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The alleviation effect of iron on cadmium phytotoxicity in mangrove A. marina. Alleviation effect of iron on cadmium phytotoxicity in mangrove Avicennia marina (Forsk.) Vierh
Chemosphere 226 (2019) 413e420
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The alleviation effect of iron on cadmium phytotoxicity in mangrove A. marina. Alleviation effect of iron on cadmium phytotoxicity in mangrove Avicennia marina (Forsk.) Vierh Li Jian a, b, Liu Jingchun a, *, Yan Chongling a, Du Daolin b, Lu Haoliang a a b
Key Laboratory of Ministry of Education for Coastal and Wetland Ecosystems, Xiamen University, Xiamen, 361102, China Institute of Environment and Ecology, School of the Environment and Safety Engineering, Jiangsu University, Zhenjiang, 212013, China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Fe promoted Fe plaque to immobilise more Cd and decrease Cd absorption. Fe improved competition between Fe and Cd, and facilitated a decrease in Cd toxicity. Fe caused LMWOA secretion increase and played a crucial role in Cd detoxification. Significant positive correlation existed between Fe concentration and Cd tolerance. Fe application accelerated the growth and enhanced Cd tolerance of A. marina.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 31 October 2018 Received in revised form 8 March 2019 Accepted 26 March 2019 Available online 27 March 2019
Cd has high activity and bioavailability and is a poisonous element to plants. As a critical ecosysterm, mangroves are subjected to serious Cd pollution. In this research, the hypothesis was presented that improving Fe bioavailability would alleviate Cd phytotoxicity to Avicennia marina (Forsk.) Vierh. To test this, we examined the effect of four exogenous Fe and three Cd concentrations on A. marina. The results showed that a significant positive correlation excited between moderate exogenous Fe concentration and Cd tolerance of A. marina. Moderate exogenous Fe concentration directly or indirectly promoted the formation of Fe plaque, which immobilised more Cd on the root surface and decreased Cd absorption in roots. Furthermore, an exogenous Fe application increased plant biomass and Fe accumulation in A. marina tissues. This improved the competition between Fe and Cd within the plants. Therefore, an Fe application facilitated a decrease in Cd toxicity within A. marina. Simultaneously, a moderate Fe concentration caused an increase in low-molecular-weight organic acid (LMWOA) secretion from the roots. Meanwhile, Cd can be chelated/complexed by LMWOAs. It also played a crucial role in Cd detoxification in A. marina. In conclusion, Fe application accelerated the growth and enhanced Cd tolerance of A. marina. Therefore, improving Fe bioavailability will protect mangroves from Cd contamination. © 2019 Elsevier Ltd. All rights reserved.
Handling Editor: T. Cutright Keywords: Mangrove Cadmium Iron plaque Radial oxygen loss Low molecular weight organic acid
* Corresponding author. E-mail address: [email protected] (L. Jingchun). https://doi.org/10.1016/j.chemosphere.2019.03.172 0045-6535/© 2019 Elsevier Ltd. All rights reserved.
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1. Introduction Mangroves are critical zones that protect the intertidal belt of tropical and subtropical regions. As an ecological habitat, they provide an abundance of plants and animals that play a crucial role in maintaining coastal ecological balance. However, various pollutants drain from human habitation towards the mangroves, some of which are prone to accumulate in mangrove sediments, such as organic contaminants, organochlorine compounds, polycyclic aromatic hydrocarbons (Bhattacharya et al., 2003) as well as heavy metals Cd, Cu, Pb and Cr etc. (MacFarlane et al., 2003; Silva et al., 2006; Wu et al., 2011; Bayen, 2012; Li et al., 2016a, 2016b). Unlike organic contaminants, heavy metals cannot be degraded by microorganisms and they are instead absorbed and accumulated by plants and benthic macrofauna (Saha et al., 2006). They are then transferred into food chains/webs where they finally endanger human health. Thus, heavy metals present more lasting harm than organic contaminants. Mangroves are subjecting to serious heavy metal pollution and Cd concentration of 87 mg/g$dw was detected in mangrove sediments (Bayen, 2012). As a critical ecosysterm with an abundant biodiversity, mangroves supply plentiful food source for animals including human being, and they play a crucial role in fisheries. Hence, heavy metal pollution in mangrove is closely related to the health of coastal ecosystems. The white mangrove Avicennia marina is a pioneer mangrove species in southeast China, and it has been reported to have a high accumulation of heavy metals (Li et al., 2015, 2016a; Siswanti et al., 2016). It accumulates Cd, Cu, Zn and Pb in root tissues at levels equal to or above the surrounding sediment under field conditions (MacFarlane et al., 2003; Nowrouzi et al., 2012; Li et al., 2015). Because Cd has a high activity and bioavailability, A. marina could absorb Cd2þ through apoplastic and symplasm transport readily (Li et al., 2015). In symplastic translocation, there are specific or generic ionophores or channel proteins for Cd absorption in the roots. Once absorbed into the roots, Cd competes with a divalent cation, such as Ca2þ or Mg2þ/Fe2þ, for ionophores or channel proteins (Welch et al., 1999). In plants, Cd competes with the divalent cation, participates in plant metabolic process and then interferes with plant development, finally resulting in poor growth and lower biomass (Li et al., 2015). The geochemical cycling of Fe plays an important role in form transformation and bioavailability of elements (Li et al., 2017). As an essential activator of multiple enzymes in the process of chlorophyll synthesis and as the main component of Fe plaque, Fe is an essential element for plant growth and facilitates tolerance to contaminants, especially in wetland plants (Li et al., 2018). Photosynthesis by chlorophylls produces oxygen within the leaves. Some of the oxygen is transported into gas-filled spaces and the aerenchyma of the cortex by axial diffusion, then into nonporous tissues and the rhizosphere by radial diffusion (Sorrell, 2003; Lin et al., 2018). This is known as radial oxygen loss (ROL). ROL forms micro-aerobic surroundings, accelerates Fe oxidation precipitation and further develops Fe plaque on the root surface. It is considered to be one of the predominant factors affecting metal tolerance (Liu et al., 2009). Fe plaque, found on the root surface of wetland plants, can reduce or promote the accumulation of metal pollutants (Pi et al., 2010). As a coating on the root surface, Fe plaque functions as a physical ‘barrier’ co-precipitating and immobilising heavy metals (Tripathi et al., 2014), thus the bioavailability and absorption of heavy metals is reduced in plant roots (Pi et al., 2011). In addition, Fe competes with heavy metals for sensitive metabolic sites (Kuo, 1986) and has a positive effect on heavy metal detoxification within plants. For these reasons, Fe is a critical element for heavy metal tolerance in plants. However, there are few reports to provide the evidence of Fe element alleviating Cd phytotoxicity.
