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The influence of flavonoid amendment on the absorption of cadmium in Avicennia marina roots
Ecotoxicology and Environmental Safety 120 (2015) 1–6
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The influence of flavonoid amendment on the absorption of cadmium in Avicennia marina roots Jian Li, Haoliang Lu, Jingchun Liu, Hualong Hong, Chongling Yan n Key Laboratory of Ministry of Education for Coastal and Wetland Ecosystems, Xiamen University, Xiamen 361102, China
art ic l e i nf o
a b s t r a c t
Article history: Received 9 February 2015 Received in revised form 23 April 2015 Accepted 5 May 2015
Flavonoid is a key factor for the tolerance to cadmium in plants. Concentration-dependent kinetics experiment was conducted to investigate the influence of flavonoid amendment on the Cd2 þ uptake in Avicennia marina (Forsk) Vierh. roots. We found that compared with the control, saturation concentration and maximal absorption rate of Cd was higher under flavonoid amendment (po 0.05). When roots were exposed to ion transport inhibitor (LaCl3), flavonoid amendment also facilitated Cd transport in roots. Flavonoids had no influence on Cd2 þ uptake in root cell walls. In conclusion, flavonoids enhance the tolerance to Cd and have a significant stimulative effect on symplasm transport of Cd in A. marina roots. Ca2 þ -channel was not the unique means of symplasm transport for Cd2 þ absorption. & 2015 Elsevier Inc. All rights reserved.
Keywords: Cadmium Flavonoid Mangrove Avicennia marina
1. Introduction Mangrove forests located in the intertidal zone of tropical and subtropical regions, are known as specific ecological habitats, and have been regarded as playing a key role in maintaining the coastal ecological balance (Du et al., 2013; Liu et al., 2010). However, mangroves are subjected to environmental pollutants, especially heavy metal contaminants, such as Cd, Cr, Cu, Pb, Hg (Cuong et al., 2005; Wu et al., 2011). A. marina, being the pioneer mangrove species in the southeast of China, was found to accumulate Cd, Cu, Pb and Zn in root tissues under field conditions with levels equal to or greater than the surrounding sediment concentrations (MacFarlane and Burchett, 2002; MacFarlane et al., 2003; Nowrouzi et al., 2012). Cd has drawn more attention because of its high activity and bioavailability, which can interfere with plant metabolic processes, resulting in poor growth and lower biomass. The plants absorb Cd2 þ through apoplastic and symplasm transport, nevertheless, there are specific or generic ionophore or channel proteins into root cells. Cd2 þ can be taken in through Ca2 þ and Mg2 þ /Fe2 þ channels, and compete with divalent cations (Welch et al., 1999). Mangrove plants have a high content of phenolic compounds (Rahim et al., 2007; Rahim et al., 2008) mainly containing flavonoids and tannins. As one kind of secondary metabolites, flavonoids account for 37% of secondary metabolites (Narasimhan et al., n
Corresponding author. E-mail address: [email protected] (C. Yan).
http://dx.doi.org/10.1016/j.ecoenv.2015.05.004 0147-6513/& 2015 Elsevier Inc. All rights reserved.
2003). Besides being free radical scavengers, flavonoid can chelate heavy metal depending on their different molecular structure (Korkina, 2007), thus inhibit lipid peroxidation and Fenton reaction (Chen et al., 1990), where heavy metals cause lipid peroxidation and free radical imbalance (Korkina, 2007). Stingu et al. (2012) indicated that phenolic compounds made a critical difference in tolerance and bioaccumulation of Avena sativa L. as chelator and solubilizer of heavy metals (Stingu et al., 2012); Keilig and Ludwig-Muller (2009) made a similar conclusion, pointed out that flavonoids were a key factor for the tolerance in Arabidopsis thaliana. Arora et al. (1998) tested the inhibition of flavonoids in the liposome membrane system by lipid peroxidation induced by Fe3 þ or peroxide radical. They found that all the tested flavonoids had higher antioxidation efficiency to peroxidation induced by metal ion than those induced by peroxy radical (Arora et al., 1998). The substituent group mode of flavonoids B-ring influences its antioxidation ability significantly. Certain hydroxy substituents on the A-ring may compensate and become a larger determinant of the antioxidant efficacy when B-ring cannot contribute to the antioxidant activity of the flavonoids. These are determining factors for flavonoids’ antioxidation ability (Arora et al., 1998). All these authors have suggested that the chelation of flavonoids makes much critical difference to their antioxidation. The anti-lipid peroxidation capability of flavonoids is the cause of the activity of scavenging free radical and metal chelation. The absorption of Cd2 þ is impacted by divalent cations (Zn2 þ , Ca2 þ , Fe2 þ etc.) (Lombi et al., 2002; Zhao et al., 2002) and root exudates (flavonoids and low molecular weight organic acids etc.) (Roth et al., 2012). Cd2 þ is absorbed through an iron transporter
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protein in the plasma membrane; where the absorption and transport of Cd2 þ in roots is increasingly involved. Fe2 þ affect the absorption of Cd2 þ by competing binding site and transport agent with Cd2 þ (Charlatchka and Cambier, 2000; Davranche and Bollinger, 2000; Pi et al., 2011). Flavonoids as chelators take on a transporter role to influence the absorption of Cd2 þ ; with the same effect on Zn2 þ , Fe2 þ etc. A large number of studies reported that flavonoids are able to function as chelators for metals (Aherne and O’Brien, 2000; Brown et al., 1998; Korkina, 2007; Michalak, 2006; Soczynska-Kordala et al., 2001). They are the main components of root exudates in response to Fe-deficiency (Cesco et al., 2010; Olsen et al., 1981), which act as a Fe2 þ -carrier to transport Fe2 þ . The classic Michaelis–Menten enzyme kinetics model has been widely applied to absorption of metals in plant roots. The Michaelis constant Km (half-saturation constant) shows the affinity of roots system with metal, while Vmax (maximal rate of velocity) represents the capability of roots for absorbing metal (Zhao et al., 2002, 2006). The model is described as
V = (Vmax[S ])/(Km + [S ] ) Zhao et al. (2002) utilized the classic Michaelis–Menten equation to study concentration-dependent kinetics of Cd2 þ uptake under Fe-deficiency in Thlaspi caerulescens. They suggested that Vmax was usually more focused on than Km, for the reason of absorption velocity deciding the uptake and transport ability of Cd2 þ in plants. Cd can replace necessary elements to plants growth, e.g. Cu, in various cyto-plasmic and membrane proteins (Yan et al., 2015) and then cause plant poisoning. However, Cd accumulation in leaves of A. marina is much less than that in root, even undetectable (Wu et al., 2014). A. marina accumulate heavy metals Cd in root tissues with levels equal to or greater than the surrounding sediment concentrations, especially in fine nutritive roots (Wu et al., 2014). Thus the researches on the absorption of Cd in A. marina roots are especially crucial. To our knowledge, few researches have reported on the influence of flavonoids on concentration-dependent kinetics of Cd2 þ absorption in A. marina roots. In the present study, A. marina, one of the dominant mangrove species in the southeast of China was selected as the representative species because of its high content of phenolic compounds, abundance and well-developed root system. The aim being: (i) to investigate the concentration-dependent kinetics of Cd2 þ absorption in A. marina roots; (ii) to evaluate the influence of flavonoids on concentration-dependent kinetics of Cd2 þ absorption in A. marina roots, and (iii) to determine the physiological role of flavonoids in Cd2 þ uptake and accumulation in mangrove seedlings.
