth was allowed under greenhouse conditions: photoperiod of 11–13 h Light:Dark, temperature of 22 ± 2◦ C and 65 ± 5% relative air humidity. Soil moisture was kept at 35 ± 5% with deionized water, according to field capacity.
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No. of experimental sets I
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The plants were divided through three different experimental sets, according to Zn concentration (0, 900 and 1800 mg kg−1). Each set consisted of three different NaCl treatments (0, 60 and 120 mmol L−1), with three plants per treatment (each plant is considered one replicate). These treatments were chosen to be within the range of salinity and Zn tolerated by B. juncea (Sridhar et al. 2005; Taylor et al. 2010). Each treatment Id is composed by its concentration in Zn and NaCl, respectively (e.g. “900/60” corresponds to the treatment composed by 900 mg Zn kg−1 and 60 mmol NaCl L−1). The complete experimental design is displayed in Table 1. For soil contamination, ZnSO4 ·7H2 O (zinc sulfate 7-hydrate) was applied at concentrations of 900 and 1800 mg Zn kg−1 according to the experimental design. The pots were fed with 30 mL of NaCl solution with concentrations of 0, 60 and 120 mmol L−1 (0.001, 6.0 and 12.0 dS m−1, respectively) on alternate days. To compensate evapotranspiration loss, 20 mL of deionized water were supplied to each pot every other day. Plants, 3 replicates per treatment, were harvested 90 days after sowing to posterior analysis. Plant Growth and Development At the end of the experiment, each plant was divided in root and shoot, and carefully washed with deionized water in order to remove any dust deposits and surface substrate. Fresh biomass weight (FW) was determined immediately and dry biomass weight (DW) was assessed after oven drying for 48 h at 80◦ C and cooled down to room temperature. Water content (WC,%) was calculated according to the formula: WC = (FW − DW)/FW × 100. Length of roots and shoots was measured, and leaf area was determined (Leaf Area Meter CI-202, CID Inc, Camas, Washington, USA). Determination of Zn and Na in the Plant Tissue At the end of the experiment, tissues of each plant, divided by root and shoot, were ground and air-dried. Then, 0.5 g of each dried sample was digested in an acid mixture (5 mL 65% HNO3 and 3 mL 30% H2 O2 ) in open vessels on a hot plate, followed by filtration with Whatman No. 42 filter paper (Jones 2001). Zn and Na contents in the filtrate were determined by using inductively coupled plasma-optical emission spectrometry (ICP-OES; PerkinElmer Optima 4300 DV, PerkinElmer, Waltham, Massachusetts, USA).
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Analytical Measurements Chlorophyll content. Chlorophyll content was determined as described by Lichtenthaler (1987). 10 mg of fresh leaves samples were ground in 2 mL 80% (v/v) acetone. The homogenate was centrifuged at 3000 × g for 10 min (Hettich Zentrifugen EBA 12R, A. Hettich, Tuttlingen, Germany). The absorbance of supernatant was measured at 663.2 and 646.8, maximum absorbance of chlorophyll a and b, respectively, against 80% acetone as a blank (WPA Lightwave S2000, WPA, Cambridge, United Kingdom). Equations for calculating the chlorophylls a and b content are as shown below. Ca (μg mL−1 FW) = 12.25A663.2 − 2.79A646.8
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Cb (μg mL
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FW) = 21.50A646.8 − 5.10A663.2
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Total chlorophyll content was estimated by totaling chlorophyll a and b contents. According to Lichtentaler and Buschmann (2001) the quantification of Chlorophyll content relative to dry weight is more suitable as a reference system, thus the results were expressed as mg g−1 leaf DW [Eq. (3)]. Chla + b (μgmL−1 FW) × solvent volume (mL) × 1000 (3) Chltotal (mgg−1 DW) = leaf sample equivalent DW (g) Chlorophyll fluorescence. Fresh leaves were randomly chosen from each plant and their chlorophyll fluorescence was measured with a Hansatech pulse modulated fluorimeter (Hansatech Fluorescence Monitoring System, Hansatech Instruments Ltd, Norfolk, England). Hydrogen Peroxide (H2 O2 ). The levels of H2 O2 were estimated according to Novo and Gonz´alez (2013). At the end of the experiment, one hundred mg of fresh leaves samples were homogenized at 4◦ C with liquid nitrogen and 2 mL 0.1% (w/v) trichloroacetic acid (TCA). The homogenate was centrifuged at 12,000 × g for 15 min and 0.5 mL of the supernatant was added to 0.5 mL 10 mmol potassium phosphate buffer (pH 7.0) and 1 mL 1 M potassium iodide (KI). Absorbance was read at 390 nm. For the blank, 0.5 mL of 0.1% (w/v) trichloroacetic acid (TCA), 0.5 mL of 10 mmol potassium phosphate buffer pH 7.0 and 1 mL of 1 M KI were used. The content of H2 O2 was calculated by comparison with a standard calibration curve, plotted by using different known concentrations of H2 O2 . Statistical data. The Kolmogorov–Simirnov and Levene’s test were used to ensure the normality assumption and the homogeneity of variances, respectively. One-way analysis of variance (ANOVA) and Tukey test were applied for post-hoc comparisons between groups (p ≤ 0.05). However, for some data groups with unequal variance, the KruskalWallis test and Mann-Whitney U-test (p ≤ 0.05) were then used due to their conservative nature and relevance to the data set with unequal variance (Quinn and Keough 2002). All statistical analyses were performed using the IBM SPSS Statistics 19.0, SPSS Inc., New York, USA software package. RESULTS Length measurements of B. juncea plants grown in the 0 and 900 mg Zn kg−1 sets, displayed no significant differences (p ≤ 0.05) among the correspondent NaCl treatments for both root and shoot (Figure 1a, 1b). However, in the 1800 mg Zn kg−1 set, root and shoot length of plants fed with deionized water were higher than those watered with the
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Figure 1 Length of (A) root and (B) shoot of Brassica juncea plants grown in the various Zn/NaCl treatments. Values followed by different letters in each set differ significantly at p ≤ 0.05, n = 3.
