Phenolic metabolism and related heavy metal tolerance mechanism in Kandelia Obovata under Cd and Zn stress

Ecotoxicology and Environmental Safety 169 (2019) 134–143

Contents lists available at ScienceDirect

Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv

Phenolic metabolism and related heavy metal tolerance mechanism in Kandelia Obovata under Cd and Zn stress

T

Shan Chena, Qiang Wanga, Haoliang Lua, Junwei Lia, Dan Yanga, Jingchun Liua, ⁎ Chongling Yana,b, a b

Key Laboratory of Ministry of Education for Coastal and Wetland Ecosystems, Xiamen University, Xiamen 361102, PR China State Key Laboratory of Marine Environmental Science, Xiamen University, Xiamen 361102, PR China

A R T I C LE I N FO

A B S T R A C T

Keywords: Chlorophyll content Kandelia obovata Metabolism Phenolic

In the present study, a set of pot culture experiments was conducted to reveal how the metabolism process of phenolic compounds was affected by cadmium (Cd) and zinc (Zn) and to further uncover heavy metal tolerance mechanisms in Kandelia obovata. After 60d of treatment, the biomass and chlorophyll a content in the leaves were suppressed, but total phenolic compounds in roots and leaves were improved by the increasing gradient of Cd or Zn concentrations; Total phenolic compounds significantly increased by 3.6–44.6% in the roots, and by 0.4–126.6% in the leaves. At the meantime, the activity of Shikimate dehydrogenase (SKDH), cinnamyl alcohol dehydrogenase (CAD), and polyphenol oxidase (PPO) in the roots increased by 11.2–307.6%, 12.4–175.4% and − 2.7–392.8%, and the results were 3.4–69.5%, 1.7–40.0%, 16.0–99.7% in the leaves. Higher toxicity of Cd than Zn, as well as slight alleviating effect of 100 mg kg−1 Zn on 2.5 mg kg−1 Cd were found. Additionally, a significantly positive correlation coefficients for relationship between phenolic metabolism related enzyme activity and Cd/Zn contamination levels was found, and leaf SKDH, leaf CAD, and leaf PPO activities were moderately correlated with leaf Cd (r = 0.39, r = 0.43, and r = 0.57, respectively) and leaf Zn (r = 0.44, r = 0.41, r = 0.19, respectively) content, which indicate that Cd and Zn play a previously unrecognized but major role in phenolic compounds synthesis, transport, and metabolism in K. obovata. The results also provided evidence that the application of high levels of Cd and Zn was accompanied by three phenolic metabolism pathways participating in heavy metal tolerance process.

1. Introduction Mangrove ecosystems are located in a transitional zone between terrestrial and aquatic ecosystems, and mangrove forests are fragile ecological sensitive zones distributed throughout tropical and subtropical regions (Chen et al., 2010; Huang and Wang, 2010). In recent years, due to the increasing worldwide population, as well as the development of urban industrial and agricultural production (M.B. Ali, 2006; I. Ali, 2006; Ali et al., 2011; Basheer, 2018), environmental pollution by heavy metals has drawn acute attention in mangrove wetlands (Das et al., 2016; Gupta and Ali, 2013; Zhu et al., 2018). These forests have a high capacity to retain pollutants and therefore become sinks for pollutants from river or marine flows (Ali et al., 2014; Dai et al., 2018), thus increasing pressure on the mangrove environment. According to the original study, K. obovata can accumulative Cd and Zn in roots, leaves and stems (Cheng et al., 2010; Du et al., 2014). MacFarlane found K.obovata transferred most metals exhibiting a

⁎

continuous decline from roots to leaf tissues, while translocation of Zn and Cu to aboveground parts was high (G.R.MacFarlane, 2000). Cadmium (Cd) is one of the most toxic heavy metals with high mobility (Alharbi et al., 2018; Ali et al., 2018; Javed et al., 2017). The toxicity of Cd results in inhibition of plant growth and root elongation, photosynthesis decline, loss of water balance, nutrient reduction, lipid peroxidation, protein degradation, and second metabolite production by reducing chlorophyll production and enzyme activity (Jia et al., 2016; Jiang et al., 2017). It has been reported that Cd can substitute Fe in several proteins, thus increasing free Fe iron, indirectly resulting in more hydroxyl radicals production through the Fenton reaction, which finally causes plant tissues broken. A recent study showed that phenolic compounds as a protective mechanism against stress caused by Cd in blueberry plantlets (Manquian-Cerda et al., 2018). Zinc (Zn) is an essential element in plants as well as being an important heavy metal pollution element in mangrove systems (Ni et al., 2005). Zinc plays a fundamental role in the stabilization and protection of the biological

Corresponding author at: Key Laboratory of Ministry of Education for Coastal and Wetland Ecosystems, Xiamen University, Xiamen 361102, PR China. E-mail address: [email protected] (C. Yan).

https://doi.org/10.1016/j.ecoenv.2018.11.004 Received 19 June 2018; Received in revised form 30 October 2018; Accepted 1 November 2018 0147-6513/ © 2018 Elsevier Inc. All rights reserved.

Ecotoxicology and Environmental Safety 169 (2019) 134–143

S. Chen et al.

culture medium. The culture experiment was carried out in a greenhouse, located at Xiamen University, South East of China, without artificial control of light and temperature (60–80% relative humidity, 25 ± 5 °C temperature and 12 h light/dark at 800–1400 µmol photons m−2 s−1) (Dai et al., 2017) lasting 60 d. Seedlings with similar heights, quantity and two pairs of leaves were selected for the following experiments.

membranes against oxidative and peroxidative injuries (Ali et al., 2012; Zeng et al., 2011). Conversely, high levels of Zn can result in limited germination of plants and reduced root development, which induces plant senescence (Li et al., 2015); however, no one has reported that the relationship between this phenomenon and the phenolic metabolism process. Therefore, in this context, we will discuss how phenolic metabolism throws light on the mechanisms reducing Cd/Zn toxicity. Plants can synthesize more phenolic acids and flavonoids under heavy metal stress (Jia et al., 2016; Zhang et al., 2013). Phenolic compounds play various roles in defending plants from adverse environmental conditions, including cell wall structural changes, channeling and storing carbon compounds, and establishing flower color and contributing to certain flavors. Plant phenolic compounds can remove the harmful reactive oxygen species (ROS) and chelate heavy metals by hydroxyl (-OH) and carboxylic acid (-COOH) (Ali et al., 2017; Ali and Jain, 2004; Sytar et al., 2013). Plant polyphenols have many heterogeneous groups,e.g., coumarins, lignins, flavonoids, phenolic acids, and tannins (Krol et al., 2015; Melida et al., 2011). Some are unique but some are common among plants, and are often present in relatively high concentrations. The electron-donating, deprotonation equilibrium and radicals scavenging activity of phenolic compounds are determined by their chemical structure, type, position, and number of functional groups (Manquian-Cerda et al., 2018). Cd and Zn not only affect plant growth, but also change the metabolism of phenolic compounds (Manquian-Cerda et al., 2016). Phenolic metabolism in plants is a complex process related to at least five different interaction pathways (Fourcroy et al., 2016). The glycolytic pathway can produce phosphoenolpyruvate, the pentose phosphate pathway participates in erythrose-4-phosphate production, the shikimate pathway can synthesize phenylalanine, general phenylpropanoid metabolism can generate activated cinnamic acid derivatives and lignin, as well as various specific flavonoid pathways (Austin and Noel, 2003; Catherine A. Rice-Evans, 1996; Hrazdina, 1993; Winkel-Shirley, 2001). Phenolic compounds metabolism-related enzymes are closely linked to phenolic compounds accumulation and activity. Shikimate dehydrogenase (SKDH) participates in providing a substrate for PAL (phenylalanine ammonia-lyase); cinnamyl alcohol dehydrogenase (CAD) provides precursors for the biosynthesis of lignin, and polyphenol oxidases (PPOs) supplies polymerized phenols of a melanin-like nature (Dauwe et al., 2007; Kováčik et al., 2009). Polyphenol oxidases (PPOs) act as quenchers of photooxidation in chloroplasts. Furthermore, owing to the vacuolar location of PPO substrates, PPOs potentially synthesize specialized metabolites and respond to environmental factors (Anne Vissers et al., 2017; Araji et al., 2014; Pourcel et al., 2010; Ruuhola et al., 2018). The metabolism and accumulation of phenolic compounds in plants can be changed under biotic and abiotic stress (Krol et al., 2015). Revealing the phenolic metabolism under Cd and Zn stress would provide insight into the toxic detoxification mechanisms of mangrove plants in the contaminated environments. The objectives of the present study are to evaluate (1) how phenolic metabolism of K. obovata are affected by Cd and Zn stress; (2) to found possible detoxification role of phenolic compounds exposed to Cd and Zn stress.