2. Material and methods 2.1. Plant material Mature A. marina propagules were collected from the Jiulong River mangrove natural reserve (24°24' N, 117°55' E), Fujian China, in September 2013. Complete undamaged propagules with testa intact and high vitality were chosen for pre-cultivation in sea sand. Sea sand used was prewashed with concentrated HCl and rinsed thoroughly with tap water (Liu et al., 2009). All propagules selected had comparable sizes (25 7 2 g). The propagules were inserted into the 4 L polyethylene seedling pots. Polyethylene seedling pots were filled with nutrient solution and black polyethylene beads to prevent light exposure of the nutrient solution. Cultured the propagules for 2 months to be 3–4 euphyllas. In
general, seedlings were grown in a modified Hogland nutrient solution containing the following macronutrients in mg/l: KNO3, 707.70; NH4H2PO4, 230.04; MgSO4, 240.72; H3BO3, 2.868; CuSO4 5H2O, 0.08; ZnSO4 7H2O, 0.22; MnSO4 H2O, 1.55; (NH4)6Mo7O24 4H2O, 0.61; FeSO4, 5.57; EDTA.2Na, 7.45; Ca(NO3)2, 1180.76. The nutrient solution was renewed once every 3 days. The seedlings were placed in a greenhouse with daily temperature of 20–29.5 °C, relative humidity of 59–80% and light intensity of 800– 1400 umol photons m 2s 1 (Yan and Tam, 2011). 2.2. Experimental design and sample culture All seedlings were divided into two groups, group-A treated without flavonoids and group-B treated with flavonoids respectively, were used for the following 5 processes. The group just treated with different Cd concentration was stated as “control”. Step 1. Seedlings in group-A were inserted into 18 black polyethylene 1 L cups (four seedlings per cup) filled with isometric Hogland nutrient solution. This was adjusted to a concentration of 5 mg/l Cd2 þ . The same operation was performed on group-B with added flavonoids (Catechin: gallic acid ¼1:2) to 5 mg/l. Three duplicate cups for group-A and group-B were cultivated for 1, 2, 4, 8, 16, 32 h, respectively. Roots were rinsed thoroughly, then cut out and freeze dried in a vacuum for 48 h. Step 2. Seedlings in group-A were inserted into 18 black polyethylene cups (four seedlings per cup) filled with isometric Hogland nutrient solution. This was adjusted to a concentration of Cd2 þ to 0, 2.5, 5, 10, 20, and 40 mg/l. The same operation process was performed on group-B with adjusting flavonoids concentration (Catechin: gallic acid ¼1:2) to 5 mg/l. Three duplicate cups for group-A and group-B were cultivated. The period was decided by step 1. Roots were rinsed thoroughly, then cut out and freeze dried in a vacuum for 48 h. Step 3. Root cell wall preparations that maintained the morphologic and geometric characteristics of intact roots were obtained. Root systems were immersed in methanol: chloroform (2:1, v/v) solutions for 3 d and rinsed in several changes of distilled water for 1 d. This treatment has been shown to yield lipid-free cell wall preparations in maize while maintaining the structure and morphologic characteristics of an intact roots (Hart et al., 1992). Culture and harvest was consistent with step 2. Step 4. Root systems were immersed in a solution containing 0.2 mmol/l LaCl3 for 30 min (Cohen et al., 1998), then rinsed thoroughly. Culture and harvest was consistent with step 2. Step 5. The root were immersed in a solution containing 20umol/l CCCP (Carbonylcyanidem-chlorophenyl-hydrazone) for 30 min (Cohen et al., 1998), then rinsed thoroughly. Culture and harvest was consistent with step 2. Cd concentration was determined by atomic absorption spectrophotometry (AAS, Model AA-6800, Shimadzu, Kyoto) with 90– 94% percentage recovery and 1 ug/kg detection limit after digestion operated by the description of Soto-Jimenez and Paez-Osuna (2001). The concentration factor was calculated as the ratio of the metal concentrations in the plants (in micrograms per gram dry weight (DW)) and the concentrations in the solution (in milligrams per liter). Reagent blanks and standard references of plant material (GBW-07603) (from the National Research Center for Standards in China) were included to verify the accuracy and precision of the digestion procedure and subsequent analysis. All reagents were Merck analytical grade or Suprapur quality, and all materials (bottles, filters etc,) were acid-cleaned (14% (v/v) nitric acid) and rinsed with deionized water prior to use. Deionized water was
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used in preparing stock solutions and in each step of the procedures. 2.3. Analysis of Cd concentration in roots The data were analyzed using a statistical package, SPSS version 13.0 and Systat sigmaplot version 12.0. The concentration of flavonoids and Cd in nutrient solution as independent variables, Cd concentration in roots as dependent variable, a one-way analysis of variance (ANOVA) was carried out. All results presented and discussed are based on mean values and standard deviation (S.D.) of three replications, and were tested by the Duncan's Multiple Range Test and least significant difference (LSD).