60 and 120 mmol NaCl L−1 solutions, with significant differences (p ≤ 0.05) between the salt treatments for the shoot (Figure 1a, 1b). Dry biomass weight results exhibited a pronounced decline as the Zn content of the different sets increased (Figure 2a, 2b). While root dry weight was significantly higher (p ≤ 0.05) on the 0/0 and 0/60 treatments, and no differences were registered in the remaining sets (Figure 2a), the shoot dry weight values were higher for 0/0 and 0/60 in the first Zn set, and higher for the saltless treatments in the 900 and 1800 mg Zn kg−1 sets (Figure 2b). The downward slope in Figure 3a depicts the reduction in leaf area with increasing concentrations of NaCl and Zn. The plants treated with deionized water displayed higher
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Figure 2 Dry weight of (A) root and (B) shoot of Brassica juncea plants grown in the various Zn/NaCl treatments. Values followed by different letters in each set differ significantly at p ≤ 0.05, n = 3.
photochemical efficiency than those fed with the 60 and 120 mmol NaCl L−1 solutions on every set (Figure 3b). The concentrations of hydrogen peroxide in B. juncea plants developed in the 0 and 900 mg Zn kg−1 sets, showed no significant differences (p ≤ 0.05) among the three salinity treatments. Notwithstanding, the contents of this ROS in the 1800 mg Zn kg−1 set, were topped by the 120 mmol NaCl L−1 treatment, followed by the 1800/60 treatment, and lastly by the deionized solution (Figure 4a). In the first set, leaf Chlorophyll contents in the plants of the 0/60 and 0/120 treatments were significantly reduced by 24.4% and 35.2%, respectively. The results in the other two sets revealed significant differences (p ≤ 0.05)
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Figure 3 Leaf Area (A) and photochemical efficiency (B) of Brassica juncea plants grown in the various Zn/NaCl treatments. Values followed by different letters in each set differ significantly at p ≤ 0.05, n = 3.
solely between the 0 and the 120 mmol NaCl L−1 treatments, with reductions of 22.8% and 27.2% in 900/120 and 1800/120, respectively (Figure 4b). Unlike the first set, in which no significant differences (p ≤ 0.05) in the Zn contents of the root were registered among the three treatments, the roots of the plants in the 900 and 1800 mg Zn kg−1 sets exhibited significantly higher Zn accumulation results with the increment in salt (Figure 5a). Analogously to the root, the Zn concentrations in the shoot of the zincless set presented no significant differences (p ≤ 0.05) within the three treatments. However, the shoot Zn content in the 900 mg Zn kg−1 set was greater in the 60 and 120 mmol NaCl L−1 treatments, and more elevated in the 1800/60 treatment of the third
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Figure 4 Hydrogen peroxide (A) and chlorophyll content (B) of Brassica juncea plants grown in the various Zn/NaCl treatments. Values followed by different letters in each set differ significantly at p ≤ 0.05, n = 3.
set (Figure 5b). Both plant parts denoted higher Zn accumulation results with the increase of the Zn concentrations in each set. The Na content of the root and shoot described different results in the various sets. In the first set, Na concentrations in the root were more elevated in the 0/60 treatment than in 0/0, but statistically equal (p ≤ 0.05) to those of 0/120, while in the 900 mg Zn kg−1 set the 900/60 treatment was pronouncedly higher than the 0 and 120 mmol NaCl L−1 treatments. Finally the root Na content in the 1800 mg Zn kg−1 set was peaked by the B. juncea plants fed with the 120 mmol NaCl L−1 solution, followed by those of the saltless treatment and lastly by the plants subjected to the 1800/60 treatment (Figure 5c). With regard to the shoot
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Figure 5 Zn concentrations in root (A) and shoot (B), and Na concentrations in root (C) and shoot (D) of Brassica juncea plants grown in the various Zn/NaCl treatments. Values followed by different letters in each set differ significantly at p ≤ 0.05, n = 3. (Continued)
Na contents, the 0 and 900 mg Zn kg−1 sets displayed an increasing accumulation trend that matched the growth in NaCl concentrations of the treatments in each set. Oppositely, the 1800 mg Zn kg−1 set not only showed an overall smaller Na concentration in the shoot, but also a different accumulation pattern with the 1800/60 treatment exhibiting significantly higher concentrations than 1800/0, and the 120 mmol NaCl L−1 treatment being statistically equal to the previous two (Figure 5d). When biomass differences were considered, and Zn uptake was represented on a per pot basis (herein designated as harvestable amount), the content of Zn was higher in 0/0
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and 0/60 than in the 120 mmol NaCl L−1 treatment for the 0 mg Zn kg−1 set, and greater in the plants treated with the 60 mmol NaCl L−1 solution than in those watered with deionized water and 120 mmol NaCl L−1 solution in the remaining two sets (Figure 6). DISCUSSION Salinity and Zn share a wide range of toxic effects that include growth inhibition, chlorophyll loss and oxidative damage (Ebbs and Uchill 2008; Siddiqui, Mohammad, and Khan 2009; Nagajyoti, Sreekanth, and Lee 2010). A glance at certain ecophysiological parameters like the shoot dry weight, leaf area, or photochemical efficiency, suggests the
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Figure 6 Harvestable amount of Zn in Brassica juncea plants grown in the various Zn/NaCl treatments. Values followed by different letters in each set differ significantly at p ≤ 0.05, n = 3.