2.2. Cd and Zn treatments of seedlings in soil culture After sea sand culture for two months, a soil experiment with twelve different treatments was performed to explore the interaction between Cd and Zn on the K. obovata. The surface sediment（0–20 cm）used in the experiments was collected from the same sampling site. The soil type was sediment and the soil texture was silt (75.39 ± 7.62% dry weight [DW]) and clay (23.06 ± 1.91% DW), with pH (soil water ratio, m/v = 1:5) of 6.63 ± 0.04, salinity of 19.2 ± 1.20%, and content of TOC 0.76–1.49%, SOC of 4.48~6.48%, Zn of 241.21–301.76 mg kg−1, and Cd of 0.237 mg kg−1. The collected sediment was treated with different amounts of Zn and Cd by the addition of ZnSO4·7H2O at levels of 0, 100, and 500 mg kg−1 DW and CdCl2·2.5H2O at levels of 0, 2.5, 5, and 20 mg kg−1 DW, sediment without applied Zn and Cd was used as the control, indicated by Cd0Zn0, Cd2.5Zn0, Cd5Zn0, Cd20Zn0, Cd0Zn100, Cd2.5Zn100, Cd5Zn100, Cd20Zn100, Cd0Zn500, Cd2.5Zn500, Cd5Zn500, Cd20Zn500, respectively. Before the experiments, the treated sediments were thoroughly mixed every day for one month to retain homogenized and distilled water was added to maintain freshness. The chosen seedlings were transplanted into 3.0 L plastic pots contained about 3 kg of the treated sediment. The pots were arranged in the same experimental greenhouse. After 60 days of treatments, plants samples were collected for further analyses. 2.3. Sampling After 60 days of soil culture, the K. obovata was carefully transferred from the soil and then washed with tap water until all sediment on the root surface was removed. All plants were separated into roots, leaves and stems; and then placed in clean envelopes. A portion of the samples was freeze-dried for the determination of total phenolic compounds. A portion of the samples were collected and treated with liquid nitrogen to detect enzyme activities. A portion of the samples were collected to measure the chlorophyll content. The remainder portion of fresh roots, leaves, and stems were heated at 110 °C for 15 min, then dried at 70 °C overnight. Later, dry samples were ground using an agate mortar for analysis of Cd and Zn concentration and biomass. 2.4. Biochemical analysis 2.4.1. Detection of total biomass Dry weight of roots, stems and leaves of each K. obovata were measured by electronic balance, and total biomass was summated by dry weight of roots, stems and leaves.

2. Materials and methods 2.4.2. Detection of Cd and Zn concentration The concentrations of Cd and Zn in roots, leaves and stems of the plant were analyzed by ICP-MS（Agilent 7500 ICP-MS, USA ）after a modified (Mongkhonsin et al., 2016) nitric-perchloric (HNO3-HClO4) acid digestion method. The blank reagent and the material GBW 07603 (GSV-2) were used as the reference standards to ensure the accuracy of the digestion process. The recovery rate of Cd and Zn was 98–102% and 99–102%, respectively.

2.1. Plant materials and culture conditions Undamaged propagules of K. obovata were collected from the Natural Mangrove Reserve of Zhang Jiang Estuary (117°24′–117°30′E, 23°53′–23°56′N) in Yun Xiao County, Fujian Province, China. Selection criteria for the hypocotyls included growing well, had a similar size, and had strong vitality. The propagules were disinfected with 10% KMnO4 for 1.5 h and then rinsed with distilled water before being planted in the pot. Four hypocotyls were planted in each pot. The propagules were irrigated by Hoagland solution with 10% NaCl every 3 days. Sea sands washed thoroughly with tap water were used as the

2.4.3. Detection of phenolic compounds Total phenolic compounds content in different parts of K. obovata was determined by the Folin-Ciocalteu reagent method (Singleton and 135

Ecotoxicology and Environmental Safety 169 (2019) 134–143

S. Chen et al.

Rossi, 1965). Samples of 2 g freeze-dried roots, stems, and leaves were ground into powder under liquid nitrogen, and 25 mL of 20% ethanol was added. The samples were then placed in an 80 °C water bath to undergo extraction for 30 min, after which the leaching solution was filtered and the residue washed with 20% ethanol. The filtrate volume was 100 mL as the test solution. Gallic acid was made into a 0.61 mg mL−1 standard solution with 20% methanol. Quantities of 0.2, 0.4, 0.6, 0.8, and 1.0 mL of standard solution in 10 mL were used to prepare the gallic acid working solutions. Each 0.4 mL gallic acid standard solution and test solution were transferred to 10 mL centrifuge tubes. Then, 1 mL of 0.2 mol L−1 FolinCiocalteu solution and 2 mL of 15% Na2CO3 solution were added to the centrifuge tubes, the volume was made up to 8 mL with distilled water, and thoroughly mixed. After reaction at 25 °C for 2 h, the absorbance of the reaction solution was measured at 765 nm. The addition of 0.4 mL of 20% ethanol instead of the sample was used as the blank controls. Total phenolic compounds values were calculated by the regression equation of the standard gallic acid solution.

Table 1 Two-way ANOVA analysis of Biomass, Cd, Zn, chlorophylls, enzyme activity and phenolic contents in the tissues of K. obovata under Cd and Zn stress. Main effects

Biomass Cd Content

Zn Content

Phenolic compounds

Chlorophylls

2.4.4. Detection of chlorophyll a, chlorophyll b and total chlorophyll content Mature leaves with no midrib were cut into small pieces, then weighed 0.2 g of leaf sample. Small amounts of calcium carbonate and quartz were added into a mortar with the leaf, then homogenizing in 95% ethanol until the tissue became white. The homogenate was filtered through a funnel with filter paper, and the filtrate diluted with distilled water up to 25 mL in a volumetric flask. The absorbance of the filtrate was measured spectrophotometrically at 645 nm and 663 nm. Chlorophyll content was calculated by the equation of Sae-Lee et al. (2012).