In fact, bioavailable Fe is restrictive on mangrove plant growth (Alongi, 2010). Thus, the hypothesis was presented that improving Fe bioavailability would alleviate Cd phytotoxicity to Avicennia marina (Forsk.) Vierh. In this study, different concentrations of exogenous Fe were used to evaluate the mechanisms of Fe influence on Cd tolerance in mangrove plants as follows: (1) plant physiological response in vivo, (2) physicochemical impact on the environment of root surface and (3) plant defence behaviour. 2. Materials and methods 2.1. Plant materials Mature A. marina propagules were collected from the Jiulong River Estuary Mangrove Natural Reserve (24 240 N, 117 550 E), Zhangzhou city, Fujian province, China, in September 2015. Complete propagules with high vitality were chosen for pre-cultivation in sea sands (Liu et al., 2009). Propagules of comparable size (2.5 ± 0.5 cm in diameter) were inserted into polyethylene seedling pots with a modified Hoagland's 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$7H2O, 0.22; MnSO$H2O, 1.55; (NH4)6Mo7O24$4H2O, 0.61; FeSO4, 5.57; EDTA$2Na, 7.45; Ca(NO3)2, 1180.76 (Li et al., 2015). The nutrient solution was renewed every 3 d and the propagules were cultured for about 2 months until they had 3e4 euphyllas. These seedlings were placed in a greenhouse with a daily temperature of 20 C-29.5 C, relative humidity of 59%e80% and light intensity of 800e1400 mmol photons/m2/s (Yan and Tam, 2011). 2.2. Experimental design and sample culture The whole experimental process included repeat five times culture and each culture contained Fe plaque induction and Cd stress. The first stage was Fe plaque induction for 2 d. Four concentration gradients in triplicate were used for Fe plaque induction using FeSO4$7H2O (pH ¼ 6.0): no exogenous Fe (0.02 mmol/l Fe in nutrient solution), low-dose Fe (0.6 mmol/l Fe), mid-dose Fe (1.2 mmol/l Fe) and high-dose Fe (1.8 mmol/l Fe). The second stage was Cd stress for 6 d. Three concentration gradients in triplicate were used for Cd stress using CdCl2 (pH ¼ 6.0): no Cd stress (0 mg/ l), low Cd stress (0.5 mg/l) and high Cd stress (2 mg/l) (Liu et al., 2007a, 2007b). These seedlings were cultivated in a greenhouse for 40 d after which the seedlings were washed with tap water and kept intact for analyses. Seedlings were then divided into three parts for ROL analysis, the collection of low-molecular-weight organic acids (LMWOAs) and chlorophyll concentration detection. 2.3. Sample analysis 2.3.1. Radial oxygen loss (ROL) detection in A. marina roots At harvest, part of the seedlings was used to test ROL. ROL detection was performed referring to Liu et al. (2009). The absorbance was measured at 527 nm by an ultraviolet (UV)-visible spectrophotometer in the deoxygenated box filled with N2 gas after incubation for 24 h. The roots, leaves and stems were separated, washed with tap water, dried and weighed. ROL was calculated by the following formula:
ROLðumol O2 =d=plantÞ ¼ cðy zÞ=4 where, c ¼ initial volume of Titanium (III) citrate (l); y ¼ concentration of Ti3þ blank (without plants) (mmol/l);
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z ¼ concentration of Ti3þ after incubation for 24 h (mmol/l).
2.3.2. Low-molecular-weight organic acid (LMWOA) determination in A. marina root exudates and Fe plaque extraction from A. marina root surfaces Part of the seedlings was used for LMWOA detection. LMWOAs were collected according to Lu et al. (2007) and analysed by high performance liquid chromatography as described by Cawthray (2003). The seedling roots after LMWOA collection were used to extract Fe plaque based on the DCB technique of Li et al. (2016a). Residual roots and all of the leaves and stems were separated, washed with tap water and then freeze-dried. The dried roots, stems and leaves were finely ground in an agate mortar for analysis. 2.3.3. Measurement of chlorophyll concentration in A. marina leaves Fresh 0.5 g samples of A. marina leaves were homogenised in 95% alcohol and then filtered in the dark until a final 25 ml extract was obtained. The absorbance of the supernatant was recorded at 645 and 663 nm by a UVevisible spectrophotometer. The concentration of chlorophyll a (chl a) and chlorophyll b (chl b) (mg/l) in the supernatant was calculated using the following equations (Li, 2000):
Fig. 1. The biomass of root, stem and leaf in A. marina under Cd and Fe stress. In the same Fe treatment, values with different lowercase letters indicate significant differences (P < 0.05) among Cd concentrations based on one-way ANOVAs (n ¼ 3).
increasing Cd concentration (n ¼ 12, P < 0.01) and also showed a significant negative correlation (n ¼ 36, P < 0.01).
Chlorophyll a (mg/g FW) ¼ (12.7A663 2.69A645) V/(1000 FW). Chlorophyll b (mg/g FW) ¼ (22.9A645 4.68A663) V/(1000 FW).
3.2. Accumulation of Fe and Cd in A. marina tissues
Dry plant samples (about 0.5 g) were digested as described by Soto-Jimenez and Paez-Osuna (2001). 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 analytical procedure. 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 deionised water prior to use. Fe and Cd concentrations in the samples were detected by flame atomic absorption spectrometry (AAS, Model AA-6800, Shimadzu, Kyoto) with a 99%e 103% recovery for Fe and 98%e101% for Cd.
In A. marina roots, Cd accumulation increased with Cd stress and an exogenous Fe application (n ¼ 9, P < 0.05), and there was a highly positive correlation between them (n ¼ 36, P < 0.01) (Fig. 2).
2.4. Statistical analysis Statistical analyses were performed using SPSS version 19.0 (Armonk, NY: IBM Corp.). One-way and two-way analysis of variance were applied to examine significant effects of Fe and Cd concentration (independent variables). All results are reported as mean values and standard deviation from triplicate samples, and differences were tested by the Ducan's Multiple Range Test and least significant difference. A p value < 0.05 was considered statistically significant. 3. Results 3.1. Joint effect of Fe and Cd on A. marina tissue growth A. marina root and leaf biomass increased with an Fe application (Fig. 1; n ¼ 9, P < 0.05). And a remarkable positive correlation was found to exist between biomass and Fe concentrations (n ¼ 36, P < 0.01). The addition of Fe had no significant effect on stem growth (n ¼ 9, P > 0.05). Under low Cd stress (0.5 mg/l Cd), root and leaf biomass showed no considerable differences compared with the control (n ¼ 12, P > 0.05), but biomass decreased dramatically (n ¼ 12, P < 0.05) when the seedlings were exposed to a high concentration of Cd (2 mg/l Cd). Stem biomass decreased with
Fig. 2. The tissue distributions of Fe and Cd in A. marina under Cd and Fe stress.
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This was different from roots where Cd accumulation decreased with an Fe application to stems and leaves (Fig. 2; n ¼ 9, P < 0.05), and there was a significant negative correlation with an Fe treatment (n ¼ 36, P < 0.01). Most of Cd were accumulated in the roots with the highest concentration of 17.44 ± 1.04 mg/kg$DW. The percentage of Cd accumulation in A. marina tissues was reduced in the order root > stem > leaf. Regardless of Cd stress, Fe concentration intensified significantly with an increasing Fe application in A. marina roots (Fig. 2; n ¼ 9, P < 0.05). The highest Fe concentration was found in the roots under high-dose Fe supply, it's 11,970.39 ± 1637.90 mg/kg$DW. Although Cd exposure stimulated more Fe accumulation in the roots (n ¼ 12, P < 0.05), no remarkable differences were found between Cd stress and Fe accumulation in the roots (n ¼ 12, P > 0.05). In A. marina stems, Fe absorption increased with an increasing exogenous Fe concentration (Fig. 2; n ¼ 9, P < 0.05), and Fe concentration had a highly positive correlation with exogenous Fe in plants (n ¼ 36, P < 0.01). Cd stress resulted in Fe transport hindrance in the stems (n ¼ 12, P < 0.05). Fe absorption increased significantly with increasing exogenous Fe, regardless of Cd exposure in the leaves (n ¼ 9, P < 0.05). Fe concentration revealed a remarkable positive correlation with Fe treatment in the leaves (n ¼ 36, P < 0.01). The percentage of Fe accumulation in the tissues decreased in the order root > stem > leaf (Fig. 2).
synthesis (P > 0.05), and there were no correlations between them (Fig. 3; n ¼ 36, P > 0.05).
3.4. Joint effect of Fe and Cd on ROL from entire roots of A. marina The amount of ROL was reduced by the addition of Cd (Fig. 4; n ¼ 12, P < 0.05). The highest and lowest release were 25.3274 ± 2.70 mmol O2/d/plant and 6.7178 ± 1.26 mmol O2/d/plant under 0 mg/l and 2 mg/l Cd stress, respectively. It showed a remarkable negative correlation with a Cd treatment (n ¼ 36, P < 0.01). Despite of increase under low-dose Fe treatment (n ¼ 9, P < 0.05), ROL was impeded by mid-dose and high-dose Fe. ROL was the lowest with the high-dose Fe (n ¼ 9, P < 0.05).
