Exogenous phosphorus treatment facilitates chelation-mediated cadmium detoxification in perennial ryegrass (Lolium perenne L.)

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Exogenous phosphorus treatment facilitates chelation-mediated cadmium detoxification in perennial ryegrass (Lolium perenne L.) Hui Jiaa, Deyi Houa,*, David O’Connora, Shizhen Pana, Jin Zhua, Nanthi S. Bolanb, Jan Mulderc a

School of Environment, Tsinghua University, Beijing 100084, China Global Centre for Environmental Remediation, ATC Building, Faculty of Science and Information Technology, The University of Newcastle, Callaghan, NSW 2308, Australia c Faculty of Environmental Sciences and Natural Resource Management, Norwegian University of Life Sciences, P.O. Box 5003, 1432 Ås, Norway b

G R A P H I C A L A B S T R A C T

A R T I C LE I N FO

A B S T R A C T

Editor: R Teresa

Cadmium (Cd) is an on-going environmental pollutant associated with hindered plant growth. In response, plants possess various strategies to alleviate Cd stress, including reactive oxygen species (ROS) scavenging and chelation-mediated Cd detoxification. The present study examined the Cd defense mechanism of perennial ryegrass (Lolium perenne L.), taking into account the effect of exogenous phosphorus (P) input. It was found that despite triggering antioxidant enzyme activity, Cd stress heightened lipid peroxidation levels. Exogenous P input partially mitigated the lipid peroxidation impact and decreased the levels of superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) antioxidant enzymes, revealing reduced ROS-scavenging activity. Importantly, notable relationships were determined between the amount of Cd uptake in the root and the amount of non-protein thiols (R2 = 0.914), glutathione (R2 = 0.805) and phytochelatins (R2 = 0.904) in proportion to the amount of exogenous P applied. The levels of amino acids proline and cysteine were also enhanced by exogenous P input showing their influence in alleviating Cd stress. Overall, it is reported that Cd detoxification in ryegrass plants can be stimulated by exogenous P input, which facilitates chelation-mediated Cd detoxification processes.

Keywords: Plant toxicity Oxidative stress Antioxidant enzymes Non-protein thiols Cd

Abbreviations: CAT, catalase; DW, dry weight; GSH, glutathione; MDA, malondialdehyde; NPT, non-protein thiol; PCs, phytochelatins; POD, peroxidase; ROS, reactive oxygen species; SOD, superoxide dismutase ⁎ Corresponding author. E-mail address: [email protected] (D. Hou). https://doi.org/10.1016/j.jhazmat.2019.121849 Received 26 September 2019; Received in revised form 16 November 2019; Accepted 7 December 2019 0304-3894/ © 2019 Elsevier B.V. All rights reserved.

Please cite this article as: Hui Jia, et al., Journal of Hazardous Materials, https://doi.org/10.1016/j.jhazmat.2019.121849

Journal of Hazardous Materials xxx (xxxx) xxxx (Lovelock et al., 2004) (Yin et al., 2016) (Du et al., 2014) This study

(Fang et al., 2012)

(Dai et al., 2017)