SKDH CAD PPO

Root Stem Leaf Total Root Stem Leaf Total Root Stem Leaf Total phenolic content a b a/b a+b Root Leaf Root Leaf Root Leaf

Cd application

Zn application

Interaction Cd×Zn

0.000 0.000 0.000 0.000 0.000 0.757 0.000 0.000 0.000 0.000 0.169 0.000 0.000

0.000 0.000 0.726 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.008 0.000 0.000

0.001 0.000 0.002 0.000 0.016 0.000 0.000 0.006 0.000 0.000 0.786 0.000 0.001

0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000

0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000

0.000 0.001 0.032 0.000 0.000 0.000 0.000 0.001 0.002 0.001

Note: No significance (P > 0.05). Significant differences (p < 0.05) based on ANOVAs. All analysis results were calculated by a two-way ANOVA.

2.4.5. Detection of enzyme activities Activities of phenolic compounds metabolism-related enzymes, including SKDH, CAD, and PPO were detected using the method described by Kováčik et al. (2009). Briefly, approximately 0.5 g of roots, leaves, and stems were homogenized in pH 7.0 and 50 mM potassium phosphate buffer at 4 °C, respectively. The homogenates were then centrifuged at 15,000 ×g for 15 min at 4 °C. SKDH activity was detected in 0.1 M Tris-HCl buffer (pH 9). The reaction mixture consisted of 1.45 mL of 2 mM shikimic acid, 1.45 mL of 0.5 mM NADP, and 0.1 mL of supernatant. SKDH activity was calculated using molar absorptivity with absorbance at 340 nm. CAD activity was measured in 0.1 M TrisHCl buffer (pH 8.8). The assay mixture contained 1.45 mL of 1 mM appropriate coniferyl alcohol, 1.45 mL of 1 mM NADP and 0.1 mL of enzyme extract. Measurements and calculations were performed as for SKDH. PPO activity was measured in a solution consisting of 2.85 mL of 50 mM potassium phosphate buffer (pH 7.0), 50 µL of 60 mM catechol, and 0.1 mL of supernatant. The absorbance was detected at 420 nm.

Fig. 1. Total biomass of K. obovata plants after treatment with Cd and Zn for 60 d. The different letters (a-g) are significant differences according to Duncan's new multiple range test (p < 0.05). Values with the same letters are equivalent; different letters mean significant differences (p < 0.05) based on one-way ANOVA. The data are given as the mean ± SD (n = 12).

2.5. Statistical analysis All statistical analyses were performed by SPSS 25.0 software. Oneway ANOVA was used to evaluate differences of biomass, phenolic acids content, chlorophyll, Cd/Zn content and enzymes. Two-way ANOVA was used to evaluate main effects of Cd/Zn. Pearson Correlation analysis was used for detecting relationships between them. All results were related to calculating mean values, standard deviation (Hamada et al., 2015), the Duncan and least significant difference (LSD) at p = 0.05. All figures were plotted using Excel 2016 software.

Cd and Zn (Table 1). As shown in Fig. 1, in the case of Cd only treatments, the biomass significantly decreased with the increases of Cd supply. Compared to the control (22.537 g·plant−1), the biomass in treatment of 2.5 mg kg−1 Cd, 5 mg kg−1 Cd and 20 mg kg−1 Cd significantly decreased by 12.8%, 27.6% and 29.8%, respectively. There was no difference in biomass between 100 mg kg−1 Zn only treatment (22.497 g·plant−1) and the control, but the biomass in 500 mg kg−1 Zn only treatment was 21.314 g·plant−1, which was lower than the control. In the case of combined treatments, the biomass in the 2.5 mg kg−1 Cd with 100 mg kg−1 Zn was 22.524 g·plant−1, the value was similar with the control and significantly higher by 4.4% than 2.5 mg kg−1 Cd only treatment, and by 13.6% than 2.5 mg kg−1 Cd with 500 mg kg−1 Zn combined treatment. The addition of 100 mg kg−1 Zn stimulated an increase in plant biomass in the 2.5 mg kg−1 Cd treatment, whereas no

3. Results 3.1. Effects of Cd and/or Zn on plant total biomass The total biomass of K. obovata significantly changed in the different Cd and Zn treatment, and there were significant interactions between 136

Ecotoxicology and Environmental Safety 169 (2019) 134–143

S. Chen et al.

Fig. 2. Cd and Zn concentrations in K. obovata tissues exposed to different Cd and Zn treatments. The different letters (a-k) in root, total Cd content, total Zn content, letters (A-F) in stem and letters (a´-f´) in leaf are significant differences according to Duncan's new multiple range test (p < 0.05). Values with the same letters are equivalent; different letters mean significant differences (p < 0.05) based on one-way ANOVA. The data are given as the mean ± SD (n = 12).

positive effects were shown in the 5 and 20 mg kg−1 Cd treatments. Higher toxicity of Cd over Zn as well as antagonistic effects of Zn to Cd were found.

simulated the uptake of Cd. The highest Cd content (0.778 mg kg−1) was observed in seedlings exposed to combined treatments of 20 mg kg−1 Cd with 500 mg kg−1 Zn, which was significantly higher by 22.7% and 22.3% than that of the 20 mg kg−1 Cd only and 20 mg kg−1 Cd with 100 mg kg−1 Zn treatment, respectively. While, the application of Cd inhibited the uptake of Zn in 100 mg kg−1 Zn treatment, and the results were reversed in 500 mg kg−1 Zn treatment (Fig. 2B). Similar to Cd content, the highest Zn content(198.6 mg kg−1) was also recorded in combined treatments of 20 mg kg−1 Cd with 500 mg kg−1 Zn, which was 2.51 times and 2.77 times as higher as that in the 0 mg kg−1 Zn and 100 mg kg−1 Zn with 20 mg kg−1 Cd treatment, respectively. As shown in Fig. 2C and D, in different parts of the plant, the

3.2. Cd and Zn contents in roots, stems, leaves and total plant The contents of Cd and Zn accumulated in the roots, leaves and total plant were significantly affected by Cd and Zn treatments (P < 0.05), and there were significant interactions between Cd and Zn (Table 1 and Fig. 2). As shown in Fig. 2A, the Cd content in the whole plant increased with Cd levels under different Zn treatments, and the applied Zn 137

Ecotoxicology and Environmental Safety 169 (2019) 134–143

S. Chen et al.

difference was found in stems among Cd treatments (Table 1 and Fig. 3). As shown in Fig. 3D, after the addition of 2.5, 5, or 20 mg kg−1 Cd only, total phenolic compounds content in K.obovata seedlings increased with Cd levels by 7.6–11.0% under different Zn treatments, and the content increased with Zn levels by 7.5–9.1% under different Cd treatments. After Cd/Zn treatment, phenolic compounds content in the roots of the plant ranged from 8.18 mg kg−1 to 11.51 mg kg−1, and by 3.6–44.6% higher than the control. The highest phenolic compounds content exhibited at 20 mg kg−1 Cd with 500 mg kg−1 Zn combined treatment, which was significantly higher than that of the 20 mg kg−1 Cd or 500 mg kg−1 Zn only treatments (Fig. 3A). In the stems, phenolic compounds content slightly varied from 11.94 mg kg−1 to 12.43 mg kg−1, and averaged at 12.19 mg g−1 (Fig. 3B). In the leaves, phenolic compounds contents ranged from 8.38 to 18.92 mg g−1, which was by 0.4–126.6% higher than the control, and totally increased with Cd levels, higher phenolic compounds content was obviously observed in all the 500 mg kg−1 Zn treatments, whereas there was no difference between 0 or 2.5 mg kg−1 Cd treatment combined with 100 mg kg−1 Zn and the control (Fig. 3C).