3.3. Combined effect of Fe and Cd on chlorophyll synthesis in A. marina leaves At a low dose of exogenous Fe (0.6 mmol/l Fe) and a mid-dose of exogenous Fe (1.2 mmol/l Fe), the chl a concentration was remarkably higher than that of the control and high-dose Fe (1.8 mmol/l Fe) (Fig. 3; n ¼ 9, P < 0.05). High-dose Fe (1.8 mmol/l Fe) was adverse to chl a formation. The synthesis of chl a was inhibited by Cd stress (n ¼ 12, P < 0.05). A significant negative correlation was found between chl a concentration and Cd treatment (n ¼ 36, P < 0.01). Neither the Fe application nor Cd stress had any considerable effect on chl b
Fig. 4. ROL from the entire roots in A. marina under Cd and Fe stress. In the same Fe treatment, values with different lowercase letters indicate significant differences (P < 0.05) among Cd concentrations based on one-way ANOVAs (n ¼ 3).
Fig. 3. Chlorophyll concentrations in A. marina leaves under Cd and Fe stress.
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3.5. Joint effect of Fe and Cd on Fe precipitation and Cd immobilisation in Fe plaque from A. marina root surfaces In the present study, the Fe application resulted in more Fe oxidation and precipitation on A. marina roots, and DCB-Fe concentrations rose with an increasing Fe application in DCB extracts (Fig. 5; n ¼ 9, P < 0.05). They also showed a remarkable positive correlation (n ¼ 36, P < 0.01). However, Cd stress impeded Fe precipitation on the roots (n ¼ 12, P < 0.05), and a negative correlation was observed between DCB-Fe concentration and Cd exposure (n ¼ 36, P < 0.05). DCB-Cd accumulation in Fe plaque was highly positively correlated with an Fe application (n ¼ 36, P < 0.05) (Fig. 5). Interestingly, under low level Cd stress (0.5 mg/l Cd), DCB-Cd concentration increased with an Fe application (n ¼ 3, P < 0.05), while Cd precipitation was hampered by Fe when seedlings were exposed to a high level of Cd (2 mg/l Cd). DCB-Cd under low level Cd stress showed the highest concentration when Fe was added (n ¼ 3, P < 0.05). Whereas no correlation existed between DCB-Cd concentration and Cd stress concentration (n ¼ 36, P > 0.05). 3.6. Joint effect of Fe and Cd on LMWOAs in A. marina root exudates There was no correlation between exogenous Fe concentration and oxalic acid concentration in root exudates (Fig. 6; n ¼ 36, P > 0.05). Oxalic acid exudation was stimulated by Cd exposure (n ¼ 12, P < 0.05) and showed a positive correlation with Cd stress (n ¼ 36, P < 0.01). Tartaric, fumaric, succinic and citric acid concentrations increased with an Fe application (Fig. 6; n ¼ 9, P < 0.05), while the concentrations were dramatically reduced under the high-dose Fe treatment (n ¼ 9, P < 0.05). Compared with the control treatment, low level Cd stress did not induce a considerable increase in tartaric acid (n ¼ 12, P > 0.05), but a high Cd exposure stimulated more tartaric acid secretion in the roots (n ¼ 12, P < 0.05). By contrast with the tartaric acid results, concentrations of fumaric, succinic and citric acid were remarkably increased under low level Cd stress (n ¼ 12, P < 0.05). 3.7. Relationship between exogenous Fe supply and Cd tolerance in A. marina There was a significant positive correlation between low-dose and mid-dose exogenous Fe supply and Cd tolerance of A. marina. In terms of Cd concentration, the positive correlation coefficients were as follows: R2 ¼ 0.8793 (P < 0.0001) under 0 mg/l of Cd stress (included high-dose Fe supply), R2 ¼ 0.9681 (P < 0.0001) under
Fig. 6. LMWOA concentrations in the root exudate of A. marina under Cd and Fe stress. In the same Fe treatment, values with different lowercase letters indicate significant differences (P < 0.05) among Cd concentrations based on one-way ANOVAs (n ¼ 3).
0.5 mg/l of Cd stress and R2 ¼ 0.921(P < 0.0001) under 2.0 mg/l of Cd stress (Fig. 7).
4. Discussion Heavy metals inhibit plant growth, especially Cd with its high activity and bioavailability (Li et al., 2016a). In the present study, Cd exposure impeded the growth of roots, stems and leaves in A. marina. While the results suggest that an Fe application significantly alleviates root and leaf growth inhibited by Cd in A. marina, there was no correlation between stem biomass and Fe application (Fig. 1). Because Fe competes with heavy metals for sensitive metabolic sites within leaves, and an Fe application has a remarkable effect on Fe concentration in leaves (n ¼ 36, P < 0.01), Cd stress had no significant effect on Fe concentration in leaves (n ¼ 36, P > 0.05). An exogenous Fe treatment promotes an increase in DCB-
Fig. 5. Fe and Cd concentrations in Fe plaque on the root surface of A. marina under Cd and Fe stress. In the same Fe treatment, values with different lowercase letters indicate significant differences (P < 0.05) among Cd concentrations based on one-way ANOVAs (n ¼ 3).
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Fig. 7. Correlation analysis between low and middle dose Fe supplies and Cd tolerance of A. marina under Cd and Fe stress [(total biomass) with metals/(total biomass) without metals]. A significant positive correlation between exogenous Fe concentration and A. marina tolerance was discovered. Correlation under 0 mg/l of Cd stress is R2 ¼ 0.8793, P < 0.0001 (included high-dose Fe supply); under 0.5 mg/l of Cd stress is R2 ¼ 0.9681, P < 0.0001; under 2.0 mg/l of Cd stress is R2 ¼ 0.921, P < 0.0001.