3. Results 3.1. Time-dependent kinetics of Cd2 þ absorption in the root system. There was no significant difference between first hour and the second hour (Fig.1). Flavonoids have no significant influence on the uptake of Cd2 þ in root systems in the first two periods. Nevertheless, the concentration of Cd2 þ increased with the following period (p o0.05). Compared with group-A (the control), group-B had higher values in any point of 4 h, 8 h, 16 h and 32 h (p o0.05). Cd uptake was consistent with the increasing trends over time (Fig. 1), even though the concentration of Cd2 þ in roots with added flavonoids was higher than the control. Considering the stability and efficiency of the experiment, 32 h was chosen. 3.2. Concentration-dependent kinetics of Cd2 þ absorption in root systems Concentrations of Cd2 þ absorption for different periods are shown in Fig.2. A kinetic model consisting of saturation and linear components was suitably to describe Cd2 þ in the roots of group-A (r2 ¼0.9985). For roots of group-B, a linear-plus-saturation function or a linear function described the kinetics of Cd2 þ equally well (Fig. 2; r2 ¼0.9969). The kinetic parameters (Km and Vmax) for the saturation system (Fig. 2) fitted the data to computer generated linear-plus-saturation equations. Saturation Cd2 þ for group-A and group-B roots exhibited significantly different Km values (p o0.05), at 25.15 and 39.14 ug/kg. Group-B plants exhibited a nearly 63.9% higher Km for absorption. Moreover, the
Fig. 1. Curve fits of Cd2 þ time-dependent kinetics in A. Marina roots.
Fig. 2. Curve fits of Cd2 þ concentration-dependent kinetics in A. Marina roots under different CdCl2 stress. V0, V1 represent rate of Cd2 þ absorption with addition and non-addition of flavonoids respectively.
group-B roots exhibited a nearly 51.1% higher Vmax for absorption (1066.01 versus 769.23 ug/kg (DW)/h, p o0.05). The slopes of the linear components were similar, 20.01 and 17.12 ug/kg (DW)/h /uM for group-B and group-A roots respectively. 3.3. Concentration-dependent kinetics of Cd2 þ uptake in the root cell walls The linear component reflected apoplastic binding of Cd2 þ in the cell walls that was not removed during the desorption period. The kinetics of Cd2 þ absorption (or binding) in morphologically intact root cell walls was examined in Fig.3. A linear model was adequately to describe Cd2 þ binding to root cell wall preparations of both group-A and group-B seedlings (r2 ¼0.9878 for group-A and r2 ¼0.9867 for group-B roots).The group-A and group-B roots exhibited a similar Vmax for absorption (2552.61 versus 2323.98 ug/kg (DW)/h). The slope of the concentration-dependent Cd2 þ binding in the cell wall preparations was greater than that of the linear component for intact roots in group-A (22.31 versus 17.12 ug/kg (DW)/h /uM).
Fig. 3. Curve fits of Cd2 þ concentration-dependent kinetics in A. Marina root cell walls under different CdCl2 stress. V0, V1 represent rate of Cd2 þ absorption with addition and non-addition of flavonoids respectively.
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Fig. 4. Curve fits of Cd2 þ concentration-dependent kinetics in A. Marina roots treated with LaCl3 under different CdCl2 stress. V0, V1 represent rate of Cd2 þ absorption with addition and non-addition of flavonoids respectively, and Vʹ0, Vʹ1 represent rate of Cd2 þ absorption with addition and non-addition of flavonoids under LaCl3 treatment.
3.4. Concentration-dependent kinetics of Cd2 þ in roots treated with LaCl3 LaCl3, a Ca2 þ -channel blocker, inhibited kinetic transport components (Fig. 4). A linear-plus-saturation function was the best fit (r2 ¼0.9668, r2 ¼0.9727 for group-A and group-B, respectively) with a reduced slope (po 0.05), 7.09 ug/kg (DW)/h /uM, versus 17.12 ug/kg (DW)/h /uM for the control in group-A. This result is considered as a model in which La3 þ may occupy Cd2 þ -binding site from the cell walls, compete with Cd2 þ for a plasma membrane transporter and decrease the slope of the linear component accompanying the reduced Vmax (p o0.05), 385.35 ug/kg (DW)/h versus 769.23 ug/kg (DW)/h, for control, and inhibiting the saturation component. Group-B roots exhibited a much higher Vmax (642.52 ug/kg (DW)/h) compared with group-A (385.35 ug/kg (DW)/h, po 0.05) in response to exposure to LaCl3, however, a lower one than group-B in 3.2 (1066.01 ug/kg (DW)/h, po 0.05). 3.5. Concentration-dependent kinetics of Cd2 þ in roots treated with CCCP Treatment with the respiratory inhibitor of protonophore CCCP specifically abolished the saturation Cd2 þ -component and clearly exhibited the linear component (Fig. 5). The best-fit model was a linear function (r2 ¼ 0.9827 and r2 ¼ 0.9856 in group-A and group-B respectively, for CCCP-treated roots), providing additional evidence that saturation Cd2 þ uptake represents Cd2 þ transportermediated across the root-cell plasma membrane. Flavonoids would not facilitate absorption of Cd2 þ in roots because of the approximate Vmax inhibiting respiration. 3.6. Absorption of Cd2 þ in surface growth. Compared with the group treated without flavonoids, the group of flavonoid amendments showed higher concentration of Cd2 þ in A. marina roots system (p o0.05), however, no remarkable difference was exhibited in surface growth of plants (Data not shown). 4. Discussion Divalent cation transport proteins would be non-specific
Fig. 5. Curve fits of Cd2 þ concentration-dependent kinetics in A. Marina roots treated with CCCP under different CdCl2 stress. V0, V1 represent rate of Cd2 þ absorption with addition and non-addition of flavonoids.