averaged Cd contents over all the treatments showed as root (1.351 mg kg−1) > leaf (0.309 mg kg−1) > stem (0.259 mg kg−1), and Zn contents performed as root (215.9 mg kg−1) > stem (111.3 mg kg−1) > leaf (87.7 mg kg−1). The contents of Cd accumulated in the roots and leaves increased with Cd levels under different Zn treatments, but Cd contents in stem did not change obviously among Zn treatments (Table 1). In roots, either at 5 or 20 mg kg−1 Cd levels, the 100 mg kg−1 Zn treatment elevated Cd uptake by 9.8% and 24.1% than that in the Cd only treatments (Fig. 2C), but the highest Cd content in root was 2.370 mg kg−1, which was measured in the 20 mg kg−1 Cd with 500 mg kg−1 Zn combined treatment. The value was 1.65 times and 1.33 times as higher as that of 0 mg kg−1 Zn and 100 mg kg−1 Zn with 20 mg kg−1 Cd treatment, and 2.66 times as that of control. Similar change trends in Cd content were found in leaves, with the contents ranged from 0.107 mg kg−1 to 0.768 mg kg−1. However, the effects of Cd treatments on Zn content were different from that of Zn on Cd. Zn content in root mainly depended on Zn levels, and applied Cd did not significantly affect Zn content (Table 1); Zn content in leaves decreased with increasing Cd supply, and similar results were found in stems at 0 mg kg−1 and 100 mg kg−1 Zn levels, but the result was converse at 500 mg kg−1 Zn levels (Fig. 2D). The Zn content of root in the combined treatment of 20 mg kg−1 Cd with 100 mg kg−1 Zn was 153.7 mg kg−1, there was no different from those of no Zn treatments, but it was significantly lower than the other treatments. The highest Zn content both in roots and stems were detected in the 20 mg kg−1 Cd with 500 mg kg−1 Zn combined treatment, with the value of 342.3 mg kg−1 and 169.5 mg kg−1, respectively, but the highest value in leaves (116.9 mg kg−1) was tested in the 5 mg kg−1 Cd with 500 mg kg−1 Zn combined treatment, 20 mg kg−1 Cd limited Zn uptake in leaves at 500 mg kg−1 Zn (Fig. 2D).

3.4. Chlorophyll content The results of chlorophyll a, chlorophyll b, chlorophyll a/b and total chlorophyll were significantly different between Cd or Zn treatments ( P < 0.05), and there were significant interactions between Cd or Zn (Table 1). Cd or Zn only treatments exhibited significant decreases in chlorophyll a content in K. obovata leaves (Fig. 4A), but no distinct difference was found in chlorophyll b content (Fig. 4B). However, at the same concentration of Zn treatment, chlorophyll a content experienced a slight increase first and then a slow fall with increasing concentrations of Cd and peaked in the 2.5 mg kg−1 Cd with 100 mg kg−1 Zn combined treatment (Fig. 4A). Similar results were observed in chlorophyll b content (Fig. 4B). This means that low dosage of Cd enhanced the synthesis of chlorophyll a and chlorophyll b in the plant, whereas

3.3. Content of phenolic compounds in different parts of the plant Phenolic compounds content in roots, leaves and total of the plant showed substantial difference between Cd or Zn treatments, and there was significant interaction between Cd or Zn, but no significant

Fig. 3. Total phenolic acids content in leaves, stems and roots of K. obovata exposed to different Cd and Zn treatments. The different letters (a-h) are significant differences according to Duncan's new multiple range test (p < 0.05). Values with the same letters are equivalent; different letters mean significant differences (p < 0.05) based on one-way ANOVA. The data are given as the mean ± SD (n = 12).

138

Ecotoxicology and Environmental Safety 169 (2019) 134–143

S. Chen et al.

Fig. 4. Effect of different Cd and Zn treatments on chlorophyll content in leaves of K. obovata. The different letters (a-f) are significant differences according to Duncan's new multiple range test (p < 0.05). Values with the same letters are equivalent; different letters mean significant differences (p < 0.05) based on one-way ANOVA. The data are given as the means ± SD (n = 12).

100 mg kg−1 Zn combination with 5 and 20 mg kg−1 Cd, whereas no difference was found in CAD activities between the treatment of 100 mg kg−1 Zn combined with 2.5 mg kg−1 Cd and the control (Fig. 5B). The PPO activities in the K. obovata roots significantly affected by Cd and Zn additions. Compared to the control, in the treatments without Zn supply, no difference in PPO activities were found among 0, 2.5 and 5 mg kg−1 Cd treatment; under 100 or 500 mg kg−1 Zn supply, PPO activities both enhanced in 5 mg kg−1 and 20 mg kg−1 Cd treatments, and the highest PPO activity (7.74 UA mg−1) was measured in the 20 mg kg−1 Cd with 500 mg kg−1 Zn combined treatment, which was by 392.8% higher than the control (1.57 UA mg−1), and the PPO activities in 0, 2.5 mg kg−1 Cd treatment with 100 mg kg−1 Zn treatments were slightly lower by 1.6–2.7% than that of the control. The PPO activity in the leaves rapidly increased in the 5 and 20 mg kg−1Cd with 100 mg kg−1Zn or without Zn treatment, as well as all the treatment with 500 mg kg−1 Zn, but no stimulate effect of Cd supply was found in the 500 mg kg−1 Zn treatment (Fig. 5C).

higher dosage Cd treatments resulted in pigment degradation. The value of chlorophyll a/b represents the light capturing energy ability of K. obovata. There was no difference in chlorophyll a/b values between the control treatment and the 2.5 mg kg−1 Cd or 100 mg kg−1 Zn only treatments; however, lower values were found in the 20 mg kg−1 Cd with 500 mg kg−1 Zn combined treatment and the 500 mg kg−1 Zn only treatment (Fig. 4C). All Cd concentrations combined with 100 mg kg−1 Zn treatments exhibited higher total chlorophyll content than that in the respective Cd or Zn only treatments (Fig. 4D). This demonstrates that the light capturing energy ability of K. obovata was decreased by the increasing concentrations in the Cd and Zn treatments.

3.5. Enzyme activities related to phenolic compounds metabolism The activities of SKDH, CAD and PPO both in roots and leaves differed with Cd and Zn applications ( P < 0.05), and there were significant interactions between Cd or Zn (Table 1). Compared to the control, SKDH activity in the roots of K. obovata seedlings was obviously enhanced by 11.2–307.6% under individual as well as the combined treatments with Cd and Zn, while the SKDH activity in the leaves significantly increased by 3.4–69.5%, no differences were found among the treatments with 100 mg kg−1 Zn only, 2.5 mg kg−1 Cd with 100 mg kg−1 Zn and the control (Fig. 5A). The CAD activities in the roots increased with Cd and Zn levels, and a sharply stimulation effects by 20 mg kg−1 Cd was found at 500 mg kg−1 Zn levels. Under Cd/Zn treatment, CAD activities increased by 12.4–175.4%, with the highest CAD activity recorded at 740.4 nmol min−1 mg−1. The value was 1.82 times as higher as that of 500 mg kg−1 Zn only treatment, and 5.02 times as higher as the control. As well as, the CAD activities in those exposure with 2.5 and 5 mg kg−1 Cd with 100 mg kg−1 Zn supply, were by 59.5% and 35.8% higher than that without Zn treatment, respectively (Fig. 5B). The CAD activities in the leaves were enhanced by 1.7~40.0% with Cd/Zn treatment. Significant differences were found after the addition of 500 mg kg−1 Zn and