Fe, Cd precipitation and accumulation on the root surfaces (Liu et al., 2007b). This hampers Cd uptake and transport to aboveground tissue and decreases Cd accumulation in leaves and stems of A. marina (Fig. 2), thus alleviating Cd toxicity. It's indicated that Cd competes with divalent cation (such as Fe2þ), inhibits protochlorophyllide reductase and affects aminolaevulinic acid synthesis in plants (Stobart et al., 1985). This results in failure of the chlorophyll synthesis and a reduction of chlorophyll concentration in mangrove plants (Chen et al., 2014; Wang et al., 2014). Chen et al. (2014) suggested that Cd hampered all kinds of chlorophyll formation. While our data reveals that Cd stress inhibits chl a synthesis, it did not significantly affect chl b synthesis (Fig. 3). This might be different depending on the mangrove plant species. As an essential activator of multiple enzymes in the chlorophyll synthesis process, Fe competes with heavy metals for sensitive metabolic sites within leaves (Kuo, 1986; Liu et al., 2007a). Exogenous Fe supply has competitive effects on Cd bioavailability and induces an increase in chl a concentration. However, excess Fe would cause a decrease in chl a concentration, which is unfavourable to plants (Fig. 3). Therefore, low-dose and mid-dose exogenous Fe alleviates Cd toxicity and restores chl a synthesis. ROL is a critical process from the root to the rhizosphere that directly utilises oxidation or indirectly aerobe oxidation for plant detoxification (Revsbech et al., 1999), and this enhances plant tolerance to flooding stress (Colmer, 2003). While it was reported that poisonous As compound hampered ROL of Qryza sativa L. (Wu et al., 2013) and excess Cu ion inhibited ROL of Bruguiera gymnorrhiza and Rhizophora stylosa (Cheng et al., 2012). Except for the toxic action of Cd in roots, Cd exposure impeded leaf growth and chlorophyll synthesis (Figs. 1 and 3). ROL is positively correlated with the photosynthetic rate, leaf biomass and aboveground biomass (Lai et al., 2012), which is an important reason for ROL decrease in A. marina (Fig. 4). Because of ROL, a tiny aerobic environment formed that impelled Fe oxidation and increased Fe plaque formation. Whereas, excess Fe plaque would prevent oxygen release from the
roots. Therefore, compared with other Fe treatments, more oxygen was released under a low Fe treatment (n ¼ 9, P < 0.05, Fig. 4). The formation of Fe hydroxide reacts with other metals and phytotoxic metal is then immobilised. Thus, Fe plaque formation is an adaptive behaviour of plants to a stressful environment (Kuo, 1986; Pi et al., 2010; Wu et al., 2013). Because of increasing Fe source, Fe concentrations increased significantly with Fe treatments, regardless of Cd stress in DCB extracts (Fig. 5). This is consistent with the report on O. sativa (Liu et al., 2007b). ROL is a crucial factor for Fe plaque formation. Cd stress significantly inhibited oxygen release from roots, indirectly decreased immobilisation of Fe and then DCB-Fe in Fe plaque decreased in the roots (Fig. 5). Under low-level Cd stress, Fe plaque was the pool of Cd absorption and precipitation (Fig. 5). Interestingly, an Fe application impeded Cd accumulation in Fe plaque under high-level Cd exposure and Cd was excluded from entering the plants (Fig. 5). It could be considered that root absorption plays a more important role than physicochemical absorption and precipitation of Fe plaque when high level Cd is added (Liu et al., 2007b). Cd toxicity to plants is a result of the high reactivity between Cd and sulfhydryl groups (Polle and Schuetzenduebel, 2004). This stimulates the production of enzymes and consumption of antioxidative systems, leading to oxidative stress. Hence, Cd toxicity affects root functions and induces different exudations from roots (Lu et al., 2007). It was reported that citric and malic acid were secreted from the roots as a detoxification mechanism in Zea mays and Triticum aestivum when plants were stressed under high Al levels (Jones and Kochian, 1996). K. obovata secrets more malic acid than citric acid under high Cd stress (30 and 50 mg/kg) (Lu et al., 2007). In this study, Cd induced a larger amount of oxalic, tartaric, fumaric, succinic and citric acid secretion in A. marina root exudates, indicating that Cd complexation by these LMWOAs acts as the same detoxifying mechanism for Cd (Fig. 6) (Lu et al., 2007). In addition, a similar range in types of LMWOAs were identified in other plants (Chen et al., 2001; Lu et al., 2007; Xie et al., 2013),
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including oxalic, formic, fumaric, malic, succinic, citric, maleic, tartaric, acetic and lactic acid. As dicarboxylic acid, the secretion of oxalic, tartaric, fumaric and succinic acid are promoted in response to Cd exposure. This is an adaptive behaviour of plants to adverse conditions, especially to Cd (Lu et al., 2007). Meanwhile, tricarboxylic citric acid responds similarly to dicarboxylic acid under Cd stress, participating in the detoxification of Cd (Fig. 6). As secondary metabolites, LMWOAs are secreted from plant roots to the rhizosphere. In previous researches, abundant oxalic and succinic acid are found to exude from roots to rhizosphere soils in temperate rain forests (Chen et al., 2001). A low- and mid-dose Fe supply promoted LMWOA exudation in roots, except for oxalic acid (Fig. 6). Although oxalic acid exudation showed no correlation with Fe supply, Cd exposure stimulated the secretion of oxalic acid. Oxalic acid concentration accounted for 73%e92% of all five kinds of LMWOAs (Fig. 6). Similar to the data from Lu et al. (2007), oxalic acid exudation is positively correlated with Cd stress. It plays a critical role in detoxification in plants. Root exudates are photosynthetic products that are formed in leaves, transported to roots and secreted into the rhizosphere. For the range of exogenous Fe used in this experiment, low- and mid-dose Fe treatments promoted chlorophyll concentration increase (Fig. 3). Tartaric, fumaric, succinic and citric acid exudates were considerably changed under an Fe treatment (Fig. 6), and were correlated with chlorophyll concentration (P < 0.05). Hence, moderate exogenous Fe provides a more powerful competitiveness with Cd, promotes more chlorophyll formation and then more LMWOAs are produced and released from the roots to the rhizosphere. In addition, Cd can be chelated/ complexed by LMWOAs. This also alleviates the stress from an adverse environment in A. marina roots (Jones and Kochian, 1996). A significant positive correlation was found between moderate exogenous Fe supply and Cd tolerance of A. marina (Fig. 7). This further demonstrates that an exogenous Fe supply alleviates Cd phytotoxicity in A. marina. 5. Conclusion The mitigation effect of Fe on Cd stress was reflected in three ways in the mangrove A. marina. Firstly, moderate exogenous Fe directly contributed to more Fe oxidation and precipitation on the root surface, and more Cd immobilisation was then discovered in Fe plaque. Secondly, exogenous Fe supply facilitated A. marina growth and increased chlorophyll concentration in leaves. Then, more chlorophyll induced an increase in photosynthesis and further caused an increase in ROL. This increase in ROL resulted in more Fe oxidation and precipitation, more Fe plaque formation and more Cd immobilisation on the root surface. Cd immobilisation on the root surface significantly restrained Cd absorption, transportation and accumulation in A. marina. Exogenous Fe increased Fe accumulation in A. marina tissues, improved Fe competition with Cd and further alleviated Cd phytotoxicity in A. marina. Finally, an exogenous Fe supply increased chlorophyll concentration in the leaves, and more LMWOAs were produced and released into the rhizosphere. Meanwhile, Cd can be chelated/complexed by LMWOAs. It also played a crucial role in Cd detoxification in A. marina. In conclusion, a moderate Fe supply accelerates growth and enhances Cd tolerance in A. marina. Improving Fe bioavailability may be a optional approach to protect mangroves from heavy metal contamination. Acknowledgements This work was jointly funded by Major Program of National Natural Science Foundation of China (31530008), National Natural Science Foundation of China (31570414, 31800429), Natural Science
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The effects of sulfur amendments on the geochemistry of sulfur, phosphorus and iron in the mangrove plant (Kandelia obovata (S. L.)) rhizosphere. Mar. Pollut. Bull. 114, 733e741. Li, J., Du, Z., Zou, C.B., Dai, Z., Du, D., Yan, C., 2018. The mutual restraint effect between the expansion of Alternanthera philoxeroides (Mart.) Griseb and cadmium mobility in aquatic environment[J]. Ecotoxicol. Environ. Saf. 148 (C), 237e243. Liu, H.J., Zhang, J.L., Christie, P., Zhang, F.S., 2007a. Influence of external zinc and phosphorus supply on Cd uptake by rice (Oryza sativa L.) seedlings with root surface iron plaque. Plant Soil 300, 105e115. Liu, H.J., Zhang, J.L., Zhang, F.S., 2007b. Role of iron plaque in Cd uptake by and translocation within rice (Oryza sativa L.) seedlings grown in solution culture. Environ. Exp. Bot. 59, 314e320. Liu, Y., Tam, N., Yang, J., Pi, N., Wong, M., Ye, Z., 2009. Mixed heavy metals tolerance and radial oxygen loss in mangrove seedlings. Mar. Pollut. Bull. 58, 1843e1849. Lin, Y.S., Fan, J., Yu, J.F., Jiang, S., Yan, C.L., Liu, J.C., 2018. Root activities and arsenic translocation of Avicennia marina (Forsk.) Vierh seedlings influenced by sulfur and iron amendments[J]. Mar. Pollut. Bull. 135, 1174e1182. Lu, H., Yan, C., Liu, J., 2007. Low-molecular-weight organic acids exuded by mangrove (Kandelia candel (L.) Druce) roots and their effect on cadmium species change in the rhizosphere. Environ. Exp. Bot. 61, 159e166. MacFarlane, G.R., Pulkownik, A., Burchett, M.D., 2003. Accumulation and distribution of heavy metals in the grey mangrove, Avicennia marina (Forsk.)Vierh.: biological indication potential. Environ. Pollut. 123, 139e151. Nowrouzi, M., Pourkhabbaz, A., Rezaei, M., 2012. Bioaccumulation and distribution of metals in sediments and Avicenna marina tissues in the Hara Biosphere Reserve, Iran. Bull. Environ. Contam. Toxicol. 89, 799e804. Pi, N., Tam, N., Wong, M., 2011. Formation of iron plaque on mangrove roots receiving wastewater and its role in immobilization of wastewater-borne pollutants. Mar. Pollut. Bull. 63, 402e411. Pi, N., Tam, N.F.Y., Wong, M.H., 2010. Effects of wastewater discharge on formation of Fe plaque on root surface and radial oxygen loss of mangrove roots. Environ. Pollut. 158, 381e387. Polle, A., Schuetzenduebel, A., 2004. Heavy metal signalling in plants: linking cellular and organismic responses. In: Hirt, H., Shinozaki, K. (Eds.), Plant