1. Introduction Heavy metal contamination of the soil environment is a chronic issue in many places throughout the world (Jin et al., 2019; Peng et al., 2019; Zhang et al., 2019). Each year, more than 13,000 metric tons (13 Gg) of Cd are released to the natural environment via anthropogenic activities (Gallego et al., 2012). Key sources include impure of phosphate fertilizers, irrigation with polluted wastewater, metallurgy, waste incineration, traffic, and cement manufacturing (Seshadri et al., 2016; Shah et al., 2001); elevated Cd can also be derived from lithogenic origins (Gallego et al., 2012). This has resulted wide-spread soil contamination. In China, for example, approximately 7 % of agricultural soils have elevated levels of Cd (MEP, 2014; Hou and Li, 2017). Plant roots take up Cd in the freely available Cd2+ form, which is likely translocated through the xylem in vascular plants (Rizwan et al., 2016). It has been widely established that Cd uptake hinders plant growth, with many studies published on the impacts of Cd stress, particularly for rice and other grass plants (Gallego et al., 2012; Dąbrowski et al., 2013). One way that taken up Cd causes damages to plants is by triggering the release of reactive oxygen species (ROS), which raise plant oxidative stress levels (Choppala et al., 2014). The overproduction and accumulation of ROS, known as ‘oxidative burst’, indicates incompatible interaction between Cd and plant cells (Romero-Puertas et al., 2004). For instance, Cd2+ indirectly activates NADPH oxidase on cell membranes, leading to the release of superoxide radicals (O2%−) and hydrogen peroxide (H2O2). Oxidative stress is known to increase plant malondialdehyde (MDA) levels (Dai et al., 2018), which signifies heightened lipid peroxidation (Tian et al., 2019), affecting cellular membranes, lipoproteins, and other lipid containing molecules. Plants possess various strategies to alleviate Cd stress (Choppala et al., 2014), including ROS-scavenging (Daud et al., 2013; Jia et al., 2016) and chelation-mediated detoxification (Mendoza-Cózatl et al., 2010). Antioxidant enzymes, such as superoxide dismutase (SOD), quench O2%− and convert it to H2O2 and O2. Peroxidase (POD) and catalase (CAT) are involved in converting reactive H2O2 and O2 to benign H2O (Gill and Tuteja, 2010). The amino acid proline (Sharma et al., 1998; Zouari et al., 2016) is associated with activation of cellular antioxidant systems, and scavenging ROS under oxidative stress (Zouari et al., 2016). The application of exogenous proline has been shown to enhance the antioxidant system of young date palms (Phoenix dactylifera L.) (Zouari et al., 2016). In chelation-mediated detoxification, thiol compounds with high affinity for Cd are thought to hold a key role in reducing Cd stress (Zouari et al., 2016; Zhang et al., 2013). Plant thiols are divided into protein thiols (PSHs) and non-protein thiols (NPTs). The major NPT form is glutathione (γ-glutamyl-cysteinyl-glycine; GSH), which derives from the sulfur-containing amino acid cysteine (Mendoza-Cózatl et al., 2010). GSH is normally present in low concentrations (10–30 μM free cysteine (Zagorchev et al., 2013)), and is commonly measured to monitor plant thiol status (Yang and Guan, 2017). Phytochelatins (PCs) are GSH-derived peptides induced by Cd stress (Bhargava et al., 2005), which directly function in the Cd detoxification processes (MendozaCozatl et al., 2008) through PC-Cd chelation (Greger et al., 2016; Dai et al., 2017; Nishikawa et al., 2006). In general, higher levels of GSH and PCs are associated with more efficient Cd detoxification processes in plants (Zagorchev et al., 2013). Heavy metals can be stabilized within soils by sorption, precipitation, and complexation reactions with soil constituents, which reduces their bioavailability to plants (Seshadri et al., 2016; O’Connor et al., 2019a). However, the bioavailable fraction is increased by the presence of certain elements (Du et al., 2014; Dong et al., 2007). For instance, Yin et al. (2016) found that exogenous P input changes the chemical form of Cd, thus increasing its bioavailability to spinach (Spinacia oleracea L.). Therefore, it may be expected that Cd stress levels would increase with exogenous input of elements such as N, Zn, Fe, Se, and P,

Promoted plant growth and hydraulic conductance P decreased Cd accumulations in Spinach cultivars Increased the content of root exudates Increased thiols content, and antioxidant enzyme activity in roots

Hydroponic

Field hydroponic Soil hydroponic

Arabidopsis (Arabidopsis thaliana)

Dwarf mangroves (Rhizophora mangle L.) Spinach (Spinacia oleracea L.) Mangrove (Kandelia obovata S.L.) Perennial ryegrass (Lolium perenne L.)