3.6. Correlation among biomass; SKDH, CAD, and PPO activities; and chlorophyll, phenolic compounds, Cd, and Zn contents Pearson product-moment correlation coefficient analysis was applied to the combined treatment seedlings. As shown in Table 2, the plant biomass had a positive correlation with chlorophylls (r = 0.73), but showed a negative correlation with total phenolic compounds (r = 0.88), total Cd ( r = -0.76), total Zn (r = -0.34), leaf SKDH (r = -0.67), leaf CAD (r = -0.66), leaf PPO (r = -0.72), root SKDH (r = -0.77), root CAD (r = -0.76) and root PPO (r = -0.69) under separate Cd and/or Zn treatments. Similar negative correlations were found between total chlorophylls and total phenolic compounds (r = -0.75), total Cd (r = 0.77), total Zn (r = -0.37), the activities of SKDH (r = -0.54), CAD (r = -0.42)and PPO (r = -0.49) in leaf, and SKDH (r = -0.78), CAD (r = -0.75) and PPO (r = -0.67) in root. 139

Fig. 5. Activities of enzymes related to phenolic metabolism such as SKDHshikimate dehydrogenase, CAD-cinnamyl alcohol dehydrogenase, PPO-polyphenol oxidase in K. obovata plants after 60 days of cadmium (Cd) or zinc (Zn) excess. The different letters (a-i) in leaf and letters (A-F) in root are significant differences according to Duncan's new multiple range test (p < 0.05). Values with the same letters are equivalent; different letters mean significant differences (p < 0.05) based on one-way ANOVA. Data are means ± SD (n = 12).

Phenolic compounds in roots, leaves and total plant were highly positively correlated to the activities of SKDH, CAD, and PPO in root and leaf, Cd contents in root, leaf and total plant and Zn contents in roots. In addition, phenolic compounds in leaf and total plant were positively correlated to total Zn content. Moreover, Cd and Zn contents in leaves exhibited positively correlated to their contents in roots, whereas Cd contents in leaf had a negative correlation with Zn content in leaves, and Zn contents in leaves negatively correlated to Cd content in root (Table 2). Thus, Cd and Zn treatments inhibited the growth of K. obovata, stimulated the accumulation of phenolic compounds and activated the relative enzyme activities of phenolic metabolism, and moderate antagonism effect between Cd and Zn was observed.

140

1.00

0.73 * * 1.00

TC

RPAC

− 0.80 − 0.78 0.54 * * 1.00

LPAC

− 0.81 − 0.54 1.00

− 0.88 − 0.75 − 0.88 0.82 * * 1.00

TPAC

− 0.67 − 0.54 0.81 * * 0.61 * * 0.82 * * 1.00

Leaf SKDH − 0.66 − 0.42 0.85 * * 0.56 * * 0.80 * * 0.87 * * 1.00

Leaf CAD − 0.72 − 0.49 0.92 * * 0.52 * * 0.83 * * 0.76 * * 0.82 * * 1.00

Leaf PPO − 0.77 − 0.78 0.83 * * 0.74 * * 0.89 * * 0.87 * * 0.81 * * 0.81 * * 1.00

Root SKDH

− 0.76 − 0.75 0.84 * * 0.64 * * 0.85 * * 0.80 * * 0.74 * * 0.79 * * 0.89 * * 1.00

Root CAD

− 0.69 − 0.67 0.86 * * 0.60 * * 0.85 * * 0.93 * * 0.90 * * 0.83 * * 0.94 * * 0.88 * * 1.00

Root PPO

− 0.72 − 0.78 0.63 * * 0.64 * * 0.70 * * 0.39 * 0.43 * 0.57 * * 0.64 * * 0.77 * 0.53 * * 1.00

Leaf Cd

0.11 0.19 0.24 − 0.24 0.01 0.44 * * 0.41 * 0.19 0.23 0.26 * * 0.41 * − 0.25 1.00

Leaf Zn

− 0.76 − 0.82 0.71 * * 0.67 * * 0.77 * * 0.54 * * 0.52 * * 0.71 * * 0.75 * * 0.88 * * 0.67 * * 0.92 * * − 0.16 1.00

Root Cd

− 0.48 − 0.47 0.74 * * 0.40 * 0.67 * * 0.89 * * 0.86 * * 0.72 * * 0.80 * * 0.81 * * 0.93 * * 0.35 * 0.64 * * 0.52 * * 1.00

Root Zn

− 0.76 − 0.77 0.65 * * 0.71 * * 0.74 * * 0.50 * * 0.48 * * 0.62 * * 0.70 * * 0.77 * * 0.57 * * 0.92 * * − 0.30 0.92 * * 0.38 * 1.00

Total Cd

− 0.34 − 0.37 0.61 * * 0.25 0.51 * * 0.77 * * 0.74 * * 0.55 * * 0.67 * * 0.72 * * 0.83 * * 0.25 0.78 * * 0.38 * 0.95 * * 0.21 1.00

Total Zn

Note: *Correlation is significant at the 0.05 level (2-tailed); **Correlation is significant at the 0.01 level (2-tailed); TC-Total chlorophylls; LPAC-Leaf phenolic compound content; RPAC-Root phenolic compound content; TPAC-Total phenolic compound content.

Biomass TC LPAC RPAC TPAC Leaf SKDH Leaf CAD Leaf PPO Root SKDH Root CAD Root PPO Leaf Cd Leaf Zn Root Cd Root Zn Total Cd Total Zn

Biomass

Table 2 Pearson product-moment correlation coefficients for relationships between biomass, total chlorophyll content, phenolic compound content, leaf and root SKDH, leaf and root CAD, leaf and root PPO, Cd and Zn (n = 12).

S. Chen et al.

Ecotoxicology and Environmental Safety 169 (2019) 134–143

Ecotoxicology and Environmental Safety 169 (2019) 134–143

S. Chen et al.

Fig. 6. SKDH-shikimate dehydrogenase; PPO-polyphenol oxidase; CAD-cinnamyl alcohol dehydrogenase; POX-phenolic peroxidase; PAL-phenylalanine ammonialyase.

4. Discussion

previous studies on the accumulation of heavy metal in mangrove plants (Huang and Wang, 2010). Thus it can be seen, Zn and/or Cd treatments affected the distribution of Cd and Zn content and the metabolism of phenolic compounds in plants (ANOVA, P < 0.05, Table 1). Mangrove plants appear to have a special mechanism which limits the translocation of metals to the aerial parts, and transport most of the Cd in the roots and the absorption rate in roots is reduced with increased concentration of Cd (Jiang et al., 2017; Kovacik et al., 2009), with approximately 76–84% of the Cd was distributed in the roots (Xie et al., 2013). Heavy metal retention in the roots can be a strategy for protecting the more sensitive aerial parts from the deleterious effect induced by heavy metal stress (Huang and Wang, 2010). Under separate applications, the Cd and Zn content in roots and leaves of plant increased consistently with increasing concentrations of metal treatments. In the combined treatments, the presence of Cd significantly reduced Zn content in K. obovata organs, indicating an antagonistic interaction between Cd and Zn (Table 2). The accumulation of heavy metals in K. obovata generally leads to variations in physiological and metabolic processes. In the low concentration treatment of 100 mg kg−1 Zn, Zn mainly participates in oxidative stress to ease toxicity induced by other heavy metals; however, at high concentrations of Zn treatment, Zn can be toxic to plants and induces oxidative stress by generating free radicals and ROS (reactive oxygen species) (Garg and Kaur, 2013). A schematic diagram of the distribution of heavy metals in roots, leaves and stems is shown in Fig. 6. Phenolic compounds can chelate heavy metals or remove free radicals attenuating oxidative damage under heavy metal stress (Manquian-Cerda et al., 2016). Phenolic compounds are also a type of secondary metabolite that together with others, participate in plant defense activity (I. Ali et al., 2006; M.B. Ali et al., 2006). So, the level of phenolic compounds is often used to assess the impact of environmental stresses in plants. This could be the reason why the content of phenolic compounds increased with increasing concentrations of Cd and Zn in our experiment. Plants should accumulate more phenolics to scavenge free radicals or binding heavy metals (I. Ali et al., 2006; M.B. Ali et al., 2006; Chao et al., 2009). In this case, phenolic compounds became involved in one of the defensive systems that the plants used against Cd and Zn stress.