concurrent toxicity of Zn and NaCl. Moreover, the occurrence of synergistic effects on plants between trace metals and other stresses such as drought or salinity has already been reported by other authors (Barcel´o and Poschenrieder 1990; Bauddh and Singh 2012; Novo and Gonz´alez 2013). Nonetheless, the length and dry weight of the root were essentially driven by the different Zn concentrations, in contrast with the results obtained for the same assessments in the shoot, wherein salinity appeared to play an important role among the different sets. A quick reaction to the increment of the external osmotic pressure, and a slower response due to the accumulation of Na+ in leaves, constitute the two phases responsible for the reduction of the shoot growth, whose sensibility to salt is higher than that verified in the root growth (Munns and Tester 2008). The results of this experiment coincide with the findings of other studies, in which leaf area of B. juncea plants decreases with increasing concentrations of NaCl (Siddiqui, Mohammad, and Khan 2009; Javid, Ford, and Nicolas 2012). Impairment of the photosynthetic system as result of chlorophyll loss and consequent diminution of the photochemical efficiency, in B. juncea plants subjected to increasing salinity levels, has been extensively observed (Siddiqui, Mohammad, and Khan 2009; Ahmad et al. 2012; Mittal, Kumari, and Sharma 2012). Although chlorosis and photoinhibitory damage can be ascribed to Zn stress (Nagajyoti, Sreekanth, and Lee 2010), the chlorophylls and Fv/Fm measurements indicate the impact of salt on their reduction. Furthermore, in the 900 and 1800 mg Zn kg−1 sets, the application of NaCl solutions of gradual concentration lessened the photochemical efficiency to values below the average of healthy plants (Bj¨orkman and Demmig 1987). Hydrogen peroxide produced as a result of NaCl-induced oxidative harm and also as a product of superoxide dismutase activity is a highly damaging ROS, principally at high salt concentrations, and has been referred as a potential biochemical indicator of salinity tolerance (Ashraf and Harris 2004; Munns and Tester 2008; Joshi, Saxena, and Arora 2010). The outcome of the hydrogen peroxide assay in the first two sets agrees with previously
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published data that shows an effective response of B. juncea against the production of this ROS (Ahmad et al. 2012). The dependence on the coordinated increase in the activities of antioxidative enzymes like superoxide dismutase and ascorbate peroxidase has been evoked to explain the mechanism used by plants to prevent the reduction of metal ions, the synthesis of hydroxyl radicals and the elimination of active oxygen species (Ashraf and Harris 2004; Joshi, Saxena, and Arora 2010). The significant difference between the various treatments in the 1800 mg Zn kg−1 set may derive from the combined effect of the high content of Zn and the increasing concentrations of salt, leading to the production of more H2 O2 as the cumulative stress is more harmful to the B. juncea plants. The rising concentrations of Zn across the three sets seem to concur with the increase of the root Zn content, still the observed impact of salt in each set may play a far more important role for the uptake process. An increase in the mobility of Zn due to the presence of NaCl, most specifically Cl−, has been proposed (Guevara-Riba et al. 2005; Acosta et al. 2011), and thus rising salinity may enhance Zn transport to the root by making it more available in the rhizosphere, similarly to the effect of chelating agents on this element in other experiments involving B. juncea (Ebbs and Kochian 1998; Quartacci et al. 2006). A primary analysis to the weakening of the shoot ecophysiological properties as result of the synergistic effect between Zn and salinity stress, seemed determinant for the translocation of Zn to the aerial part, nevertheless a closer look at the results reveals a progressive gain of the shoot Zn content, most especially in the 60 mmol NaCl L−1 treatment of the 1800 mg Zn kg−1 set. In this set, the B. juncea plants were subjected to the highest Zn stress, as marked by the decay in their ecophysiological, photosynthetic and antioxidative activity. The substantial decrease of the Na content in the root and shoot shown in the third set may have arisen from a defensive strategy through which the B. juncea plants limit the uptake of sodium to avoid further damage, while still accumulating Zn. The tolerance to the joint effect of Zn and salinity exhibited by the B. juncea plants fed with the 60 mmol NaCl L−1 solution, allowed this treatment to accumulate a considerable quantity of Zn, that not only largely exceeds the harvestable amount of Zn per plant verified in other B. juncea studies wherein NaCl was absent and chelating agents were applied (Ebbs and Kochian 1998; Quartacci et al. 2006), but also surpasses the literature reports about the harvestable amount per plant of the well-known Zn hyperaccumulator Thlaspi caerulescens in saltless conditions (Mcgrath, Shen, and Zhao 1997; Escarr´e et al. 2000; Whiting et al. 2001). This comparison with the heavyweight champion of Zn accumulation is exceptionally important if we consider that when subjected to a 50 mmol NaCl L−1 treatment, T. caerulescens suffered a reduction of 44% in terms of shoot Zn concentration, in addition to the strong inhibition of the shoot growth (Comino et al. 2005). Further investigation about the mobilization of Zn in soil due to NaCl addition, as well as about the process that leads to the exclusion of Na uptake in the presence of elevated concentrations of Zn, is critical to better comprehend the processes behind the results of this study and bring new insights about the possibility of using controlled amounts of salt to enhance the extraction of Zn with B. juncea. CONCLUSION In spite of the observed decline in certain ecophysiological and biochemical parameters, the data presented in this paper reveals the aptitude of B. juncea to tolerate the synergistic effect of salinity and Zn stress within the range of the assessed concentrations. More importantly, moderate to moderately high NaCl concentrations increased the uptake