transport media, in addition to Fe2 þ and Ca2 þ , transport more divalent cations, e.g. Cu2 þ , Zn2 þ , Mg2 þ (Cohen et al., 2004). Cd2 þ Compete with Fe2 þ and Ca2 þ to enter plants through transport media resulted in abnormal growth of the hosts. In this research, we studied the absorption characteristics of Cd2 þ under flavonoid amendments in the A. marina roots system. The concentrationdependent kinetics for Cd2 þ was graphically complex but could be resolved into a linear component and a saturation component. These represented an apoplast transport of Cd2 þ -binding cell walls and a transmembrane transport of cross-root cell plasma membrane exhibiting Michaelis–Menten kinetics (Zhao et al., 2002, 2006). Vmax would reflect influence of flavonoid amendments on absorption of Cd2 þ in the A. marina roots system, because Vmax represents the capability for roots absorbing Cd2 þ , while Km shows the affinity of its root system with Cd2 þ . After the second period, group-B had higher values in any point compared with group-A (Fig.1). This showed that flavonoids facilitate the absorption of Cd2 þ in roots. 32 h was critically different from that reported as 20 min in pea and wheat (Cohen et al., 1998; Hart et al., 1998) for the period of different concentrations of Cd2 þ absorption under different conditions in root systems. The enormous variance here reflects a stronger Cd2 þ absorption and accumulation capability in A. marina roots. The group of flavonoid amendments showed a significant difference from the control for Michaelis constant, exhibiting a nearly 51.1% higher Vmax (Fig. 2). This showed that a large number of transport proteins in the root-cell plasma membrane were inactive. Thus flavonoid amendments facilitated a large increase in a high-affinity, saturation Cd2 þ -absorption system. Active transport was the major route for Cd2 þ absorption in roots, because the saturation uptake represented carrier-mediated transport across the root-cell plasma membrane. It attested to a catalytic role of flavonoids for Cd2 þ transmembrane transport in root system. The similar slopes of the linear components provided additional evidence of flavonoids acting on transmembrane transport and facilitating a large increase in a saturation Cd2 þ -absorption system. A proposed mechanism for flavonoid contribution to increased iron availability through the formation of Fe complexes/chelates is summarized in Fig.6. (Cesco et al., 2010). Flavonoids can also chelate heavy metals depending on various molecular structures (Keilig and Ludwig-Muller, 2009; Korkina, 2007). We can infer that Cd2 þ competed with other divalent cations, chelated with flavonoids and entered into the cytomembrane, in which the flavonoids act as a carrier.
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Fig. 6. Proposed mechanisms of the contribution of flavonoids to iron availability in the rhizosphere, and its acquisition by plants (adapted from Cesco et al. (2010)).
Kinetics of Cd2 þ absorption (or binding) was examined here in morphologically intact root cell walls (Fig. 3). A linear model adequately described Cd2 þ binding to root cell wall preparations of both the groups treated methanol: chloroform. The similar Vmax showed that flavonoid amendments have not facilitated an increase in Cd2 þ root cell walls binding. The linear component reflected in the cell walls that was not removed during desorption period. It showed that flavonoid amendments have not facilitated apoplastic binding of Cd2 þ . The difference of slope between the cell wall preparations and the intact roots in group-A could be attributed to an exposure of potential binding sites in the root cell wall preparations resulting from removal of the root symplast (Ditomaso et al., 1992). In the experiment of concentration-dependent kinetics of Cd2 þ in roots treated with LaCl3, 0.2 mmol/l La3 þ inhibited both plasma membrane transport and cell wall-blinding of Cd2 þ efficiently, similar to Ca2 þ (Fig. 4). Increasing concentration of Ca2 þ was reported to inhibit from Cd2 þ absorption because Cd2 þ would be transported through the Ca2 þ -channel (White, 2000). Our finding agreed with Zhao et al. (2002) researching the response of T. caerulescens to LaCl3. They found that 50 umol/l LaCl3 or 5 mmol/l CaCl2 decreased absorption of Cd2 þ in T. caerulescens. This would result from inhibition of La3 þ /Ca2 þ and the formation of Cd2 þ -Clcomplex reducing Cd2 þ activity. This proves that an antagonism effect was present between La3 þ /Ca2 þ and Cd2 þ . Our results showed that flavonoid amendments have facilitated absorption of Cd2 þ in roots treated with a Ca2 þ -channel blocker. Perfus-Barbeoch et al. (2002) demonstrated that plants absorbed Cd2 þ through Ca2 þ -channel by way of patch clamp technique. However, Lindberg et al. (2004) found Cd2 þ was transported by a
mechanism of non-selective cation channels. We inferred that other channel (or channels) has been used to transport the Cd2 þ in the A. marina roots except for Ca2 þ -channel. Roots treated with the respiratory inhibitor protonophore CCCP showed a similar kinetic function (Fig. 5). This demonstrates further flavonoid amendments have only acted on transmembrane transport, and that the Ca2 þ -channel was not the unique system of symplasm transport for Cd2 þ absorption in A. marina roots. No remarkable difference was exhibited in surface growth of plants. The reason might be more time is required for the roots to transport Cd2 þ to stems and leaves.
5. Conclusion A Michaelis–Menten kinetic model consisting of saturation and linear components have represented transmembrane transport in roots and apoplastic transport in root cell walls. It was suited to describe Cd2 þ absorption in mangrove plant A. marina roots under the influence or non-influence of flavonoids. Compared to the control, Km and Vmax was higher under the influence of flavonoids. Demonstrating that flavonoids enhance the tolerance of plant to Cd. Nevertheless, flavonoids showed no influence on the uptake of Cd2 þ in root cell walls. The same result was achieved in roots treated by CCCP. These results showed that flavonoids act on symplasm transport when roots take up the heavy metal Cd2 þ , but do not affect apoplastic transport. In the roots treated with the ion transport inhibitor (LaCl3), Km and Vmax was higher compared with the group without flavonoid amendments, and lower than the control, which demonstrated that the Ca2 þ -channel was not the unique means of
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symplasm transport for Cd2 þ absorption in A. marina roots.
Acknowledgments This work was supported by National Important Scientific Research Programme of China (2013CB956504) and National Natural Science Foundation of China (31370516, 31170471). We thank Prof. John Merefield for assistance with English grammar. Dr. Yu J.Y. was greatly acknowledged for valuable advice; Dr. Zhang J.C., Dr. Yan W.F. and Dr. Jia H. and Dr. Yang J.J. were also sincerely acknowledged for the co-operation of sample cultures.