30 mg/kg 15, 30 mg/L 60 mg/kg 20, 80 mg/L

Hydroponic

3.1 mg/L

Cd bound to the cell wall

Reduce malonaldehyde content, increased cell wall polysaccharide content Increased polysaccharide content and degree of demethylesterification P significantly reduced internal nutrient use Enhanced the distribution of Cd Reduced Cd translocation from root to shoot Increased toxicity defense and antioxidant capacity

Soil pot

Chinese flowering cabbage (Brassica parachinensis L.) Mangrove (Avicennia marina)

13, 56, 108 mg/ kg 30, 90 mg/L

Decreased root elongation, induced Cd immobilization

(Qiu et al., 2011) Reduced health risk

(Wang et al., 2015) hydroponic Rice (Oryza sativa L.)

52 mg/L

Soil pot Pakchoi (Brassica chinensis L.)

303 mg/kg

Formation of Cd-protein deposits, and vacuole sequestrated high Cd, involved in Cd detoxification Improved level of antioxidative- related gene expression in the Cd-treated rice tissues Decreased longitudinal Cd transportation.

Decrease MDA contents, and alleviated Cd stress

(Xue et al., 2014)

(Jiang et al., 2007)

Reduced numbers of damaged chloroplasts, and increased chlorophyll content Decreased Cd entering into root protoplast of Pakchoi P-Cd chelates was formed in maize roots 7.75 mg/L Hydroponic Maize (Zea mays L. cv Shendan 16)

P dose Growth condition Plant

Table 1 Effects of P addition on plant response mechanism under Cd-induced stress.

Mechanism

Effect

Ref.

H. Jia, et al.

2

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Fig. 1. Ryegrass growth (A) and root P concentrations (B) for different treatments after 28 d. Lowercase letters represent significant differences (P < 0.05).

2. Materials and methods

Table 2 Cadmium contents in ryegrass plant tissue and translocation factors (TF) under different treatments. Treatments

Cd0P0 Cd0P20 Cd0P80 Cd0.8P0 Cd0.8P20 Cd0.8P80 Cd4P0 Cd4P20 Cd4P80

Cd concentration (mg/kg DW)

2.1. Hydroponic experiments

TF

Ryegrass root

Ryegrass shoot

– – – 33.52 ± 1.28A 38.32 ± 0.99B 42.86 ± 1.61C 106.41 ± 4.10D 123.29 ± 2.65E 140.66 ± 3.58F

– – – 12.89 12.90 14.38 32.71 35.38 39.40

± ± ± ± ± ±

0.57a 0.81a 0.36b 2.17c 2.49d 1.48e

– – – 0.38 0.35 0.34 0.31 0.29 0.28

± ± ± ± ± ±

Commercially available perennial ryegrass (Lolium perenne L.) seeds (purity > 96 %) were obtained from YuCong Seed Company (Jiangsu, China). The seeds were sterilized with H2O2 (30 %, V/V) for 30 min, and then rinsed several times with distilled water before germinated in nutrient solution using germination discs. Healthy seedlings of ∼10 mm height were selected and cultured in Hoagland nutrient solution (N 200 mg/L, K 270 mg/L, Ca 160 mg/L, S 770 mg/L, Cl 0.65–2.5 mg/L, Mg 98.6 mg/L, Fe 1120 mg/L (Hoagiand, 1933), see table S1). Based on pre-experiments and a review of related literature (Dai et al., 2018, 2017; Du et al., 2014), various concentrations of Cd and P were applied as follows: 0, 0.8, and 4 mg/L of Cd as CdCl2·2.5H2O and 0, 20, and 80 mg/L of P as KH2PO4. For reference (Hoagiand, 1933), Hoagland plant nutrient solution normally contains 31 mg/L of P as 1 M KH2PO4. The treatments were denoted as CdxPy, where x is the Cd concentration (mg/L), and y is the P concentration (mg/L) in the applied nutrient solution. Nutrient solutions were maintained at pH 6.5 with 0.1 M HCl, and were replaced every 3 d. Three individual plants (n = 3) were grown in each of the nine treatment solutions. Plant cultures were grown for 28 d in a controlled environment of 23 °C ( ± 3 °C) and 75 % ( ± 3 %) relative humidity, and subjected to artificial aeration (2 L/min) and a photosynthetic photon flux density of 800−1400 μmol m−2 s−1 (Mingji et al., 2009; Liu et al., 2009). The solute distributions in the nine treatment solutions were modeled using Visual MINTEQ (version 3.0) to determine Cd2+ complexation potential, the results of which are presented in the Supporting Information. The calculations show that logKsp values for Cd solid phases (i.e. Cd3(PO4)2) range from −31.44 to −82.54, dependent on the amount of P applied (Table S2).