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of Zn to harvestable amounts that, as per our database, outdo the records of T. caerulescens, therefore classifying B. juncea as a remarkable candidate for the phytoremediation of saline soils polluted with Zn. Additional research about the mechanism that triggers the exclusion of Na uptake at a given Zn concentration threshold, in addition to a supplementary study of the impact of NaCl in the mobilization of Zn in soil, would contribute to fully understand the obtained results and consider the controlled use of salt to ameliorate the extraction of Zn in non-saline soils.
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International Journal of Phytoremediation Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/bijp20
Effect of Salinity on Zinc uptake by Brassica juncea a
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Luís A. B. Novo , Emma F. Covelo & Luís González
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Department of Plant Biology and Soil Science , University of Vigo, As Lagoas , Marcosende , Vigo , Spain Accepted author version posted online: 29 Oct 2013.Published online: 06 Jan 2014.
Click for updates To cite this article: Luís A. B. Novo , Emma F. Covelo & Luís González (2014) Effect of Salinity on Zinc uptake by Brassica juncea , International Journal of Phytoremediation, 16:7-8, 704-718, DOI: 10.1080/15226514.2013.856844 To link to this article: http://dx.doi.org/10.1080/15226514.2013.856844
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International Journal of Phytoremediation, 16:704–718, 2014 C Taylor & Francis Group, LLC Copyright ISSN: 1522-6514 print / 1549-7879 online DOI: 10.1080/15226514.2013.856844
EFFECT OF SALINITY ON ZINC UPTAKE BY BRASSICA JUNCEA Lu´ıs A. B. Novo, Emma F. Covelo, and Lu´ıs Gonz´alez
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Department of Plant Biology and Soil Science, University of Vigo, As Lagoas, Marcosende, Vigo, Spain Salinity is a major worldwide problem that affects agricultural soils and limits the reclamation of contaminated sites. Despite the large number of research papers published about salt tolerance in Brassica juncea L., there are very few accounts concerning the influence of salinity on the uptake of trace metals. In this study, B. juncea plants divided through soil sets comprising 0, 900 and 1800 mg Zn kg−1, were treated with solutions containing 0, 60 and 120 mmol L−1 of NaCl, with the purpose of observing the effect of salt on Zn uptake, and some physiological responses throughout the 90 days experiment. Increasing concentrations of NaCl and Zn produced a decline in the ecophysiological and biochemical properties of the plants, with observable synergistic effects on parameters like shoot dry weight, leaf area, or photochemical efficiency. Nevertheless, plants treated with 60 mmol L−1 of NaCl accumulated striking harvestable amounts of Zn per plant that largely exceed those reported for Thlaspi caerulescens. It was concluded that salinity could play an important role on the uptake of Zn by B. juncea. The potential mechanisms behind these results are discussed, as well as the implications for phytoremediation of Zn on saline and non-saline soils. KEY WORDS: salinity, NaCl, Zn, phytoremediation, trace metals, Brassica juncea
INTRODUCTION Salinity is a major problem in the soils throughout the world. More than 800 million hectares of land all over the world are salt affected, which accounts for more than 6% of the planet’s total land area (Munns and Tester 2008). Salinity afflicts large areas of agricultural soils that become substantially or partially unproductive, and there are signs that irrigation systems and type of irrigation water have significantly contributed to converting arable lands in saline lands (Ashraf and McNeilly 2004). According to Ghassemi, Jakeman and Nix (1995), about 20 to 27% of the world’s irrigated lands may be salt affected. However, most of this salt influenced soil has arisen from natural causes, as result of the accumulation of salts over long periods of time in arid and semiarid zones (Munns and Tester 2008). Sites contaminated with industrial waste or wastewater, are often subjected to multiple stresses like trace metals, salinity or drought (Bauddh and Singh 2012), that carry a wide range of deleterious effects to plants. Detrimental consequences of salinity to plants include the impairment of mineral nutrition, growth inhibition, water status imbalance and
Address correspondence to Lu´ıs A. B. Novo, Department of Plant Biology and Soil Science, University of Vigo. As Lagoas, Marcosende, 36310 Vigo, Spain. E-mail: [email protected] 704