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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
The influence of flavonoid amendment on the absorption of cadmium in Avicennia marina roots Jian Li, Haoliang Lu, Jingchun Liu, Hualong Hong, Chongling Yan n Key Laboratory of Ministry of Education for Coastal and Wetland Ecosystems, Xiamen University, Xiamen 361102, China
art ic l e i nf o
a b s t r a c t
Article history: Received 9 February 2015 Received in revised form 23 April 2015 Accepted 5 May 2015
Flavonoid is a key factor for the tolerance to cadmium in plants. Concentration-dependent kinetics experiment was conducted to investigate the influence of flavonoid amendment on the Cd2 þ uptake in Avicennia marina (Forsk) Vierh. roots. We found that compared with the control, saturation concentration and maximal absorption rate of Cd was higher under flavonoid amendment (po 0.05). When roots were exposed to ion transport inhibitor (LaCl3), flavonoid amendment also facilitated Cd transport in roots. Flavonoids had no influence on Cd2 þ uptake in root cell walls. In conclusion, flavonoids enhance the tolerance to Cd and have a significant stimulative effect on symplasm transport of Cd in A. marina roots. Ca2 þ -channel was not the unique means of symplasm transport for Cd2 þ absorption. & 2015 Elsevier Inc. All rights reserved.
Keywords: Cadmium Flavonoid Mangrove Avicennia marina
1. Introduction Mangrove forests located in the intertidal zone of tropical and subtropical regions, are known as specific ecological habitats, and have been regarded as playing a key role in maintaining the coastal ecological balance (Du et al., 2013; Liu et al., 2010). However, mangroves are subjected to environmental pollutants, especially heavy metal contaminants, such as Cd, Cr, Cu, Pb, Hg (Cuong et al., 2005; Wu et al., 2011). A. marina, being the pioneer mangrove species in the southeast of China, was found to accumulate Cd, Cu, Pb and Zn in root tissues under field conditions with levels equal to or greater than the surrounding sediment concentrations (MacFarlane and Burchett, 2002; MacFarlane et al., 2003; Nowrouzi et al., 2012). Cd has drawn more attention because of its high activity and bioavailability, which can interfere with plant metabolic processes, resulting in poor growth and lower biomass. The plants absorb Cd2 þ through apoplastic and symplasm transport, nevertheless, there are specific or generic ionophore or channel proteins into root cells. Cd2 þ can be taken in through Ca2 þ and Mg2 þ /Fe2 þ channels, and compete with divalent cations (Welch et al., 1999). Mangrove plants have a high content of phenolic compounds (Rahim et al., 2007; Rahim et al., 2008) mainly containing flavonoids and tannins. As one kind of secondary metabolites, flavonoids account for 37% of secondary metabolites (Narasimhan et al., n
Corresponding author. E-mail address: [email protected] (C. Yan).
http://dx.doi.org/10.1016/j.ecoenv.2015.05.004 0147-6513/& 2015 Elsevier Inc. All rights reserved.
2003). Besides being free radical scavengers, flavonoid can chelate heavy metal depending on their different molecular structure (Korkina, 2007), thus inhibit lipid peroxidation and Fenton reaction (Chen et al., 1990), where heavy metals cause lipid peroxidation and free radical imbalance (Korkina, 2007). Stingu et al. (2012) indicated that phenolic compounds made a critical difference in tolerance and bioaccumulation of Avena sativa L. as chelator and solubilizer of heavy metals (Stingu et al., 2012); Keilig and Ludwig-Muller (2009) made a similar conclusion, pointed out that flavonoids were a key factor for the tolerance in Arabidopsis thaliana. Arora et al. (1998) tested the inhibition of flavonoids in the liposome membrane system by lipid peroxidation induced by Fe3 þ or peroxide radical. They found that all the tested flavonoids had higher antioxidation efficiency to peroxidation induced by metal ion than those induced by peroxy radical (Arora et al., 1998). The substituent group mode of flavonoids B-ring influences its antioxidation ability significantly. Certain hydroxy substituents on the A-ring may compensate and become a larger determinant of the antioxidant efficacy when B-ring cannot contribute to the antioxidant activity of the flavonoids. These are determining factors for flavonoids’ antioxidation ability (Arora et al., 1998). All these authors have suggested that the chelation of flavonoids makes much critical difference to their antioxidation. The anti-lipid peroxidation capability of flavonoids is the cause of the activity of scavenging free radical and metal chelation. The absorption of Cd2 þ is impacted by divalent cations (Zn2 þ , Ca2 þ , Fe2 þ etc.) (Lombi et al., 2002; Zhao et al., 2002) and root exudates (flavonoids and low molecular weight organic acids etc.) (Roth et al., 2012). Cd2 þ is absorbed through an iron transporter
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protein in the plasma membrane; where the absorption and transport of Cd2 þ in roots is increasingly involved. Fe2 þ affect the absorption of Cd2 þ by competing binding site and transport agent with Cd2 þ (Charlatchka and Cambier, 2000; Davranche and Bollinger, 2000; Pi et al., 2011). Flavonoids as chelators take on a transporter role to influence the absorption of Cd2 þ ; with the same effect on Zn2 þ , Fe2 þ etc. A large number of studies reported that flavonoids are able to function as chelators for metals (Aherne and O’Brien, 2000; Brown et al., 1998; Korkina, 2007; Michalak, 2006; Soczynska-Kordala et al., 2001). They are the main components of root exudates in response to Fe-deficiency (Cesco et al., 2010; Olsen et al., 1981), which act as a Fe2 þ -carrier to transport Fe2 þ . The classic Michaelis–Menten enzyme kinetics model has been widely applied to absorption of metals in plant roots. The Michaelis constant Km (half-saturation constant) shows the affinity of roots system with metal, while Vmax (maximal rate of velocity) represents the capability of roots for absorbing metal (Zhao et al., 2002, 2006). The model is described as
V = (Vmax[S ])/(Km + [S ] ) Zhao et al. (2002) utilized the classic Michaelis–Menten equation to study concentration-dependent kinetics of Cd2 þ uptake under Fe-deficiency in Thlaspi caerulescens. They suggested that Vmax was usually more focused on than Km, for the reason of absorption velocity deciding the uptake and transport ability of Cd2 þ in plants. Cd can replace necessary elements to plants growth, e.g. Cu, in various cyto-plasmic and membrane proteins (Yan et al., 2015) and then cause plant poisoning. However, Cd accumulation in leaves of A. marina is much less than that in root, even undetectable (Wu et al., 2014). A. marina accumulate heavy metals Cd in root tissues with levels equal to or greater than the surrounding sediment concentrations, especially in fine nutritive roots (Wu et al., 2014). Thus the researches on the absorption of Cd in A. marina roots are especially crucial. To our knowledge, few researches have reported on the influence of flavonoids on concentration-dependent kinetics of Cd2 þ absorption in A. marina roots. In the present study, A. marina, one of the dominant mangrove species in the southeast of China was selected as the representative species because of its high content of phenolic compounds, abundance and well-developed root system. The aim being: (i) to investigate the concentration-dependent kinetics of Cd2 þ absorption in A. marina roots; (ii) to evaluate the influence of flavonoids on concentration-dependent kinetics of Cd2 þ absorption in A. marina roots, and (iii) to determine the physiological role of flavonoids in Cd2 þ uptake and accumulation in mangrove seedlings.