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Norton, G.J., Nautiyal, C.S., 2014. Roles for root iron plaque in sequestration and uptake of heavy metals and metalloids in aquatic and wetland plants. Metallomics 6, 1789e1800. Wang, Y., Zhu, H., Tam, N.F.Y., 2014. Effect of a polybrominated diphenyl ether congener (BDE-47) on growth and antioxidative enzymes of two mangrove plant species, Kandelia obovata and Avicennia marina , in South China. Mar. Pollut. Bull. 85, 376e384. Welch, R.M., Hart, J.J., Norvell, W.A., Sullivan, L.A., Kochian, L.V., 1999. Effects of nutrient solution zinc activity on net uptake, translocation, and root export of cadmium and zinc by separated sections of intact durum wheat (Triticum turgidum L-var durum) seedling roots. Plant Soil 208, 243e250. Wu, C., Li, H., Ye, Z., Wu, F., Wong, M.H., 2013. Effects of as levels on radial oxygen loss and as speciation in rice. Environ. Sci. Pollut. Res. Int. 20, 8334e8341. Wu, H., Ding, Z., Liu, Y., Liu, J., Yan, H., Pan, J., Li, L., Lin, H., Lin, G., Lu, H., 2011. Methylmercury and sulfate-reducing bacteria in mangrove sediments from Jiulong River Estuary, China. J. Environ. Sci. 23, 14e21. Xie, X., Weiss, D.J., Weng, B., Liu, J., Lu, H., Yan, C., 2013. The short-term effect of cadmium on low molecular weight organic acid and amino acid exudation from mangrove (Kandelia obovata (S., L.) Yong) roots. Environ. Sci. Pollut. Res. Int. 20, 997e1008. Yan, Z.Z., Tam, N.F.Y., 2011. Temporal changes of polyphenols and enzyme activities in seedlings of Kandelia obovata under lead and manganese stresses. Mar. Pollut. Bull. 63, 438e444.
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The alleviation effect of iron on cadmium phytotoxicity in mangrove A. marina. Alleviation effect of iron on cadmium phytotoxicity in mangrove Avicennia marina (Forsk.) Vierh Li Jian a, b, Liu Jingchun a, *, Yan Chongling a, Du Daolin b, Lu Haoliang a a b
Key Laboratory of Ministry of Education for Coastal and Wetland Ecosystems, Xiamen University, Xiamen, 361102, China Institute of Environment and Ecology, School of the Environment and Safety Engineering, Jiangsu University, Zhenjiang, 212013, China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Fe promoted Fe plaque to immobilise more Cd and decrease Cd absorption. Fe improved competition between Fe and Cd, and facilitated a decrease in Cd toxicity. Fe caused LMWOA secretion increase and played a crucial role in Cd detoxification. Significant positive correlation existed between Fe concentration and Cd tolerance. Fe application accelerated the growth and enhanced Cd tolerance of A. marina.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 31 October 2018 Received in revised form 8 March 2019 Accepted 26 March 2019 Available online 27 March 2019
Cd has high activity and bioavailability and is a poisonous element to plants. As a critical ecosysterm, mangroves are subjected to serious Cd pollution. In this research, the hypothesis was presented that improving Fe bioavailability would alleviate Cd phytotoxicity to Avicennia marina (Forsk.) Vierh. To test this, we examined the effect of four exogenous Fe and three Cd concentrations on A. marina. The results showed that a significant positive correlation excited between moderate exogenous Fe concentration and Cd tolerance of A. marina. Moderate exogenous Fe concentration directly or indirectly promoted the formation of Fe plaque, which immobilised more Cd on the root surface and decreased Cd absorption in roots. Furthermore, an exogenous Fe application increased plant biomass and Fe accumulation in A. marina tissues. This improved the competition between Fe and Cd within the plants. Therefore, an Fe application facilitated a decrease in Cd toxicity within A. marina. Simultaneously, a moderate Fe concentration caused an increase in low-molecular-weight organic acid (LMWOA) secretion from the roots. Meanwhile, Cd can be chelated/complexed by LMWOAs. It also played a crucial role in Cd detoxification in A. marina. In conclusion, Fe application accelerated the growth and enhanced Cd tolerance of A. marina. Therefore, improving Fe bioavailability will protect mangroves from Cd contamination. © 2019 Elsevier Ltd. All rights reserved.
Handling Editor: T. Cutright Keywords: Mangrove Cadmium Iron plaque Radial oxygen loss Low molecular weight organic acid
* Corresponding author. E-mail address: [email protected] (L. Jingchun). https://doi.org/10.1016/j.chemosphere.2019.03.172 0045-6535/© 2019 Elsevier Ltd. All rights reserved.
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1. Introduction Mangroves are critical zones that protect the intertidal belt of tropical and subtropical regions. As an ecological habitat, they provide an abundance of plants and animals that play a crucial role in maintaining coastal ecological balance. However, various pollutants drain from human habitation towards the mangroves, some of which are prone to accumulate in mangrove sediments, such as organic contaminants, organochlorine compounds, polycyclic aromatic hydrocarbons (Bhattacharya et al., 2003) as well as heavy metals Cd, Cu, Pb and Cr etc. (MacFarlane et al., 2003; Silva et al., 2006; Wu et al., 2011; Bayen, 2012; Li et al., 2016a, 2016b). Unlike organic contaminants, heavy metals cannot be degraded by microorganisms and they are instead absorbed and accumulated by plants and benthic macrofauna (Saha et al., 2006). They are then transferred into food chains/webs where they finally endanger human health. Thus, heavy metals present more lasting harm than organic contaminants. Mangroves are subjecting to serious heavy metal pollution and Cd concentration of 87 mg/g$dw was detected in mangrove sediments (Bayen, 2012). As a critical ecosysterm with an abundant biodiversity, mangroves supply plentiful food source for animals including human being, and they play a crucial role in fisheries. Hence, heavy metal pollution in mangrove is closely related to the health of coastal ecosystems. The white mangrove Avicennia marina is a pioneer mangrove species in southeast China, and it has been reported to have a high accumulation of heavy metals (Li et al., 2015, 2016a; Siswanti et al., 2016). It accumulates Cd, Cu, Zn and Pb in root tissues at levels equal to or above the surrounding sediment under field conditions (MacFarlane et al., 2003; Nowrouzi et al., 2012; Li et al., 2015). Because Cd has a high activity and bioavailability, A. marina could absorb Cd2þ through apoplastic and symplasm transport readily (Li et al., 2015). In symplastic translocation, there are specific or generic ionophores or channel proteins for Cd absorption in the roots. Once absorbed into the roots, Cd competes with a divalent cation, such as Ca2þ or Mg2þ/Fe2þ, for ionophores or channel proteins (Welch et al., 1999). In plants, Cd competes with the divalent cation, participates in plant metabolic process and then interferes with plant development, finally resulting in poor growth and lower biomass (Li et al., 2015). The geochemical cycling of Fe plays an important role in form transformation and bioavailability of elements (Li et al., 2017). As an essential activator of multiple enzymes in the process of chlorophyll synthesis and as the main component of Fe plaque, Fe is an essential element for plant growth and facilitates tolerance to contaminants, especially in wetland plants (Li et al., 2018). Photosynthesis by chlorophylls produces oxygen within the leaves. Some of the oxygen is transported into gas-filled spaces and the aerenchyma of the cortex by axial diffusion, then into nonporous tissues and the rhizosphere by radial diffusion (Sorrell, 2003; Lin et al., 2018). This is known as radial oxygen loss (ROL). ROL forms micro-aerobic surroundings, accelerates Fe oxidation precipitation and further develops Fe plaque on the root surface. It is considered to be one of the predominant factors affecting metal tolerance (Liu et al., 2009). Fe plaque, found on the root surface of wetland plants, can reduce or promote the accumulation of metal pollutants (Pi et al., 2010). As a coating on the root surface, Fe plaque functions as a physical ‘barrier’ co-precipitating and immobilising heavy metals (Tripathi et al., 2014), thus the bioavailability and absorption of heavy metals is reduced in plant roots (Pi et al., 2011). In addition, Fe competes with heavy metals for sensitive metabolic sites (Kuo, 1986) and has a positive effect on heavy metal detoxification within plants. For these reasons, Fe is a critical element for heavy metal tolerance in plants. However, there are few reports to provide the evidence of Fe element alleviating Cd phytotoxicity.