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secondary stresses such as oxidative and photosynthetic damage (Ashraf and McNeilly 2004; Munns and Tester 2008; Siddiqui, Mohammad, and Khan 2009; Ahmad et al. 2012). Zinc (Zn) is an essential micronutrient that influences several metabolic processes of plants, but alike other trace metals its phytotoxicity implies growth and development inhibition, leaf chlorosis and oxidative damage to the plants (Nagajyoti, Sreekanth and Lee 2010). Phytoremediation is a useful green and inexpensive technique to mitigate the impact of trace metals in soils, especially if compared to chemical and mechanical strategies that are more intricate, costly and environmental harmful (Baker et al. 1994; Cheng 2003; Mendez and Maier 2008). A large number of species among the Brassica genus have been broadly tested for phytoremediation of trace metals. They have an intrinsic skill to tolerate these trace metals, but also to promote their stabilization in the rhizosphere and even to accumulate them in elevated concentrations in the root and shoot (Kumar et al. 1995; Ebbs and Kochian 1997). Brassica juncea (L.) Czern and Coss is noted for its aptitude to endure, stabilize, extract and accumulate metals such as Cd, Cr, Cu, Ni, Pb or Zn (Ebbs et al. 1997; Ebbs and Kochian 1998; Prasad and Freitas 2003; Bluskov et al. 2005; Meyers et al. 2008; Purakayastha et al. 2008; Dede, Ozdemir, and Dede 2012). In spite of the substantial number of research articles published about salt tolerance in B. juncea, not much has been reported about the effect of NaCl on the uptake of trace metals. The species is known to be moderately salt tolerant (Ashraf and McNeilly 2004; Siddiqui, Mohammad, and Khan 2009), and although experiments carried out to date demonstrate glycophytes cannot rival with halophytes (Zaier et al. 2010; Manousaki and Kalogerakis 2011; Tapia et al. 2011), promising results indicate its potential for the phytoextraction of trace elements like Cd or Pb under salinity stress (Zaier et al. 2010; Bauddh and Singh 2012). The aim of this study is to understand the effect of salinity stress on the uptake of Zn by Brassica juncea, assess the combined impact of Zn and salt on the plant growth, and determine the suitability of this species for phytoremediation of Zn in saline soils.
MATERIAL AND METHODS Plant Material And Experimental Design Healthy B. juncea seeds acquired at Herbiseed (Herbiseed, Berkshire, United Kingdom) were allowed to germinate in trays homogeneously filled with commercial organic substrate (pH 5.6, salinity 1.5 g L−1, 180 mg N L−1, 200 mg P2 O5 L−1, 250 mg K2 O L−1, 150 mg Mg L−1, and 120 mg S L−1) from Gramoflor (G¨artner-substrat, Gramoflor GmbH & Co. KG, Vechta, Germany), under greenhouse conditions: photoperiod of 11:13 h Light:Dark, temperature of 20 ± 2◦ C and 65 ± 5% relative air humidity. Seeds were given 14 days to germinate. Soil humidity was kept constant with deionized water almost to field capacity (Veihmeyer and Hendrickson 1931). Seedlings germinated till two fully expanded leaves, were transferred to 16 cm diameter plastic pots, filled with the same organic substrate (hereinafter referred to as soil) used in the germination process, at the rate of 3 seedlings per pot. After 14 days, plants were thinned to one per pot following uniform criteria. Growth was allowed under greenhouse conditions: photoperiod of 11–13 h Light:Dark, temperature of 22 ± 2◦ C and 65 ± 5% relative air humidity. Soil moisture was kept at 35 ± 5% with deionized water, according to field capacity.
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The plants were divided through three different experimental sets, according to Zn concentration (0, 900 and 1800 mg kg−1). Each set consisted of three different NaCl treatments (0, 60 and 120 mmol L−1), with three plants per treatment (each plant is considered one replicate). These treatments were chosen to be within the range of salinity and Zn tolerated by B. juncea (Sridhar et al. 2005; Taylor et al. 2010). Each treatment Id is composed by its concentration in Zn and NaCl, respectively (e.g. “900/60” corresponds to the treatment composed by 900 mg Zn kg−1 and 60 mmol NaCl L−1). The complete experimental design is displayed in Table 1. For soil contamination, ZnSO4 ·7H2 O (zinc sulfate 7-hydrate) was applied at concentrations of 900 and 1800 mg Zn kg−1 according to the experimental design. The pots were fed with 30 mL of NaCl solution with concentrations of 0, 60 and 120 mmol L−1 (0.001, 6.0 and 12.0 dS m−1, respectively) on alternate days. To compensate evapotranspiration loss, 20 mL of deionized water were supplied to each pot every other day. Plants, 3 replicates per treatment, were harvested 90 days after sowing to posterior analysis. Plant Growth and Development At the end of the experiment, each plant was divided in root and shoot, and carefully washed with deionized water in order to remove any dust deposits and surface substrate. Fresh biomass weight (FW) was determined immediately and dry biomass weight (DW) was assessed after oven drying for 48 h at 80◦ C and cooled down to room temperature. Water content (WC,%) was calculated according to the formula: WC = (FW − DW)/FW × 100. Length of roots and shoots was measured, and leaf area was determined (Leaf Area Meter CI-202, CID Inc, Camas, Washington, USA). Determination of Zn and Na in the Plant Tissue At the end of the experiment, tissues of each plant, divided by root and shoot, were ground and air-dried. Then, 0.5 g of each dried sample was digested in an acid mixture (5 mL 65% HNO3 and 3 mL 30% H2 O2 ) in open vessels on a hot plate, followed by filtration with Whatman No. 42 filter paper (Jones 2001). Zn and Na contents in the filtrate were determined by using inductively coupled plasma-optical emission spectrometry (ICP-OES; PerkinElmer Optima 4300 DV, PerkinElmer, Waltham, Massachusetts, USA).