2. Material and methods 2.1. Plant material Mature A. marina propagules were collected from the Jiulong River mangrove natural reserve (24°24' N, 117°55' E), Fujian China, in September 2013. Complete undamaged propagules with testa intact and high vitality were chosen for pre-cultivation in sea sand. Sea sand used was prewashed with concentrated HCl and rinsed thoroughly with tap water (Liu et al., 2009). All propagules selected had comparable sizes (25 7 2 g). The propagules were inserted into the 4 L polyethylene seedling pots. Polyethylene seedling pots were filled with nutrient solution and black polyethylene beads to prevent light exposure of the nutrient solution. Cultured the propagules for 2 months to be 3–4 euphyllas. In
general, seedlings were grown in a modified Hogland nutrient solution containing the following macronutrients in mg/l: KNO3, 707.70; NH4H2PO4, 230.04; MgSO4, 240.72; H3BO3, 2.868; CuSO4 5H2O, 0.08; ZnSO4 7H2O, 0.22; MnSO4 H2O, 1.55; (NH4)6Mo7O24 4H2O, 0.61; FeSO4, 5.57; EDTA.2Na, 7.45; Ca(NO3)2, 1180.76. The nutrient solution was renewed once every 3 days. The seedlings were placed in a greenhouse with daily temperature of 20–29.5 °C, relative humidity of 59–80% and light intensity of 800– 1400 umol photons m 2s 1 (Yan and Tam, 2011). 2.2. Experimental design and sample culture All seedlings were divided into two groups, group-A treated without flavonoids and group-B treated with flavonoids respectively, were used for the following 5 processes. The group just treated with different Cd concentration was stated as “control”. Step 1. Seedlings in group-A were inserted into 18 black polyethylene 1 L cups (four seedlings per cup) filled with isometric Hogland nutrient solution. This was adjusted to a concentration of 5 mg/l Cd2 þ . The same operation was performed on group-B with added flavonoids (Catechin: gallic acid ¼1:2) to 5 mg/l. Three duplicate cups for group-A and group-B were cultivated for 1, 2, 4, 8, 16, 32 h, respectively. Roots were rinsed thoroughly, then cut out and freeze dried in a vacuum for 48 h. Step 2. Seedlings in group-A were inserted into 18 black polyethylene cups (four seedlings per cup) filled with isometric Hogland nutrient solution. This was adjusted to a concentration of Cd2 þ to 0, 2.5, 5, 10, 20, and 40 mg/l. The same operation process was performed on group-B with adjusting flavonoids concentration (Catechin: gallic acid ¼1:2) to 5 mg/l. Three duplicate cups for group-A and group-B were cultivated. The period was decided by step 1. Roots were rinsed thoroughly, then cut out and freeze dried in a vacuum for 48 h. Step 3. Root cell wall preparations that maintained the morphologic and geometric characteristics of intact roots were obtained. Root systems were immersed in methanol: chloroform (2:1, v/v) solutions for 3 d and rinsed in several changes of distilled water for 1 d. This treatment has been shown to yield lipid-free cell wall preparations in maize while maintaining the structure and morphologic characteristics of an intact roots (Hart et al., 1992). Culture and harvest was consistent with step 2. Step 4. Root systems were immersed in a solution containing 0.2 mmol/l LaCl3 for 30 min (Cohen et al., 1998), then rinsed thoroughly. Culture and harvest was consistent with step 2. Step 5. The root were immersed in a solution containing 20umol/l CCCP (Carbonylcyanidem-chlorophenyl-hydrazone) for 30 min (Cohen et al., 1998), then rinsed thoroughly. Culture and harvest was consistent with step 2. Cd concentration was determined by atomic absorption spectrophotometry (AAS, Model AA-6800, Shimadzu, Kyoto) with 90– 94% percentage recovery and 1 ug/kg detection limit after digestion operated by the description of Soto-Jimenez and Paez-Osuna (2001). The concentration factor was calculated as the ratio of the metal concentrations in the plants (in micrograms per gram dry weight (DW)) and the concentrations in the solution (in milligrams per liter). Reagent blanks and standard references of plant material (GBW-07603) (from the National Research Center for Standards in China) were included to verify the accuracy and precision of the digestion procedure and subsequent analysis. All reagents were Merck analytical grade or Suprapur quality, and all materials (bottles, filters etc,) were acid-cleaned (14% (v/v) nitric acid) and rinsed with deionized water prior to use. Deionized water was
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used in preparing stock solutions and in each step of the procedures. 2.3. Analysis of Cd concentration in roots The data were analyzed using a statistical package, SPSS version 13.0 and Systat sigmaplot version 12.0. The concentration of flavonoids and Cd in nutrient solution as independent variables, Cd concentration in roots as dependent variable, a one-way analysis of variance (ANOVA) was carried out. All results presented and discussed are based on mean values and standard deviation (S.D.) of three replications, and were tested by the Duncan's Multiple Range Test and least significant difference (LSD).