4.1. 4.1. Effects of Cd and Zn on the growth of K. obovata The negative effects of Cd in plants is well known as inhibition of plant growth, loss of water balance, nutrient reduction, lipid peroxidation, and so on (Jia et al., 2016; Jiang et al., 2017). An interesting finding from this experiment was that the plant biomass production exhibited drastically different responses to Cd and Zn (Fig. 1). The biomass of K. obovata decreased with increasing Cd levels, whereas the addition of 100 mg kg−1 Zn enhanced the biomass in the control and 2.5 mg kg−1 Cd treatments. This was supported by the change of total chlorophyll content and the value of chlorophyll a/b (Fig. 4), and the negative correlation between Cd and Zn content in leaves of the plant (Table 2). Zinc is an essential micronutrient and involves in several important biological processes related to plant growth (Garg and Kaur, 2013). Supplementation of Zn could protect plants from oxidative stress caused by other heavy metals via its antioxidant properties (GawlikDziki et al., 2012). However, higher concentrations of Zn (500 mg kg−1 Zn) not only can be toxic to plants (Mirta Tkalec et al., 2014; Mongkhonsin et al., 2016), but also aggravated the toxic effects of Cd in plants (Fig. 1). That's because Cd and Zn are in the same group in the periodic table; they have many similar structural, geochemical, and environmental properties (Ambrosi et al., 2016; Qiu et al., 2008); therefore, they will compete for ions with each other. The results of the present study support the conclusion of Zn could alleviate the toxicity of Cd in mangrove (Bodin et al., 2013; Mongkhonsin et al., 2016), but these are different from that of low doses of Cd had a positive effect on the growth of A. paniculata seedlings (Manquian-Cerda et al., 2016). Chlorophyll in leaves of K. obovata are vital to the synthesis of photosynthetic-related pigments and enzymes in the Calvin cycle (Dai et al., 2017). Zn supply can be effective in reducing Cd damage in leaves and increasing chlorophyll content (Fig. 4), thus improving the capacity of plant photosynthesis and accumulation of biomass (Dimkpa et al., 2009).

4.2. 4.2. Response of Cd and Zn content in K. obovata to different Cd and Zn stress Increased levels of Cd and Zn treatments generally resulted in higher phenolic compounds, and Cd and Zn contents in roots, stems, and leaves of K. obovata (Figs. 2 and 3), the results was consistent with 141

Ecotoxicology and Environmental Safety 169 (2019) 134–143

S. Chen et al.

SKDH, CAD, and PPO activities, root Cd content as well as moderately positively correlated to root Zn content. Therefore, Cd and Zn treatments affected phenolic compounds metabolism in K. obovata. So we speculated that the shikimate pathway, flavonoids metabolic pathway, and benzene propane metabolic pathway might throw light on participating in alleviating Cd and Zinc toxicity in K. obovata. These specific pathways are as follows, in the shikimate pathway, shikimate is changed into phenylalanine under the catalysis of the SKDH, with phenylalanine being the precursor of the phenylalanine and flavonoid metabolic pathways. Coniferaldehyde is transformed into coniferyl alcohol under the catalysis of the CAD in the phenylalanine metabolic pathway (Abdulrazzak et al., 2006), which ultimately generates G-oligolignols that are transported to the cytoderm by the Golgi complex. Goligolignols participate in the synthesis of lignin and lead to a thickening of the cell wall, which blocks the outside metals from entering the cell (Wang et al., 2013). The flavonoid metabolic pathway converts cinnamic acid to flavonoids and tannins. Some of these flavonoids are distributed in the cytoplasm, which can cause brown discoloration under PPO catalysis (Wang et al., 2013). Some flavonoids are distributed in vacuoles participating in the chelation of heavy metals or participating in the removal of ROS under the catalysis of the POX enzyme (Michalak, 2006) to alleviate the toxicity of heavy metals. Through the above analysis, we guess that these three phenolic compounds metabolic pathways can relieve the Cd and Zn poisoning by making cell walls thicken, chelating heavy metals, removing active oxygen, and cell browning reaction. The schematic diagram of these metabolic processes is shown in Fig. 6.

4.3. Cd and Zn effects on the metabolism of phenolic compounds in different tissues of K. obovata The antioxidant molecules, such as phenolics, flavonoids and other reducing substances, participated in radical scavenging process might be a defensive mechanism to the stress caused by toxic metals (Ahmed et al., 2015; Manquian-Cerda et al., 2016). Consistent with this, in the 2.5 mg kg−1 Cd, 5 mg kg−1 Cd, and 20 mg kg−1 Cd only treatments, the phenolic compounds content displayed by 13.6%, 36.3% and 36.6% higher than that of the control in the roots, and by 1.9%, 6.3%, and 18.2% higher than that of the control in the leaves, respectively (Fig. 3). Higher phenolic compounds content in roots and leaves not only appeared to have a greater capacity to eliminate ROS, showed higher abilities to translocate and chelate phytotoxic metals in plants (Jiang et al., 2017). These chelated metal ions may be transferred to vacuoles (Catherine A. Rice-Evans, 1996). An increase in soluble phenolic compounds such as intermediates in lignin biosynthesis increase cell wall endurance by the creation of physical barriers that protect cells against the harmful action of heavy metals, as well as influence the transition of metal ions within plant tissues since the lignification probably retains a substantial portion of metals into the cell wall fraction. High concentrations of Cd and Zn, such as the 5 mg kg−1 Cd or 20 mg kg−1 Cd treatments with both Zn concentrations in K. obovata obtained a higher total phenolic compounds content than that of the low concentrations of Cd/Zn treatments (Fig. 3D). This sequestration might have effectively immobilized Cd and Zn in the mangrove roots. Therefore, phenolic compounds production and lignification in mangroves decreased the translocation of Cd and Zn from the roots to the leaves among the treatments. This explains why there were no differences in the stem phenolic compounds content among the different Zn and Cd treatments (Fig. 3B). This can be identified as an ecological protection strategy in respond to heavy metals stress. The increase in phenolic compounds under Cd and/or Zn treatments caused an increase in SKDH, CAD, PPO activities. CAD activity is related to lignin biosynthesis (Kováčik et al., 2009). Our results revealed a sharp increase in CAD activity, especially by Zn stress, which explained the stronger enhancement of lignin biosynthesis to prevent heavy metals from entering cells under metal stress condition (Kováčik et al., 2009). It has been reported that the deposition of polymerized phenols play an important role in restricting free metal ion levels by stimulating several related enzymes (Mobin et al., 2014). Polyphenol oxidases can catalyze the oxygen-dependent oxidation of phenols to quinones (Marusek et al., 2006). PPOs and their potential phenolic substrates have been proved not to occur in the same place in plant cells, with PPOs mainly found in chloroplasts, whereas phenolic compounds most accumulate in vacuole and cell wall (Araji et al., 2014). In the study, we have recorded strong enhancement of peroxidase activity preferentially in Cd and/or Zn treated in roots and leaves, which has also been reported in other plant species (Drazic and Mihailovic, 2005). It has been reported (Mobin et al., 2014) that the deposition of polymerized phenols play an important role in restricting free metal ion levels by stimulating several related enzymes. SKDH is a type of enzyme that participates in the shikimate pathway, converting simple carbohydrates into aromatic amino acids or phenylalanine. SKDH activity was preferentially enhanced in Cd/Zn exposed roots in the present study (Fig. 5A). Thus, the correlation among the biomass, chlorophyll content, SKDH, CAD, and PPO activities, total phenolic compounds, Cd, and Zn content in mangroves suggests that many factors influence the metabolism of phenolic compounds in the different Cd and Zn treatments.