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Analytical Measurements Chlorophyll content. Chlorophyll content was determined as described by Lichtenthaler (1987). 10 mg of fresh leaves samples were ground in 2 mL 80% (v/v) acetone. The homogenate was centrifuged at 3000 × g for 10 min (Hettich Zentrifugen EBA 12R, A. Hettich, Tuttlingen, Germany). The absorbance of supernatant was measured at 663.2 and 646.8, maximum absorbance of chlorophyll a and b, respectively, against 80% acetone as a blank (WPA Lightwave S2000, WPA, Cambridge, United Kingdom). Equations for calculating the chlorophylls a and b content are as shown below. Ca (μg mL−1 FW) = 12.25A663.2 − 2.79A646.8
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Total chlorophyll content was estimated by totaling chlorophyll a and b contents. According to Lichtentaler and Buschmann (2001) the quantification of Chlorophyll content relative to dry weight is more suitable as a reference system, thus the results were expressed as mg g−1 leaf DW [Eq. (3)]. Chla + b (μgmL−1 FW) × solvent volume (mL) × 1000 (3) Chltotal (mgg−1 DW) = leaf sample equivalent DW (g) Chlorophyll fluorescence. Fresh leaves were randomly chosen from each plant and their chlorophyll fluorescence was measured with a Hansatech pulse modulated fluorimeter (Hansatech Fluorescence Monitoring System, Hansatech Instruments Ltd, Norfolk, England). Hydrogen Peroxide (H2 O2 ). The levels of H2 O2 were estimated according to Novo and Gonz´alez (2013). At the end of the experiment, one hundred mg of fresh leaves samples were homogenized at 4◦ C with liquid nitrogen and 2 mL 0.1% (w/v) trichloroacetic acid (TCA). The homogenate was centrifuged at 12,000 × g for 15 min and 0.5 mL of the supernatant was added to 0.5 mL 10 mmol potassium phosphate buffer (pH 7.0) and 1 mL 1 M potassium iodide (KI). Absorbance was read at 390 nm. For the blank, 0.5 mL of 0.1% (w/v) trichloroacetic acid (TCA), 0.5 mL of 10 mmol potassium phosphate buffer pH 7.0 and 1 mL of 1 M KI were used. The content of H2 O2 was calculated by comparison with a standard calibration curve, plotted by using different known concentrations of H2 O2 . Statistical data. The Kolmogorov–Simirnov and Levene’s test were used to ensure the normality assumption and the homogeneity of variances, respectively. One-way analysis of variance (ANOVA) and Tukey test were applied for post-hoc comparisons between groups (p ≤ 0.05). However, for some data groups with unequal variance, the KruskalWallis test and Mann-Whitney U-test (p ≤ 0.05) were then used due to their conservative nature and relevance to the data set with unequal variance (Quinn and Keough 2002). All statistical analyses were performed using the IBM SPSS Statistics 19.0, SPSS Inc., New York, USA software package. RESULTS Length measurements of B. juncea plants grown in the 0 and 900 mg Zn kg−1 sets, displayed no significant differences (p ≤ 0.05) among the correspondent NaCl treatments for both root and shoot (Figure 1a, 1b). However, in the 1800 mg Zn kg−1 set, root and shoot length of plants fed with deionized water were higher than those watered with the
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Figure 1 Length of (A) root and (B) shoot of Brassica juncea plants grown in the various Zn/NaCl treatments. Values followed by different letters in each set differ significantly at p ≤ 0.05, n = 3.