3. Results 3.1. Time-dependent kinetics of Cd2 þ absorption in the root system. There was no significant difference between first hour and the second hour (Fig.1). Flavonoids have no significant influence on the uptake of Cd2 þ in root systems in the first two periods. Nevertheless, the concentration of Cd2 þ increased with the following period (p o0.05). Compared with group-A (the control), group-B had higher values in any point of 4 h, 8 h, 16 h and 32 h (p o0.05). Cd uptake was consistent with the increasing trends over time (Fig. 1), even though the concentration of Cd2 þ in roots with added flavonoids was higher than the control. Considering the stability and efficiency of the experiment, 32 h was chosen. 3.2. Concentration-dependent kinetics of Cd2 þ absorption in root systems Concentrations of Cd2 þ absorption for different periods are shown in Fig.2. A kinetic model consisting of saturation and linear components was suitably to describe Cd2 þ in the roots of group-A (r2 ¼0.9985). For roots of group-B, a linear-plus-saturation function or a linear function described the kinetics of Cd2 þ equally well (Fig. 2; r2 ¼0.9969). The kinetic parameters (Km and Vmax) for the saturation system (Fig. 2) fitted the data to computer generated linear-plus-saturation equations. Saturation Cd2 þ for group-A and group-B roots exhibited significantly different Km values (p o0.05), at 25.15 and 39.14 ug/kg. Group-B plants exhibited a nearly 63.9% higher Km for absorption. Moreover, the
Fig. 1. Curve fits of Cd2 þ time-dependent kinetics in A. Marina roots.
Fig. 2. Curve fits of Cd2 þ concentration-dependent kinetics in A. Marina roots under different CdCl2 stress. V0, V1 represent rate of Cd2 þ absorption with addition and non-addition of flavonoids respectively.
group-B roots exhibited a nearly 51.1% higher Vmax for absorption (1066.01 versus 769.23 ug/kg (DW)/h, p o0.05). The slopes of the linear components were similar, 20.01 and 17.12 ug/kg (DW)/h /uM for group-B and group-A roots respectively. 3.3. Concentration-dependent kinetics of Cd2 þ uptake in the root cell walls The linear component reflected apoplastic binding of Cd2 þ in the cell walls that was not removed during the desorption period. The kinetics of Cd2 þ absorption (or binding) in morphologically intact root cell walls was examined in Fig.3. A linear model was adequately to describe Cd2 þ binding to root cell wall preparations of both group-A and group-B seedlings (r2 ¼0.9878 for group-A and r2 ¼0.9867 for group-B roots).The group-A and group-B roots exhibited a similar Vmax for absorption (2552.61 versus 2323.98 ug/kg (DW)/h). The slope of the concentration-dependent Cd2 þ binding in the cell wall preparations was greater than that of the linear component for intact roots in group-A (22.31 versus 17.12 ug/kg (DW)/h /uM).
Fig. 3. Curve fits of Cd2 þ concentration-dependent kinetics in A. Marina root cell walls under different CdCl2 stress. V0, V1 represent rate of Cd2 þ absorption with addition and non-addition of flavonoids respectively.
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Fig. 4. Curve fits of Cd2 þ concentration-dependent kinetics in A. Marina roots treated with LaCl3 under different CdCl2 stress. V0, V1 represent rate of Cd2 þ absorption with addition and non-addition of flavonoids respectively, and Vʹ0, Vʹ1 represent rate of Cd2 þ absorption with addition and non-addition of flavonoids under LaCl3 treatment.
3.4. Concentration-dependent kinetics of Cd2 þ in roots treated with LaCl3 LaCl3, a Ca2 þ -channel blocker, inhibited kinetic transport components (Fig. 4). A linear-plus-saturation function was the best fit (r2 ¼0.9668, r2 ¼0.9727 for group-A and group-B, respectively) with a reduced slope (po 0.05), 7.09 ug/kg (DW)/h /uM, versus 17.12 ug/kg (DW)/h /uM for the control in group-A. This result is considered as a model in which La3 þ may occupy Cd2 þ -binding site from the cell walls, compete with Cd2 þ for a plasma membrane transporter and decrease the slope of the linear component accompanying the reduced Vmax (p o0.05), 385.35 ug/kg (DW)/h versus 769.23 ug/kg (DW)/h, for control, and inhibiting the saturation component. Group-B roots exhibited a much higher Vmax (642.52 ug/kg (DW)/h) compared with group-A (385.35 ug/kg (DW)/h, po 0.05) in response to exposure to LaCl3, however, a lower one than group-B in 3.2 (1066.01 ug/kg (DW)/h, po 0.05). 3.5. Concentration-dependent kinetics of Cd2 þ in roots treated with CCCP Treatment with the respiratory inhibitor of protonophore CCCP specifically abolished the saturation Cd2 þ -component and clearly exhibited the linear component (Fig. 5). The best-fit model was a linear function (r2 ¼ 0.9827 and r2 ¼ 0.9856 in group-A and group-B respectively, for CCCP-treated roots), providing additional evidence that saturation Cd2 þ uptake represents Cd2 þ transportermediated across the root-cell plasma membrane. Flavonoids would not facilitate absorption of Cd2 þ in roots because of the approximate Vmax inhibiting respiration. 3.6. Absorption of Cd2 þ in surface growth. Compared with the group treated without flavonoids, the group of flavonoid amendments showed higher concentration of Cd2 þ in A. marina roots system (p o0.05), however, no remarkable difference was exhibited in surface growth of plants (Data not shown). 4. Discussion Divalent cation transport proteins would be non-specific
Fig. 5. Curve fits of Cd2 þ concentration-dependent kinetics in A. Marina roots treated with CCCP under different CdCl2 stress. V0, V1 represent rate of Cd2 þ absorption with addition and non-addition of flavonoids.