0.02 0.01 0.02 0.01 0.01 0.00

Notes: – denotes below the detection limit. Superscript letters represent significant differences (P < 0.05).

however, several studies have shown that Cd stress levels will decrease when these elements are applied (Rizwan et al., 2016). Among these, P plays an important role in plant metabolism, being involved in photosynthesis, respiration, energy transfer, and cell division (Dai et al., 2017; Wang et al., 2010). Moreover, exogenous P participates in a number of important plant response mechanisms under Cd-induced stress (Dai et al., 2018); the various mechanisms and effects observed are summarized in Table 1. Importantly, exogenous P input is thought to facilitate the formation of detoxifying PCs-Cd chelates (Dai et al., 2018; Wang et al., 2010). In this study it was hypothesized that exogenous P can help mitigate Cd stress in perennial ryegrass (Lolium perenne L.), via a PCs-mediated Cd detoxification effect. Perennial ryegrass was selected for this study for the following reasons: (1) it has a high capacity to uptake Cd (Guo et al., 2014); (2) it has a wide-branched root system that forms a dense rhizosphere; (3) it is palatability to livestock, thus presenting a contaminant pathway to animals; (4) this species is characteristic in city green areas (Xu et al., 2014); (5) it is widely cultivated in China as turf and forage grass; and, (6) it is considered a candidate plant for the phytoremediation of Cd contaminated soil. The following factors were examined experimentally: (1) the impacts of P addition on ryegrass root Cd accumulation; (2) ryegrass response to oxidative-stress resulting from Cd stress; (3) the contribution of NPTs and PCs and GSH to Cd detoxification processes; and, (4) the role of exogenous P input on Cd stress in ryegrass plants.

2.2. Measurement of plant tissue cadmium Cadmium was extracted from plant tissues and measured according to procedures used by Shen et al. (2018). Briefly, the 28 d old plants were first separated by dissection into root and shoot parts. The separated tissues were then rinsed with deionized water, dried, ground, and passed through a nylon sieve (250 μm aperture). Samples (0.2 g) were placed in concentrated HNO3 (14.4 M; 5 mL) and H2O2 (9.8 M; 2 mL) and subjected to microwave assisted digestion (at 140 ℃ for 30 min). The extractant was diluted with ultrapure water (18.2 MΩ cm) and filtered (0.22 μm), the Cd concentration was measured by inductively coupled plasma - mass spectrometry (ICP-MS). A standard plant reagent material (GBW-07603) from the National Research Center for 3

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Fig. 2. Effects of exogenous P and Cd on antioxidant enzymes membrane lipid peroxidation in ryegrass roots: (A) superoxide dismutase (SOD), (B) catalase (CAT), (C) peroxidase (POD), and (D) malondialdehyde (MDA). Lowercase letters represent significant differences (P < 0.05).