5. Conclusions This work studied that how Cd and Zn affect the phenolic metabolism of K.obovata and what possible heavy metal tolerance mechanism of K. obovata involve in alleviating Cd and Zn toxic from the perspective of phenolic metabolism. Increased phenylpropanoid metabolism, the production of phenolic compounds, and the stimulation of cinnamyl alcohol dehydrogenase activities indicate the involvement of SKDH and CAD during lignin synthesis under Cd and Zn stress. PPO also share a role in synthesis of phenolic and lignin in K.obovata, and may participtate in removing excess ROS caused by Cd and Zn, thus playing another detoxifying role during Cd and Zn treatment. This indicates that K.obovata could induce several metabolic pathways of phenolic compounds in the improvement of Cd and Zn tolerance. These results provide strong evidence and research directions for studying the mechanism of heavy metal tolerance in mangrove ecosystems. Acknowledgement This study was funded by Major Program of National Natural Science Foundation of China (31530008 and 31870483), National Important Scientific Research Programme of China (2018YFC1406603). Science and Technology Project of Quanzhou City (2014Z120). The authors would like to acknowledge of Professor John Merefield of Exeter University, UK and Professor Ruiyu Lin of Fujian Agricultural and Forest University for assistance with article embellishing. References Abdulrazzak, N., et al., 2006. A coumaroyl-ester-3-hydroxylase insertion mutant reveals the existence of nonredundant meta-hydroxylation pathways and essential roles for phenolic precursors in cell expansion and plant growth. Plant Physiol. 140, 30–48. Ahmed, I.M., et al., 2015. Secondary metabolism and antioxidants are involved in the tolerance to drought and salinity, separately and combined, in Tibetan wild barley. Environ. Exp. Bot. 111, 1–12. Alharbi, O.M.L., et al., 2018. Health and environmental effects of persistent organic pollutants. J. Mol. Liq. 263, 442–453. Ali, I., 2006. Instrumental methods in metal ion speciation. Chromatogr. Sci. Ser. 96. Ali, I., et al., 2011. Removal of arsenate from groundwater by electrocoagulation method. Environ. Sci. Pollut. Res. 19, 1668–1676.

4.4. 4.3. The metabolic pathway of phenolic compounds response to heavy metal stress Pearson analysis performed on the root data showed that root phenolic compounds content was highly positively correlated to root 142

Ecotoxicology and Environmental Safety 169 (2019) 134–143

S. Chen et al.

to multiple heavy metals. J. Hazard Mater. 182, 848–854. Javed, M.T., et al., 2017. Cadmium spiked soil modulates root organic acids exudation and ionic contents of two differentially Cd tolerant maize (Zea mays L.) cultivars. Ecotoxicol. Environ. Saf. 141, 216–225. Jia, X., et al., 2016. Elevated CO2 affects secondary metabolites in Robinia pseudoacacia L. seedlings in Cd- and Pb-contaminated soils. Chemosphere 160, 199–207. Jiang, S., et al., 2017. Response of phenolic metabolism to cadmium and phenanthrene and its influence on pollutant translocations in the mangrove plant Aegiceras corniculatum (L.) Blanco (Ac). Ecotoxicol. Environ. Saf. 141, 290–297. Kovacik, J., et al., 2009. Phenolic metabolism of Matricaria chamomilla plants exposed to nickel. J. Plant Physiol. 166, 1460–1464. Kováčik, J., et al., 2009. Comparison of cadmium and copper effect on phenolic metabolism, mineral nutrients and stress-related parameters in Matricaria chamomilla plants. Plant Soil. 320, 231–242. Krol, A., et al., 2015. The effects of cold stress on the phenolic compounds and antioxidant capacity of grapevine (Vitis vinifera L.) leaves. J. Plant Physiol. 189, 97–104. Li, Y., et al., 2015. Subcellular distribution of Cd and Zn and MT mRNA expression in the hepatopancreas of Sinopotamon henanense after single and co-exposure to Cd and Zn. Comp. Biochem. Physiol. C Toxicol. Pharmacol. 167, 117–130. Manquian-Cerda, K., et al., 2016. Effect of cadmium on phenolic compounds, antioxidant enzyme activity and oxidative stress in blueberry (Vaccinium corymbosum L.) plantlets grown in vitro. Ecotoxicol. Environ. Saf. 133, 316–326. Manquian-Cerda, K., et al., 2018. Interactive effects of aluminum and cadmium on phenolic compounds, antioxidant enzyme activity and oxidative stress in blueberry (Vaccinium corymbosum L.) plantlets cultivated in vitro. Ecotoxicol. Environ. Saf. 150, 320–326. Marusek, C.M., et al., 2006. Comparative analysis of polyphenol oxidase from plant and fungal species. J. Inorg. Biochem. 100, 108–123. Melida, H., et al., 2011. Changes in cinnamic acid derivatives associated with the habituation of maize cells to dichlobenil. Mol. Plant. 4, 869–878. Michalak, A., 2006. Phenolic compounds and their antioxidant activity in plants growing under heavy metal stress. Pol. J. Environ. Stud. 15, 523–530. Mirta Tkalec, P.P.S., Cvjetko, Petra, Sikic, Sandra, Pavlica, Mirjana, Balen, Biljana, 2014. The effects of cadmium-zinc interactions on biochemical. PLos One 9, 1–13. Mobin, M., et al., 2014. Studies on the glyphosate-induced amino acid starvation and addition of precursors on caffeic acid accumulation and profiles in adventitious roots of Echinacea purpurea (L.) Moench. Plant Cell, Tissue Organ Cult. (PCTOC). 120, 291–301. Mongkhonsin, B., et al., 2016. Phenolic compounds responding to zinc and/or cadmium treatments in Gynura pseudochina (L.) DC. extracts and biomass. Plant Physiol. Biochem. 109, 549–560. Ni, I.H., et al., 2005. Influences of salinity on the biokinetics of Cd, Se, and Zn in the intertidal mudskipper Periophthalmus cantonensis. Chemosphere 61, 1607–1617. Pourcel, L., et al., 2010. The formation of Anthocyanic Vacuolar Inclusions in Arabidopsis thaliana and implications for the sequestration of anthocyanin pigments. Mol. Plant. 3, 78–90. Qiu, R.L., et al., 2008. Antioxidative response to Cd in a newly discovered cadmium hyperaccumulator, Arabis paniculata F. Chemosphere 74, 6–12. Rice-Evans, Catherine A., N. J. M, Paganga, George, 1996. Structure-antioxidant activity relationships of flavonoids and phenolic acids. Free Radic. Biol. Med. 20, 933–956. Ruuhola, T., et al., 2018. Effects of long-term UV-exposure and plant sex on the leaf phenoloxidase activities and phenolic concentrations of Salix myrsinifolia (Salisb.). Plant Physiol. Biochem. 126, 55–62. Sae-Lee, N., et al., 2012. Chemical qualities and phenolic compounds of Assam tea after soil drench application of selenium and aluminium. Plant Soil. 356, 381–393. Singleton, V., Rossi, J.A., 1965. Colorimetry of total phenolics with phosphomolybdicphosphotungstic acid reagents. Am. J. Enol. Vitic. 16, 144–158. Sytar, O., et al., 2013. Heavy metal-induced oxidative damage, defense reactions, and detoxification mechanisms in plants. Acta Physiol. Plant. 35, 985–999. Wang, Y., et al., 2013. Plant cell wall lignification and monolignol metabolism. Front. Plant Sci. 4, 220. Winkel-Shirley, B., 2001. Flavonoid biosynthesis. A colorful model for genetics, biochemistry, cell biology, and biotechnology. Plant Physiol. 126, 485–493. Xie, X., et al., 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, 997–1008. Zeng, X.W., et al., 2011. The differentially-expressed proteome in Zn/Cd hyperaccumulator Arabis paniculata Franch. in response to Zn and Cd. Chemosphere 82, 321–328. Zhang, G., et al., 2013. Stimulation of phenolic metabolism by silicon contributes to rice resistance to sheath blight. J. Plant Nutr. Soil Sci. 176, 118–124. Zhu, G., et al., 2018. Effects of cadmium stress on growth and amino acid metabolism in two Compositae plants. Ecotoxicol. Environ. Saf. 158, 300–308.