In fact, bioavailable Fe is restrictive on mangrove plant growth (Alongi, 2010). Thus, the hypothesis was presented that improving Fe bioavailability would alleviate Cd phytotoxicity to Avicennia marina (Forsk.) Vierh. In this study, different concentrations of exogenous Fe were used to evaluate the mechanisms of Fe influence on Cd tolerance in mangrove plants as follows: (1) plant physiological response in vivo, (2) physicochemical impact on the environment of root surface and (3) plant defence behaviour. 2. Materials and methods 2.1. Plant materials Mature A. marina propagules were collected from the Jiulong River Estuary Mangrove Natural Reserve (24 240 N, 117 550 E), Zhangzhou city, Fujian province, China, in September 2015. Complete propagules with high vitality were chosen for pre-cultivation in sea sands (Liu et al., 2009). Propagules of comparable size (2.5 ± 0.5 cm in diameter) were inserted into polyethylene seedling pots with a modified Hoagland's 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$7H2O, 0.22; MnSO$H2O, 1.55; (NH4)6Mo7O24$4H2O, 0.61; FeSO4, 5.57; EDTA$2Na, 7.45; Ca(NO3)2, 1180.76 (Li et al., 2015). The nutrient solution was renewed every 3 d and the propagules were cultured for about 2 months until they had 3e4 euphyllas. These seedlings were placed in a greenhouse with a daily temperature of 20 C-29.5 C, relative humidity of 59%e80% and light intensity of 800e1400 mmol photons/m2/s (Yan and Tam, 2011). 2.2. Experimental design and sample culture The whole experimental process included repeat five times culture and each culture contained Fe plaque induction and Cd stress. The first stage was Fe plaque induction for 2 d. Four concentration gradients in triplicate were used for Fe plaque induction using FeSO4$7H2O (pH ¼ 6.0): no exogenous Fe (0.02 mmol/l Fe in nutrient solution), low-dose Fe (0.6 mmol/l Fe), mid-dose Fe (1.2 mmol/l Fe) and high-dose Fe (1.8 mmol/l Fe). The second stage was Cd stress for 6 d. Three concentration gradients in triplicate were used for Cd stress using CdCl2 (pH ¼ 6.0): no Cd stress (0 mg/ l), low Cd stress (0.5 mg/l) and high Cd stress (2 mg/l) (Liu et al., 2007a, 2007b). These seedlings were cultivated in a greenhouse for 40 d after which the seedlings were washed with tap water and kept intact for analyses. Seedlings were then divided into three parts for ROL analysis, the collection of low-molecular-weight organic acids (LMWOAs) and chlorophyll concentration detection. 2.3. Sample analysis 2.3.1. Radial oxygen loss (ROL) detection in A. marina roots At harvest, part of the seedlings was used to test ROL. ROL detection was performed referring to Liu et al. (2009). The absorbance was measured at 527 nm by an ultraviolet (UV)-visible spectrophotometer in the deoxygenated box filled with N2 gas after incubation for 24 h. The roots, leaves and stems were separated, washed with tap water, dried and weighed. ROL was calculated by the following formula:
ROLðumol O2 =d=plantÞ ¼ cðy zÞ=4 where, c ¼ initial volume of Titanium (III) citrate (l); y ¼ concentration of Ti3þ blank (without plants) (mmol/l);
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z ¼ concentration of Ti3þ after incubation for 24 h (mmol/l).
2.3.2. Low-molecular-weight organic acid (LMWOA) determination in A. marina root exudates and Fe plaque extraction from A. marina root surfaces Part of the seedlings was used for LMWOA detection. LMWOAs were collected according to Lu et al. (2007) and analysed by high performance liquid chromatography as described by Cawthray (2003). The seedling roots after LMWOA collection were used to extract Fe plaque based on the DCB technique of Li et al. (2016a). Residual roots and all of the leaves and stems were separated, washed with tap water and then freeze-dried. The dried roots, stems and leaves were finely ground in an agate mortar for analysis. 2.3.3. Measurement of chlorophyll concentration in A. marina leaves Fresh 0.5 g samples of A. marina leaves were homogenised in 95% alcohol and then filtered in the dark until a final 25 ml extract was obtained. The absorbance of the supernatant was recorded at 645 and 663 nm by a UVevisible spectrophotometer. The concentration of chlorophyll a (chl a) and chlorophyll b (chl b) (mg/l) in the supernatant was calculated using the following equations (Li, 2000):
Fig. 1. The biomass of root, stem and leaf in A. marina under Cd and Fe stress. In the same Fe treatment, values with different lowercase letters indicate significant differences (P < 0.05) among Cd concentrations based on one-way ANOVAs (n ¼ 3).
increasing Cd concentration (n ¼ 12, P < 0.01) and also showed a significant negative correlation (n ¼ 36, P < 0.01).
Chlorophyll a (mg/g FW) ¼ (12.7A663 2.69A645) V/(1000 FW). Chlorophyll b (mg/g FW) ¼ (22.9A645 4.68A663) V/(1000 FW).
3.2. Accumulation of Fe and Cd in A. marina tissues
Dry plant samples (about 0.5 g) were digested as described by Soto-Jimenez and Paez-Osuna (2001). 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 analytical procedure. 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 deionised water prior to use. Fe and Cd concentrations in the samples were detected by flame atomic absorption spectrometry (AAS, Model AA-6800, Shimadzu, Kyoto) with a 99%e 103% recovery for Fe and 98%e101% for Cd.
In A. marina roots, Cd accumulation increased with Cd stress and an exogenous Fe application (n ¼ 9, P < 0.05), and there was a highly positive correlation between them (n ¼ 36, P < 0.01) (Fig. 2).
2.4. Statistical analysis Statistical analyses were performed using SPSS version 19.0 (Armonk, NY: IBM Corp.). One-way and two-way analysis of variance were applied to examine significant effects of Fe and Cd concentration (independent variables). All results are reported as mean values and standard deviation from triplicate samples, and differences were tested by the Ducan's Multiple Range Test and least significant difference. A p value < 0.05 was considered statistically significant. 3. Results 3.1. Joint effect of Fe and Cd on A. marina tissue growth A. marina root and leaf biomass increased with an Fe application (Fig. 1; n ¼ 9, P < 0.05). And a remarkable positive correlation was found to exist between biomass and Fe concentrations (n ¼ 36, P < 0.01). The addition of Fe had no significant effect on stem growth (n ¼ 9, P > 0.05). Under low Cd stress (0.5 mg/l Cd), root and leaf biomass showed no considerable differences compared with the control (n ¼ 12, P > 0.05), but biomass decreased dramatically (n ¼ 12, P < 0.05) when the seedlings were exposed to a high concentration of Cd (2 mg/l Cd). Stem biomass decreased with
Fig. 2. The tissue distributions of Fe and Cd in A. marina under Cd and Fe stress.