using the procedures described by Jia et al. (2016). Briefly, root tips were rinsed with deionized water, dried and ground. Samples (20 mg) were placed in potassium phosphate buffer saline (PBS) solution (0.05 M; 8 mL; pH = 7.18). Extractants were centrifuged (RCF = 10,451 × g) for 15 min at 4 °C to collect the crude enzyme solution (i.e., the supernatant). POD activity was measured according to Kochba et al. (1977). Briefly, crude enzyme solution samples (0.1 mL) were treated with PBS (0.05 M, 2.9 mL), H2O2; (9.8 M; 1 mL), guaiacol (0.05 M; 1 mL), and ultrapure water (18.2 MΩ cm; 3 mL). Mixtures were maintained at 37 °C for 5 min in a water bath (without shaking), and then analyzed by spectrophotometry at 470 nm wavelength. SOD activity was determined using the method of nitroblue tetrazolium (NBT) photoreduction as describe by Giannopolitis and Ries (1977). Briefly, crude enzyme solution samples (0.1 mL) were treated with PBS buffer (0.05 M; 1.5 mL), methionine (130 mM; 0.3 mL), NBT (750 μM; 0.3 mL), riboflavin (20 μM; 0.3 mL), and EDTA-Na2 (100 μM; 0.3 mL). The mixtures were centrifuged (RCF = 10,451 × g) for 15 min. Supernatants were analyzed by spectrophotometry at 560 nm wavelength. CAT activity was analyzed according to Aebi (1984). Briefly, crude enzyme solution samples (0.2 mL) were treated with phosphoric acid (0.1 M; 1.5 mL, pH = 7.8) and ultrapure water (18.2 MΩ cm; 1 mL), then shaken in a water bath shaker (150 rpm; 10 min; 30 °C). Supernatants were analyzed using a spectrometer at 240 nm wavelength. Enzyme activity is reported herein as enzyme units (U) per mg of protein, where 1 U is the amount of enzyme that catalyzes the conversion of one micromole of substrate per min under the conditions of the study. MDA was extracted according to the procedures described by Hodges et al. (1999). Briefly, root tips were rinsed with deionized

Fig. 3. Relationship between ryegrass biomass and malondialdehyde (MDA) content under different treatments.

Standards, China, was used to verify the experimental procedure and analysis. 2.3. Measurement of antioxidant enzymes and lipid peroxidation After 28 d of cultivation, roots were analyzed for antioxidant enzymes (POD, CAT and SOD) and lipid peroxidation (signified by MDA). Antioxidant enzymes were first extracted as a crude enzyme solution 4

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Fig. 4. Levels of free amino acids in rye grass roots after 28 d (A) proline, and (B) cysteine. Lowercase letters represent significant differences (P < 0.05).

Fig. 5. The effect of P and Cd treatments on (A) phytochelatins (PCs), (B) glutathione (GSH), and (C) non-protein thiols (NPTs).

and ground. Samples (0.2 g) were placed in a mixture of sulfosalicylic acid (3 %, w/v; 5 mL), toluene (99.99 % purity; 4 mL), and nihydrin (2.5 % in a solvent of glacial acetic acid (98 %) and phosphoric acid (6 M) in a 3:2 ratio; 2 mL). The mixtures were heated in a water bath (100 °C; 60 min) and then subject to centrifugation (RCF = 2612 × g) for 5 min. The supernatants were analyzed by spectrophotometry at 520 nm wavelength. Cysteine content in root tissue was determined according to Gupta et al. (2010). Briefly, root tips were rinsed with deionized water, dried and ground. Samples (200 mg) were mixed with perchloric acid (5 %) under an ice-cold environment (viz. 0 °C), and centrifuged (RCF = 10,451 × g) for 5 min. Supernatant samples (2 mL) were mixed with ninhydrin (2.5 % in a solvent of glacial acetic acid (98 %) and phosphoric acid (6 M) in a 3:2 ratio; 2 mL) before being subjected to spectrophotometry at 560 nm wavelength.

water, dried and ground, and samples (50 mg) placed in trichloroacetic acid (TCA) (10 % w/v; 10 mL), mixed using a water bath shaker (150 rpm; 10 min; 30 °C), and centrifuged (RCF = 1672 × g). Supernatants (2 mL) were treated with thiobarbituric acid (TBA) (0.6 %; 2 mL), heated in a water bath (100 °C; 15 min), before centrifugation (RCF = 940 × g) for 15 min. The second supernatants were subjected to spectrophotometry at 450, 532 and 600 nm wavelengths. MDA values were calculated as CMDA = 6.45(D532 − D600) − 0.56D450 according to Heath and Packer (1968).

2.4. Measurement of amino acid content Free proline content in root tissue was determined by the nihydrincolorimetry method according to the procedures described by Bates et al. (1973). Briefly, root tips were rinsed with deionized water, dried 5

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GSH values (i.e., PCs = NPT − GSH) (Bhargava et al., 2005; Dai et al., 2017).