60 and 120 mmol NaCl L−1 solutions, with significant differences (p ≤ 0.05) between the salt treatments for the shoot (Figure 1a, 1b). Dry biomass weight results exhibited a pronounced decline as the Zn content of the different sets increased (Figure 2a, 2b). While root dry weight was significantly higher (p ≤ 0.05) on the 0/0 and 0/60 treatments, and no differences were registered in the remaining sets (Figure 2a), the shoot dry weight values were higher for 0/0 and 0/60 in the first Zn set, and higher for the saltless treatments in the 900 and 1800 mg Zn kg−1 sets (Figure 2b). The downward slope in Figure 3a depicts the reduction in leaf area with increasing concentrations of NaCl and Zn. The plants treated with deionized water displayed higher
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photochemical efficiency than those fed with the 60 and 120 mmol NaCl L−1 solutions on every set (Figure 3b). The concentrations of hydrogen peroxide in B. juncea plants developed in the 0 and 900 mg Zn kg−1 sets, showed no significant differences (p ≤ 0.05) among the three salinity treatments. Notwithstanding, the contents of this ROS in the 1800 mg Zn kg−1 set, were topped by the 120 mmol NaCl L−1 treatment, followed by the 1800/60 treatment, and lastly by the deionized solution (Figure 4a). In the first set, leaf Chlorophyll contents in the plants of the 0/60 and 0/120 treatments were significantly reduced by 24.4% and 35.2%, respectively. The results in the other two sets revealed significant differences (p ≤ 0.05)
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solely between the 0 and the 120 mmol NaCl L−1 treatments, with reductions of 22.8% and 27.2% in 900/120 and 1800/120, respectively (Figure 4b). Unlike the first set, in which no significant differences (p ≤ 0.05) in the Zn contents of the root were registered among the three treatments, the roots of the plants in the 900 and 1800 mg Zn kg−1 sets exhibited significantly higher Zn accumulation results with the increment in salt (Figure 5a). Analogously to the root, the Zn concentrations in the shoot of the zincless set presented no significant differences (p ≤ 0.05) within the three treatments. However, the shoot Zn content in the 900 mg Zn kg−1 set was greater in the 60 and 120 mmol NaCl L−1 treatments, and more elevated in the 1800/60 treatment of the third
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Figure 4 Hydrogen peroxide (A) and chlorophyll content (B) of Brassica juncea plants grown in the various Zn/NaCl treatments. Values followed by different letters in each set differ significantly at p ≤ 0.05, n = 3.
set (Figure 5b). Both plant parts denoted higher Zn accumulation results with the increase of the Zn concentrations in each set. The Na content of the root and shoot described different results in the various sets. In the first set, Na concentrations in the root were more elevated in the 0/60 treatment than in 0/0, but statistically equal (p ≤ 0.05) to those of 0/120, while in the 900 mg Zn kg−1 set the 900/60 treatment was pronouncedly higher than the 0 and 120 mmol NaCl L−1 treatments. Finally the root Na content in the 1800 mg Zn kg−1 set was peaked by the B. juncea plants fed with the 120 mmol NaCl L−1 solution, followed by those of the saltless treatment and lastly by the plants subjected to the 1800/60 treatment (Figure 5c). With regard to the shoot
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Figure 5 Zn concentrations in root (A) and shoot (B), and Na concentrations in root (C) and shoot (D) of Brassica juncea plants grown in the various Zn/NaCl treatments. Values followed by different letters in each set differ significantly at p ≤ 0.05, n = 3. (Continued)
Na contents, the 0 and 900 mg Zn kg−1 sets displayed an increasing accumulation trend that matched the growth in NaCl concentrations of the treatments in each set. Oppositely, the 1800 mg Zn kg−1 set not only showed an overall smaller Na concentration in the shoot, but also a different accumulation pattern with the 1800/60 treatment exhibiting significantly higher concentrations than 1800/0, and the 120 mmol NaCl L−1 treatment being statistically equal to the previous two (Figure 5d). When biomass differences were considered, and Zn uptake was represented on a per pot basis (herein designated as harvestable amount), the content of Zn was higher in 0/0
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and 0/60 than in the 120 mmol NaCl L−1 treatment for the 0 mg Zn kg−1 set, and greater in the plants treated with the 60 mmol NaCl L−1 solution than in those watered with deionized water and 120 mmol NaCl L−1 solution in the remaining two sets (Figure 6). DISCUSSION Salinity and Zn share a wide range of toxic effects that include growth inhibition, chlorophyll loss and oxidative damage (Ebbs and Uchill 2008; Siddiqui, Mohammad, and Khan 2009; Nagajyoti, Sreekanth, and Lee 2010). A glance at certain ecophysiological parameters like the shoot dry weight, leaf area, or photochemical efficiency, suggests the
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Figure 6 Harvestable amount of Zn in Brassica juncea plants grown in the various Zn/NaCl treatments. Values followed by different letters in each set differ significantly at p ≤ 0.05, n = 3.