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This was different from roots where Cd accumulation decreased with an Fe application to stems and leaves (Fig. 2; n ¼ 9, P < 0.05), and there was a significant negative correlation with an Fe treatment (n ¼ 36, P < 0.01). Most of Cd were accumulated in the roots with the highest concentration of 17.44 ± 1.04 mg/kg$DW. The percentage of Cd accumulation in A. marina tissues was reduced in the order root > stem > leaf. Regardless of Cd stress, Fe concentration intensified significantly with an increasing Fe application in A. marina roots (Fig. 2; n ¼ 9, P < 0.05). The highest Fe concentration was found in the roots under high-dose Fe supply, it's 11,970.39 ± 1637.90 mg/kg$DW. Although Cd exposure stimulated more Fe accumulation in the roots (n ¼ 12, P < 0.05), no remarkable differences were found between Cd stress and Fe accumulation in the roots (n ¼ 12, P > 0.05). In A. marina stems, Fe absorption increased with an increasing exogenous Fe concentration (Fig. 2; n ¼ 9, P < 0.05), and Fe concentration had a highly positive correlation with exogenous Fe in plants (n ¼ 36, P < 0.01). Cd stress resulted in Fe transport hindrance in the stems (n ¼ 12, P < 0.05). Fe absorption increased significantly with increasing exogenous Fe, regardless of Cd exposure in the leaves (n ¼ 9, P < 0.05). Fe concentration revealed a remarkable positive correlation with Fe treatment in the leaves (n ¼ 36, P < 0.01). The percentage of Fe accumulation in the tissues decreased in the order root > stem > leaf (Fig. 2).
synthesis (P > 0.05), and there were no correlations between them (Fig. 3; n ¼ 36, P > 0.05).
3.4. Joint effect of Fe and Cd on ROL from entire roots of A. marina The amount of ROL was reduced by the addition of Cd (Fig. 4; n ¼ 12, P < 0.05). The highest and lowest release were 25.3274 ± 2.70 mmol O2/d/plant and 6.7178 ± 1.26 mmol O2/d/plant under 0 mg/l and 2 mg/l Cd stress, respectively. It showed a remarkable negative correlation with a Cd treatment (n ¼ 36, P < 0.01). Despite of increase under low-dose Fe treatment (n ¼ 9, P < 0.05), ROL was impeded by mid-dose and high-dose Fe. ROL was the lowest with the high-dose Fe (n ¼ 9, P < 0.05).
3.3. Combined effect of Fe and Cd on chlorophyll synthesis in A. marina leaves At a low dose of exogenous Fe (0.6 mmol/l Fe) and a mid-dose of exogenous Fe (1.2 mmol/l Fe), the chl a concentration was remarkably higher than that of the control and high-dose Fe (1.8 mmol/l Fe) (Fig. 3; n ¼ 9, P < 0.05). High-dose Fe (1.8 mmol/l Fe) was adverse to chl a formation. The synthesis of chl a was inhibited by Cd stress (n ¼ 12, P < 0.05). A significant negative correlation was found between chl a concentration and Cd treatment (n ¼ 36, P < 0.01). Neither the Fe application nor Cd stress had any considerable effect on chl b
Fig. 4. ROL from the entire roots in A. marina under Cd and Fe stress. In the same Fe treatment, values with different lowercase letters indicate significant differences (P < 0.05) among Cd concentrations based on one-way ANOVAs (n ¼ 3).
Fig. 3. Chlorophyll concentrations in A. marina leaves under Cd and Fe stress.
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3.5. Joint effect of Fe and Cd on Fe precipitation and Cd immobilisation in Fe plaque from A. marina root surfaces In the present study, the Fe application resulted in more Fe oxidation and precipitation on A. marina roots, and DCB-Fe concentrations rose with an increasing Fe application in DCB extracts (Fig. 5; n ¼ 9, P < 0.05). They also showed a remarkable positive correlation (n ¼ 36, P < 0.01). However, Cd stress impeded Fe precipitation on the roots (n ¼ 12, P < 0.05), and a negative correlation was observed between DCB-Fe concentration and Cd exposure (n ¼ 36, P < 0.05). DCB-Cd accumulation in Fe plaque was highly positively correlated with an Fe application (n ¼ 36, P < 0.05) (Fig. 5). Interestingly, under low level Cd stress (0.5 mg/l Cd), DCB-Cd concentration increased with an Fe application (n ¼ 3, P < 0.05), while Cd precipitation was hampered by Fe when seedlings were exposed to a high level of Cd (2 mg/l Cd). DCB-Cd under low level Cd stress showed the highest concentration when Fe was added (n ¼ 3, P < 0.05). Whereas no correlation existed between DCB-Cd concentration and Cd stress concentration (n ¼ 36, P > 0.05). 3.6. Joint effect of Fe and Cd on LMWOAs in A. marina root exudates There was no correlation between exogenous Fe concentration and oxalic acid concentration in root exudates (Fig. 6; n ¼ 36, P > 0.05). Oxalic acid exudation was stimulated by Cd exposure (n ¼ 12, P < 0.05) and showed a positive correlation with Cd stress (n ¼ 36, P < 0.01). Tartaric, fumaric, succinic and citric acid concentrations increased with an Fe application (Fig. 6; n ¼ 9, P < 0.05), while the concentrations were dramatically reduced under the high-dose Fe treatment (n ¼ 9, P < 0.05). Compared with the control treatment, low level Cd stress did not induce a considerable increase in tartaric acid (n ¼ 12, P > 0.05), but a high Cd exposure stimulated more tartaric acid secretion in the roots (n ¼ 12, P < 0.05). By contrast with the tartaric acid results, concentrations of fumaric, succinic and citric acid were remarkably increased under low level Cd stress (n ¼ 12, P < 0.05). 3.7. Relationship between exogenous Fe supply and Cd tolerance in A. marina There was a significant positive correlation between low-dose and mid-dose exogenous Fe supply and Cd tolerance of A. marina. In terms of Cd concentration, the positive correlation coefficients were as follows: R2 ¼ 0.8793 (P < 0.0001) under 0 mg/l of Cd stress (included high-dose Fe supply), R2 ¼ 0.9681 (P < 0.0001) under
Fig. 6. LMWOA concentrations in the root exudate of A. marina under Cd and Fe stress. In the same Fe treatment, values with different lowercase letters indicate significant differences (P < 0.05) among Cd concentrations based on one-way ANOVAs (n ¼ 3).
0.5 mg/l of Cd stress and R2 ¼ 0.921(P < 0.0001) under 2.0 mg/l of Cd stress (Fig. 7).
4. Discussion Heavy metals inhibit plant growth, especially Cd with its high activity and bioavailability (Li et al., 2016a). In the present study, Cd exposure impeded the growth of roots, stems and leaves in A. marina. While the results suggest that an Fe application significantly alleviates root and leaf growth inhibited by Cd in A. marina, there was no correlation between stem biomass and Fe application (Fig. 1). Because Fe competes with heavy metals for sensitive metabolic sites within leaves, and an Fe application has a remarkable effect on Fe concentration in leaves (n ¼ 36, P < 0.01), Cd stress had no significant effect on Fe concentration in leaves (n ¼ 36, P > 0.05). An exogenous Fe treatment promotes an increase in DCB-
Fig. 5. Fe and Cd concentrations in Fe plaque on the root surface of A. marina under Cd and Fe stress. In the same Fe treatment, values with different lowercase letters indicate significant differences (P < 0.05) among Cd concentrations based on one-way ANOVAs (n ¼ 3).
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Fig. 7. Correlation analysis between low and middle dose Fe supplies and Cd tolerance of A. marina under Cd and Fe stress [(total biomass) with metals/(total biomass) without metals]. A significant positive correlation between exogenous Fe concentration and A. marina tolerance was discovered. Correlation under 0 mg/l of Cd stress is R2 ¼ 0.8793, P < 0.0001 (included high-dose Fe supply); under 0.5 mg/l of Cd stress is R2 ¼ 0.9681, P < 0.0001; under 2.0 mg/l of Cd stress is R2 ¼ 0.921, P < 0.0001.