2.6. Statistical analysis Experimental data presented herein show the mean of three samples (three individual plants, n = 3), where ± shows the standard deviation. Plant translocation factors (TFs) were calculated according to Bolan et al. (2013) as Cdr/Cds where Cdr is the Cd concentration in root and Cds is the Cd concentration in shoot. Data subjected to analysis of variance (ANOVA) was performed at the 95 % confidence level. Statistical analyses were performed using Origin (version 9.0) and IBM SPSS (version 13.0).

3. Results 3.1. Plant biomass and Cd distribution

Fig. 6. Relationship between Cd and phytochelatins (PCs) concentrations in ryegrass root. The relationship between Cd concentrations and PCs content in the root tips for Cd0.8 and Cd4 treatments.

Ryegrass biomass and Cd distribution were examined after 28 d of growth. There was no significant difference in plant biomass between plants subjected to the lower Cd level (Cd-0.8) and plants not exposed to Cd (Cd-0 treatments). The growth of plants subjected to the higher Cd level (Cd-4) was severely hindered (P < 0.05). As would be expected for a mineral fertilizer, exogenous P input was positively correlated with increased plant biomass (P < 0.05) and increased root P concentrations (Fig. 1). The influence of exogenous P input on ryegrass resistance to Cd toxicity is revealed by differences in the accumulation, distribution and translocation of Cd in root and shoot tissues of plants subjected to various amounts of exogenous P (Table 2). In general, Cd accumulation in shoots was lower than in roots, and the accumulation of Cd in the roots and shoots of plants subjected to Cd exposure (Cd-0.8 and Cd-4 treatments) were significantly increased compared to Cd-0 treatments (P < 0.05). Moreover, exogenous P input increased the amount of Cd accumulated in the roots and shoots of plants under Cd stress. Translocation factors (TFs) (i.e., the amount of Cd transported from root to shoot) for plants subjected to 0.8 mg/L Cd decreased by 12.5 % and 12.8 % with 20 mg/L and 80 mg/L exogenous P inputs, respectively. For plants subjected to the higher Cd exposure level (4 mg/L), the proportion of translocation Cd decreased by 6.7 % and 8.9 % with 20 mg/L at 80 mg/L P inputs, respectively. In summary, while exogenous P significantly increased Cd levels in ryegrass roots and shoots, it was also associated with lower TF values.

2.5. Measurement of non-protein thiols (NPTs), glutathione (GSH), and phytochelatins (PCs) Root tissue NPT contents were determined according to the procedures described by Bhargava et al. (2005). Briefly, root tips were rinsed with deionized water, dried and ground, and samples (0.2 g) placed in sulfosalicylic acid (SSA) (5 % (w/v); 2 mL). The mixtures were shaken in a water bath shaker (150 rpm; 20 min; under an ice-cold environment viz. 0 °C), and then subjected to centrifugation (RCF = 15,049 × g) for 15 min. Supernatant samples (0.2 mL) were mixed with Tris-HCl buffer (0.2 M, 2 mL, pH 8.2) and 5,5′-Dithiobis-(2nitrobenzoic acid) (DTNB) (10 mM; 0.15 mL), shaken in a water bath shaker (150 rpm; 20 min; 30 °C) and then subjected to spectrophotometry at 412 nm wavelength. Root tissue GSH contents were determined according to Eyer and Podhradský (1986). Briefly, toot tips were rinsed with deionized water, dried and ground, and samples (0.2 g) placed in NaOH (1.0 M; 0.4 mL,) and EDTA-TCA (0.4 mL). The pH was maintained between 6.5 and 6.7 by applying NaOH (1.0 M). Solution samples (2.0 mL) were mixed with DTNB (0.6 mM; 0.1 mL) and PBS (0.1 M; 0.1 mL, pH = 7.5) before being subjected to spectrophotometry at 412 nm wavelength. Amounts of PCs in roots were calculated as the difference between the NPT and

Fig. 7. Schematic showing the role of exogenous P in Cd detoxification in ryegrass plants; the arrows indicate the decrease or increase in the levels of these components as impacted by P input. 6

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Cysteine contents also increased with increasing exogenous P input. The cysteine contents were 141.7 %, 119.4 % and 131.6 % higher in plants subjected to high P input (P80) than in plants grown without P (P0) when exposed to Cd0, Cd0.8 and Cd4, respectively. The highest cysteine level was noticed in plants grown in Cd4P80, which was 266 % greater than those grown in Cd0P0. In summary, the production of amino acids, proline and cysteine increases in ryegrass roots under Cd stress, as a constituent of the plant Cd defense strategy.