Ali, I., et al., 2012. Arsenite removal from water by electro-coagulation on zinc–zinc and copper–copper electrodes. Int. J. Environ. Sci. Technol. 10, 377–384. Ali, I., et al., 2014. Removal of arsenic species from water by batch and column operations on bagasse fly ash. Environ. Sci. Pollut. Res. Int. 21, 3218–3229. Ali, I., et al., 2017. Uptake of propranolol on ionic liquid iron nanocomposite adsorbent: kinetic, thermodynamics and mechanism of adsorption. J. Mol. Liq. 236, 205–213. Ali, I., et al., 2018. Artificial neural network modelling of amido black dye sorption on iron composite nano material: kinetics and thermodynamics studies. J. Mol. Liq. 250, 1–8. Ali, I., Jain, C.K., 2004. Advances in arsenic speciation techniques. Int. J. Environ. Anal. Chem. 84, 947–964. Ali, M.B., et al., 2006. Phenolics metabolism and lignin synthesis in root suspension cultures of Panax ginseng in response to copper stress. Plant Sci. 171, 147–154. Ambrosi, G., et al., 2016. A Biphenol-based chemosensor for Zn(II) and Cd(II) metal ions: synthesis, Potentiometric studies, and crystal structures. Inorg. Chem. 55, 7676–7687. Anne Vissers, A.K., Hilgers, Roelant J., Marina Marinea, Peter Alexander, Wierenga, Harry, Gruppen, Jean-Paul Vincken, 2017. Enzymatic browning in sugar beet leaves (beta vulgaris L.): Influence of Caffeic acid derivatives, oxidative Coupling and coupled oxidation. Agric. Food Chem. 1–35. Araji, S., et al., 2014. Novel roles for the polyphenol oxidase enzyme in secondary metabolism and the regulation of cell death in walnut. Plant Physiol. 164, 1191–1203. Austin, M.B., Noel, J.P., 2003. The chalcone synthase superfamily of type III polyketide synthases. Nat. Product. Rep. 20, 79–110. Basheer, A.A., 2018. Chemical chiral pollution: impact on the society and science and need of the regulations in the 21st century. Chirality 30, 402–406. Bodin, N., et al., 2013. Assessment of trace metal contamination in mangrove ecosystems from Senegal, West Africa. Chemosphere 90, 150–157. Chao, Y.-Y., et al., 2009. Salicylic acid-mediated hydrogen peroxide accumulation and protection against Cd toxicity in rice leaves. Plant Soil. 329, 327–337. Chen, J., et al., 2010. Diurnal variation of nitric oxide emission flux from a mangrove wetland in Zhangjiang River Estuary, China. Estuar., Coast. Shelf Sci. 90, 212–220. Cheng, H., et al., 2010. The role of radial oxygen loss and root anatomy on zinc uptake and tolerance in mangrove seedlings. Environ. Pollut. 158, 1189–1196. Dai, M., et al., 2017. Phosphorus mediation of cadmium stress in two mangrove seedlings Avicennia marina and Kandelia obovata differing in cadmium accumulation. Ecotoxicol. Environ. Saf. 139, 272–279. Dai, M., et al., 2018. Exogenous phosphorus enhances cadmium tolerance by affecting cell wall polysaccharides in two mangrove seedlings Avicennia marina (Forsk.) Vierh and Kandelia obovata (S., L.) Yong differing in cadmium accumulation. Mar. Pollut. Bull. 126, 86–92. Das, S.K., et al., 2016. Antioxidative response to abiotic and biotic stresses in mangrove plants: a review. Int. Rev. Hydrobiol. 101, 3–19. Dauwe, R., et al., 2007. Molecular phenotyping of lignin-modified tobacco reveals associated changes in cell-wall metabolism, primary metabolism, stress metabolism and photorespiration. Plant J. 52, 263–285. Dimkpa, C.O., et al., 2009. Metal-induced oxidative stress impacting plant growth in contaminated soil is alleviated by microbial siderophores. Soil Biol. Biochem. 41, 154–162. Drazic, G., Mihailovic, N., 2005. Modification of cadmium toxicity in soybean seedlings by salicylic acid. Plant Sci. 168, 511–517. Du, J., et al., 2014. Phosphorus and cadmium interactions in Kandelia obovata (S. L.) in relation to cadmium tolerance. Environ. Sci. Pollut. Res. Int. 21, 355–365. Fourcroy, P., et al., 2016. Facilitated Fe nutrition by phenolic compounds excreted by the arabidopsis ABCG37/PDR9 transporter requires the IRT1/FRO2 high-affinity root Fe (2+) transport system. Mol. Plant. 9, 485–488. G.R.MacFarlane, M.D.B., 2000. Cellular distribution of copper,lead and zinc in the grey mangrove, Avicennia marina (Forsk.) Vierh. Aquat. Bot. 68, 45–59. Garg, N., Kaur, H., 2013. Response of antioxidant enzymes, phytochelatins and glutathione production towards Cd and Zn stresses in Cajanus cajan(L.) Millsp. genotypes colonized by arbuscular mycorrhizal fungi. J. Agron. Crop Sci. 199, 118–133. Gawlik-Dziki, U., et al., 2012. Comparison of phenolic acids profile and antioxidant potential of six varieties of spelt (Triticum spelta L.). J. Agric. Food Chem. 60, 4603–4612. Gupta, V.K., Ali, I., 2013. Perspectives of water treatment, remediation and recycling. Environ. Water 205–209. Hamada, M., et al., 2015. Serinibacter tropicus sp. nov., an actinobacterium isolated from the rhizosphere of a mangrove, and emended description of the genus Serinibacter. Int. J. Syst. Evol. Microbiol. 65, 1151–1154. Hrazdina, G., 1993. Compartmentation in phenolic metabolism. Int. Symp. Nat. Phenols Plant Resist. 381, 86–96. Huang, G.Y., Wang, Y.S., 2010. Physiological and biochemical responses in the leaves of two mangrove plant seedlings (Kandelia candel and Bruguiera gymnorrhiza) exposed

143