Fe, Cd precipitation and accumulation on the root surfaces (Liu et al., 2007b). This hampers Cd uptake and transport to aboveground tissue and decreases Cd accumulation in leaves and stems of A. marina (Fig. 2), thus alleviating Cd toxicity. It's indicated that Cd competes with divalent cation (such as Fe2þ), inhibits protochlorophyllide reductase and affects aminolaevulinic acid synthesis in plants (Stobart et al., 1985). This results in failure of the chlorophyll synthesis and a reduction of chlorophyll concentration in mangrove plants (Chen et al., 2014; Wang et al., 2014). Chen et al. (2014) suggested that Cd hampered all kinds of chlorophyll formation. While our data reveals that Cd stress inhibits chl a synthesis, it did not significantly affect chl b synthesis (Fig. 3). This might be different depending on the mangrove plant species. As an essential activator of multiple enzymes in the chlorophyll synthesis process, Fe competes with heavy metals for sensitive metabolic sites within leaves (Kuo, 1986; Liu et al., 2007a). Exogenous Fe supply has competitive effects on Cd bioavailability and induces an increase in chl a concentration. However, excess Fe would cause a decrease in chl a concentration, which is unfavourable to plants (Fig. 3). Therefore, low-dose and mid-dose exogenous Fe alleviates Cd toxicity and restores chl a synthesis. ROL is a critical process from the root to the rhizosphere that directly utilises oxidation or indirectly aerobe oxidation for plant detoxification (Revsbech et al., 1999), and this enhances plant tolerance to flooding stress (Colmer, 2003). While it was reported that poisonous As compound hampered ROL of Qryza sativa L. (Wu et al., 2013) and excess Cu ion inhibited ROL of Bruguiera gymnorrhiza and Rhizophora stylosa (Cheng et al., 2012). Except for the toxic action of Cd in roots, Cd exposure impeded leaf growth and chlorophyll synthesis (Figs. 1 and 3). ROL is positively correlated with the photosynthetic rate, leaf biomass and aboveground biomass (Lai et al., 2012), which is an important reason for ROL decrease in A. marina (Fig. 4). Because of ROL, a tiny aerobic environment formed that impelled Fe oxidation and increased Fe plaque formation. Whereas, excess Fe plaque would prevent oxygen release from the
roots. Therefore, compared with other Fe treatments, more oxygen was released under a low Fe treatment (n ¼ 9, P < 0.05, Fig. 4). The formation of Fe hydroxide reacts with other metals and phytotoxic metal is then immobilised. Thus, Fe plaque formation is an adaptive behaviour of plants to a stressful environment (Kuo, 1986; Pi et al., 2010; Wu et al., 2013). Because of increasing Fe source, Fe concentrations increased significantly with Fe treatments, regardless of Cd stress in DCB extracts (Fig. 5). This is consistent with the report on O. sativa (Liu et al., 2007b). ROL is a crucial factor for Fe plaque formation. Cd stress significantly inhibited oxygen release from roots, indirectly decreased immobilisation of Fe and then DCB-Fe in Fe plaque decreased in the roots (Fig. 5). Under low-level Cd stress, Fe plaque was the pool of Cd absorption and precipitation (Fig. 5). Interestingly, an Fe application impeded Cd accumulation in Fe plaque under high-level Cd exposure and Cd was excluded from entering the plants (Fig. 5). It could be considered that root absorption plays a more important role than physicochemical absorption and precipitation of Fe plaque when high level Cd is added (Liu et al., 2007b). Cd toxicity to plants is a result of the high reactivity between Cd and sulfhydryl groups (Polle and Schuetzenduebel, 2004). This stimulates the production of enzymes and consumption of antioxidative systems, leading to oxidative stress. Hence, Cd toxicity affects root functions and induces different exudations from roots (Lu et al., 2007). It was reported that citric and malic acid were secreted from the roots as a detoxification mechanism in Zea mays and Triticum aestivum when plants were stressed under high Al levels (Jones and Kochian, 1996). K. obovata secrets more malic acid than citric acid under high Cd stress (30 and 50 mg/kg) (Lu et al., 2007). In this study, Cd induced a larger amount of oxalic, tartaric, fumaric, succinic and citric acid secretion in A. marina root exudates, indicating that Cd complexation by these LMWOAs acts as the same detoxifying mechanism for Cd (Fig. 6) (Lu et al., 2007). In addition, a similar range in types of LMWOAs were identified in other plants (Chen et al., 2001; Lu et al., 2007; Xie et al., 2013),
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including oxalic, formic, fumaric, malic, succinic, citric, maleic, tartaric, acetic and lactic acid. As dicarboxylic acid, the secretion of oxalic, tartaric, fumaric and succinic acid are promoted in response to Cd exposure. This is an adaptive behaviour of plants to adverse conditions, especially to Cd (Lu et al., 2007). Meanwhile, tricarboxylic citric acid responds similarly to dicarboxylic acid under Cd stress, participating in the detoxification of Cd (Fig. 6). As secondary metabolites, LMWOAs are secreted from plant roots to the rhizosphere. In previous researches, abundant oxalic and succinic acid are found to exude from roots to rhizosphere soils in temperate rain forests (Chen et al., 2001). A low- and mid-dose Fe supply promoted LMWOA exudation in roots, except for oxalic acid (Fig. 6). Although oxalic acid exudation showed no correlation with Fe supply, Cd exposure stimulated the secretion of oxalic acid. Oxalic acid concentration accounted for 73%e92% of all five kinds of LMWOAs (Fig. 6). Similar to the data from Lu et al. (2007), oxalic acid exudation is positively correlated with Cd stress. It plays a critical role in detoxification in plants. Root exudates are photosynthetic products that are formed in leaves, transported to roots and secreted into the rhizosphere. For the range of exogenous Fe used in this experiment, low- and mid-dose Fe treatments promoted chlorophyll concentration increase (Fig. 3). Tartaric, fumaric, succinic and citric acid exudates were considerably changed under an Fe treatment (Fig. 6), and were correlated with chlorophyll concentration (P < 0.05). Hence, moderate exogenous Fe provides a more powerful competitiveness with Cd, promotes more chlorophyll formation and then more LMWOAs are produced and released from the roots to the rhizosphere. In addition, Cd can be chelated/ complexed by LMWOAs. This also alleviates the stress from an adverse environment in A. marina roots (Jones and Kochian, 1996). A significant positive correlation was found between moderate exogenous Fe supply and Cd tolerance of A. marina (Fig. 7). This further demonstrates that an exogenous Fe supply alleviates Cd phytotoxicity in A. marina. 5. Conclusion The mitigation effect of Fe on Cd stress was reflected in three ways in the mangrove A. marina. Firstly, moderate exogenous Fe directly contributed to more Fe oxidation and precipitation on the root surface, and more Cd immobilisation was then discovered in Fe plaque. Secondly, exogenous Fe supply facilitated A. marina growth and increased chlorophyll concentration in leaves. Then, more chlorophyll induced an increase in photosynthesis and further caused an increase in ROL. This increase in ROL resulted in more Fe oxidation and precipitation, more Fe plaque formation and more Cd immobilisation on the root surface. Cd immobilisation on the root surface significantly restrained Cd absorption, transportation and accumulation in A. marina. Exogenous Fe increased Fe accumulation in A. marina tissues, improved Fe competition with Cd and further alleviated Cd phytotoxicity in A. marina. Finally, an exogenous Fe supply increased chlorophyll concentration in the leaves, and more LMWOAs were produced and released into the rhizosphere. Meanwhile, Cd can be chelated/complexed by LMWOAs. It also played a crucial role in Cd detoxification in A. marina. In conclusion, a moderate Fe supply accelerates growth and enhances Cd tolerance in A. marina. Improving Fe bioavailability may be a optional approach to protect mangroves from heavy metal contamination. Acknowledgements This work was jointly funded by Major Program of National Natural Science Foundation of China (31530008), National Natural Science Foundation of China (31570414, 31800429), Natural Science
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