3.2. Oxidative stress and lipid peroxidation Antioxidant enzymes and lipid peroxidation/oxidative damage in ryegrass roots were quantitatively determined as root SOD, CAT, POD, and MDA concentrations after 28 d growth. Plant generated SOD, POD and CAT are involved in the conversion of ROS to less harmful daughter products (Gill and Tuteja, 2010), whereas MDA levels were monitored to signify lipid peroxidation levels. All of these (SOD, CAT, POD, and MDA) significantly increased (P < 0.01) with increasing Cd exposure without exogenous P input (Fig. 2). The highest SOD activity (Fig. 2A) was observed in ryegrass exposed to the highest Cd level (Cd4P0), which was 78.5 % higher than the level observed in plants grown in the absence of Cd (Cd0P0). Under the application of exogenous P, SOD activity increased in plants not exposed to Cd (i.e., Cd0 treatments). On the other hand, SOD activity declined in plants under Cd stress (i.e., Cd0.8 and Cd4 treatments) with exogenous P application compared to those without P input; the exogenous P dosage rate had little effect, i.e., low P (P-20) and high P (P80) treatments were associated with similar SOD activity. CAT activity (Fig. 2B) increased by 103.4 % and 211.1 % after exposure to Cd at concentrations of 0.8 and 4 mg/L (without P application), respectively. Exogenous P increased CAT activity in plants without Cd-stress (Cd-0), but decreased its activity in plants under Cdstress in comparison to Cd-stressed plants without exogenous P. Unlike SOD activity, CAT activity decreased more under high P (P-80) treatments than low P (P-20) treatments for plants subjected to either Cd0.8 or Cd4 treatments. POD activity (Fig. 2C) increased by 182.3 % and 336.6 % after exposure to Cd at concentrations of 0.8 and 4 mg/L (without P application), respectively. As with SOD and CAT activity, exogenous P increased POD activity in plants without Cd-stress (Cd-0), although this was not significant in the case of low P (P-20) treatments. For the low P (P-20) treatments, POD activity decreased by 40.1 % and by 9.7 % compared to P0 treatments under 0.8 mg/L Cd and 4 mg/L Cd, respectively. Under high P (P 80) treatment POD activity decreased by 11.1 % and 18.8 % compared to P0 treatments under 0.8 mg/L Cd and 4 mg/L Cd, respectively. Root MDA contents (Fig. 2D) were highest (452.15 nmol/g) in plants subjected to Cd4P0 treatments. Exogenous P in Cd-0 treatments increased MDA levels by ∼6 % as compared to Cd0P0 treatments, under both the low and high P dosage rates. Under Cd stress, high P addition (P80) significantly decreased the MDA content by 6 % and 17 % compared to Cd0.8P0 and Cd4P0 treatments, respectively. The overall relationship between lipid peroxidation (i.e., MDA content) and the ryegrass biomass is shown in Fig. 3. These results show that ryegrass biomass reduction may relate to Cd-induced lipid peroxidation (Ali et al., 2015). Moreover, the figure shows that exogenous P is associated with lower MDA levels and greater biomass, thereby indicating that P input reduced Cd induced oxidant activity.