transport media, in addition to Fe2 þ and Ca2 þ , transport more divalent cations, e.g. Cu2 þ , Zn2 þ , Mg2 þ (Cohen et al., 2004). Cd2 þ Compete with Fe2 þ and Ca2 þ to enter plants through transport media resulted in abnormal growth of the hosts. In this research, we studied the absorption characteristics of Cd2 þ under flavonoid amendments in the A. marina roots system. The concentrationdependent kinetics for Cd2 þ was graphically complex but could be resolved into a linear component and a saturation component. These represented an apoplast transport of Cd2 þ -binding cell walls and a transmembrane transport of cross-root cell plasma membrane exhibiting Michaelis–Menten kinetics (Zhao et al., 2002, 2006). Vmax would reflect influence of flavonoid amendments on absorption of Cd2 þ in the A. marina roots system, because Vmax represents the capability for roots absorbing Cd2 þ , while Km shows the affinity of its root system with Cd2 þ . After the second period, group-B had higher values in any point compared with group-A (Fig.1). This showed that flavonoids facilitate the absorption of Cd2 þ in roots. 32 h was critically different from that reported as 20 min in pea and wheat (Cohen et al., 1998; Hart et al., 1998) for the period of different concentrations of Cd2 þ absorption under different conditions in root systems. The enormous variance here reflects a stronger Cd2 þ absorption and accumulation capability in A. marina roots. The group of flavonoid amendments showed a significant difference from the control for Michaelis constant, exhibiting a nearly 51.1% higher Vmax (Fig. 2). This showed that a large number of transport proteins in the root-cell plasma membrane were inactive. Thus flavonoid amendments facilitated a large increase in a high-affinity, saturation Cd2 þ -absorption system. Active transport was the major route for Cd2 þ absorption in roots, because the saturation uptake represented carrier-mediated transport across the root-cell plasma membrane. It attested to a catalytic role of flavonoids for Cd2 þ transmembrane transport in root system. The similar slopes of the linear components provided additional evidence of flavonoids acting on transmembrane transport and facilitating a large increase in a saturation Cd2 þ -absorption system. A proposed mechanism for flavonoid contribution to increased iron availability through the formation of Fe complexes/chelates is summarized in Fig.6. (Cesco et al., 2010). Flavonoids can also chelate heavy metals depending on various molecular structures (Keilig and Ludwig-Muller, 2009; Korkina, 2007). We can infer that Cd2 þ competed with other divalent cations, chelated with flavonoids and entered into the cytomembrane, in which the flavonoids act as a carrier.
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Fig. 6. Proposed mechanisms of the contribution of flavonoids to iron availability in the rhizosphere, and its acquisition by plants (adapted from Cesco et al. (2010)).
Kinetics of Cd2 þ absorption (or binding) was examined here in morphologically intact root cell walls (Fig. 3). A linear model adequately described Cd2 þ binding to root cell wall preparations of both the groups treated methanol: chloroform. The similar Vmax showed that flavonoid amendments have not facilitated an increase in Cd2 þ root cell walls binding. The linear component reflected in the cell walls that was not removed during desorption period. It showed that flavonoid amendments have not facilitated apoplastic binding of Cd2 þ . The difference of slope between the cell wall preparations and the intact roots in group-A could be attributed to an exposure of potential binding sites in the root cell wall preparations resulting from removal of the root symplast (Ditomaso et al., 1992). In the experiment of concentration-dependent kinetics of Cd2 þ in roots treated with LaCl3, 0.2 mmol/l La3 þ inhibited both plasma membrane transport and cell wall-blinding of Cd2 þ efficiently, similar to Ca2 þ (Fig. 4). Increasing concentration of Ca2 þ was reported to inhibit from Cd2 þ absorption because Cd2 þ would be transported through the Ca2 þ -channel (White, 2000). Our finding agreed with Zhao et al. (2002) researching the response of T. caerulescens to LaCl3. They found that 50 umol/l LaCl3 or 5 mmol/l CaCl2 decreased absorption of Cd2 þ in T. caerulescens. This would result from inhibition of La3 þ /Ca2 þ and the formation of Cd2 þ -Clcomplex reducing Cd2 þ activity. This proves that an antagonism effect was present between La3 þ /Ca2 þ and Cd2 þ . Our results showed that flavonoid amendments have facilitated absorption of Cd2 þ in roots treated with a Ca2 þ -channel blocker. Perfus-Barbeoch et al. (2002) demonstrated that plants absorbed Cd2 þ through Ca2 þ -channel by way of patch clamp technique. However, Lindberg et al. (2004) found Cd2 þ was transported by a
mechanism of non-selective cation channels. We inferred that other channel (or channels) has been used to transport the Cd2 þ in the A. marina roots except for Ca2 þ -channel. Roots treated with the respiratory inhibitor protonophore CCCP showed a similar kinetic function (Fig. 5). This demonstrates further flavonoid amendments have only acted on transmembrane transport, and that the Ca2 þ -channel was not the unique system of symplasm transport for Cd2 þ absorption in A. marina roots. No remarkable difference was exhibited in surface growth of plants. The reason might be more time is required for the roots to transport Cd2 þ to stems and leaves.
5. Conclusion A Michaelis–Menten kinetic model consisting of saturation and linear components have represented transmembrane transport in roots and apoplastic transport in root cell walls. It was suited to describe Cd2 þ absorption in mangrove plant A. marina roots under the influence or non-influence of flavonoids. Compared to the control, Km and Vmax was higher under the influence of flavonoids. Demonstrating that flavonoids enhance the tolerance of plant to Cd. Nevertheless, flavonoids showed no influence on the uptake of Cd2 þ in root cell walls. The same result was achieved in roots treated by CCCP. These results showed that flavonoids act on symplasm transport when roots take up the heavy metal Cd2 þ , but do not affect apoplastic transport. In the roots treated with the ion transport inhibitor (LaCl3), Km and Vmax was higher compared with the group without flavonoid amendments, and lower than the control, which demonstrated that the Ca2 þ -channel was not the unique means of
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symplasm transport for Cd2 þ absorption in A. marina roots.
Acknowledgments This work was supported by National Important Scientific Research Programme of China (2013CB956504) and National Natural Science Foundation of China (31370516, 31170471). We thank Prof. John Merefield for assistance with English grammar. Dr. Yu J.Y. was greatly acknowledged for valuable advice; Dr. Zhang J.C., Dr. Yan W.F. and Dr. Jia H. and Dr. Yang J.J. were also sincerely acknowledged for the co-operation of sample cultures.
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