concurrent toxicity of Zn and NaCl. Moreover, the occurrence of synergistic effects on plants between trace metals and other stresses such as drought or salinity has already been reported by other authors (Barcel´o and Poschenrieder 1990; Bauddh and Singh 2012; Novo and Gonz´alez 2013). Nonetheless, the length and dry weight of the root were essentially driven by the different Zn concentrations, in contrast with the results obtained for the same assessments in the shoot, wherein salinity appeared to play an important role among the different sets. A quick reaction to the increment of the external osmotic pressure, and a slower response due to the accumulation of Na+ in leaves, constitute the two phases responsible for the reduction of the shoot growth, whose sensibility to salt is higher than that verified in the root growth (Munns and Tester 2008). The results of this experiment coincide with the findings of other studies, in which leaf area of B. juncea plants decreases with increasing concentrations of NaCl (Siddiqui, Mohammad, and Khan 2009; Javid, Ford, and Nicolas 2012). Impairment of the photosynthetic system as result of chlorophyll loss and consequent diminution of the photochemical efficiency, in B. juncea plants subjected to increasing salinity levels, has been extensively observed (Siddiqui, Mohammad, and Khan 2009; Ahmad et al. 2012; Mittal, Kumari, and Sharma 2012). Although chlorosis and photoinhibitory damage can be ascribed to Zn stress (Nagajyoti, Sreekanth, and Lee 2010), the chlorophylls and Fv/Fm measurements indicate the impact of salt on their reduction. Furthermore, in the 900 and 1800 mg Zn kg−1 sets, the application of NaCl solutions of gradual concentration lessened the photochemical efficiency to values below the average of healthy plants (Bj¨orkman and Demmig 1987). Hydrogen peroxide produced as a result of NaCl-induced oxidative harm and also as a product of superoxide dismutase activity is a highly damaging ROS, principally at high salt concentrations, and has been referred as a potential biochemical indicator of salinity tolerance (Ashraf and Harris 2004; Munns and Tester 2008; Joshi, Saxena, and Arora 2010). The outcome of the hydrogen peroxide assay in the first two sets agrees with previously
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published data that shows an effective response of B. juncea against the production of this ROS (Ahmad et al. 2012). The dependence on the coordinated increase in the activities of antioxidative enzymes like superoxide dismutase and ascorbate peroxidase has been evoked to explain the mechanism used by plants to prevent the reduction of metal ions, the synthesis of hydroxyl radicals and the elimination of active oxygen species (Ashraf and Harris 2004; Joshi, Saxena, and Arora 2010). The significant difference between the various treatments in the 1800 mg Zn kg−1 set may derive from the combined effect of the high content of Zn and the increasing concentrations of salt, leading to the production of more H2 O2 as the cumulative stress is more harmful to the B. juncea plants. The rising concentrations of Zn across the three sets seem to concur with the increase of the root Zn content, still the observed impact of salt in each set may play a far more important role for the uptake process. An increase in the mobility of Zn due to the presence of NaCl, most specifically Cl−, has been proposed (Guevara-Riba et al. 2005; Acosta et al. 2011), and thus rising salinity may enhance Zn transport to the root by making it more available in the rhizosphere, similarly to the effect of chelating agents on this element in other experiments involving B. juncea (Ebbs and Kochian 1998; Quartacci et al. 2006). A primary analysis to the weakening of the shoot ecophysiological properties as result of the synergistic effect between Zn and salinity stress, seemed determinant for the translocation of Zn to the aerial part, nevertheless a closer look at the results reveals a progressive gain of the shoot Zn content, most especially in the 60 mmol NaCl L−1 treatment of the 1800 mg Zn kg−1 set. In this set, the B. juncea plants were subjected to the highest Zn stress, as marked by the decay in their ecophysiological, photosynthetic and antioxidative activity. The substantial decrease of the Na content in the root and shoot shown in the third set may have arisen from a defensive strategy through which the B. juncea plants limit the uptake of sodium to avoid further damage, while still accumulating Zn. The tolerance to the joint effect of Zn and salinity exhibited by the B. juncea plants fed with the 60 mmol NaCl L−1 solution, allowed this treatment to accumulate a considerable quantity of Zn, that not only largely exceeds the harvestable amount of Zn per plant verified in other B. juncea studies wherein NaCl was absent and chelating agents were applied (Ebbs and Kochian 1998; Quartacci et al. 2006), but also surpasses the literature reports about the harvestable amount per plant of the well-known Zn hyperaccumulator Thlaspi caerulescens in saltless conditions (Mcgrath, Shen, and Zhao 1997; Escarr´e et al. 2000; Whiting et al. 2001). This comparison with the heavyweight champion of Zn accumulation is exceptionally important if we consider that when subjected to a 50 mmol NaCl L−1 treatment, T. caerulescens suffered a reduction of 44% in terms of shoot Zn concentration, in addition to the strong inhibition of the shoot growth (Comino et al. 2005). Further investigation about the mobilization of Zn in soil due to NaCl addition, as well as about the process that leads to the exclusion of Na uptake in the presence of elevated concentrations of Zn, is critical to better comprehend the processes behind the results of this study and bring new insights about the possibility of using controlled amounts of salt to enhance the extraction of Zn with B. juncea. CONCLUSION In spite of the observed decline in certain ecophysiological and biochemical parameters, the data presented in this paper reveals the aptitude of B. juncea to tolerate the synergistic effect of salinity and Zn stress within the range of the assessed concentrations. More importantly, moderate to moderately high NaCl concentrations increased the uptake
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of Zn to harvestable amounts that, as per our database, outdo the records of T. caerulescens, therefore classifying B. juncea as a remarkable candidate for the phytoremediation of saline soils polluted with Zn. Additional research about the mechanism that triggers the exclusion of Na uptake at a given Zn concentration threshold, in addition to a supplementary study of the impact of NaCl in the mobilization of Zn in soil, would contribute to fully understand the obtained results and consider the controlled use of salt to ameliorate the extraction of Zn in non-saline soils.
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