3.4. Non-protein thiols (NPTs), glutathione (GSH), and phytochelatins (PCs) Plant non-protein thiols (NPTs) are shown to contribute to the detoxification of Cd (Zhang et al., 2013) through chelation reactions (Greger et al., 2016) involving GSH and PCs (Dai et al., 2017; Nishikawa et al., 2006). The observed NPT, GSH, and PCs levels within the ryegrass roots are presented in Fig. 5, all of which increased under Cd stress conditions. In comparison to plants not exposed to Cd or exogenous P (Cd0P0), plants exposed to low and high levels of Cd (Cd0.8P0 and Cd4P0, respectively) had 186.4 % and 482.3 % higher levels of NPT, 97.84 % and 431.1 % higher levels of GSH, and 165.3 % and 367.4 % higher levels of PCs, respectively. The exogenous P input significantly further increased NPT, GSH and PCs levels. Plants subjected to the highest amounts of exogenous P and Cd exposure (Cd4P80) had the highest contents of each of these Cd stress related constituents. Fig. 5 shows that with increasing Cd concentration, the [NPTs]:[Cd] ratio decreased (Fig. 5C), similar results were found for [GSH]:[Cd] (Fig. 5A) and [PCs]:[Cd] (Fig. 5B) ratios. The [PCs]:[Cd] ratio for the Cd0.8P0 and Cd4P0 treated plants (without exogenous P) was in the range of 4.11–5.83:1 (Fig. 5E), which is close to reported ratios for stable PCs-Cd chelates (i.e., ∼4:1) (Mendoza-Cózatl et al., 2010; Dorcak and Krężel, 2003). Under the same level of exogenous P input, [PCs]:[Cd] ratios in roots under low-Cd treatments (i.e., Cd0.8-P0, Cd0.8-P20 and Cd0.8-P80) were higher than those under high-Cd treatments (i.e., Cd4P0, Cd4P20 and Cd4P80). This suggests that exogenous P may be involved in Cd transport and sequestration. This was also observed in the cases of [GSH]:[Cd] and [NPTs]:[Cd]. This is attributed to greater abundance of non-protein thiols under heightened Cd stress. The ratios of [GSH] + [PCs]:[Cd] were found to be > 15 for all Cd-treatments, consistent with physiologically relevant levels for stable Cd chelate formation (i.e., > 10) (Mendoza-Cózatl et al., 2010; Dorcak and Krężel, 2003). By plotting the plant thiols content against Cd concentrations, notable relationships were determined between the amount of Cd uptaken in the root and the amount of NPTs (R2 = 0.914), GSH (R2 = 0.805) and PCs (R2 = 0.904) in proportion to the amount of exogenous P applied (Fig. 6). 4. Discussion

3.3. Amino acids

Mineral P fertilizer input can serve as both a source and remedy for Cd stress. Some P fertilizers contain Cd impurities, thereby serving as a source of P; however, exogenous P input tends to decrease Cd phytotoxicity to plants (Bolan et al., 2003a,b). In this study, exogenous P input significantly increased Cd levels in ryegrass roots and shoots grown under hydroponic conditions. Possible mechanisms for greater Cd uptake include: 1) P application increased the pectin and hemicellulose content of the root cell walls, which enhanced the Cd binding ability (Dai et al., 2018; Ye et al., 2012), 2) Cd formed chelates/precipitants with stable structures (such as, Cd3(PO4)2) (Seshadri et al., 2016; Zhou et al., 2017); 3) P and Cd co-sorbed as an ion pair (Seshadri et al., 2016; Bolan et al., 2013; Cunha and Nascimento, 2009). It should be noted that these mechanisms can occur simultaneously. At the same time, it was also found that exogenous P helped mitigate Cd stress and was associated with increased plant biomass. The role that exogenous P plays in ryegrass Cd detoxification is discussed below.

Proline and cysteine levels were determined in the ryegrass roots to show the ryegrass response to Cd stress (Fig. 4). Both amino acids (proline and cysteine) significantly (P < 0.05) increased in the roots of plants under Cd stress without P addition, with the highest levels observed in plants subjected to the highest amount of Cd (Cd-4). The data suggest that both of these amino acids participate in ryegrass response to Cd stress. Under the lower Cd exposure level (Cd0.8), the proline level decreased in plants treated with the low P dose (Cd0.8P20), however, this finding is not statistically significant. In plants grown under the higher Cd exposure level (Cd4P20), the proline level significantly increased by 9.1 %, compared to plants treated with Cd4P0. The higher exogenous P level, Cd0.8P80 and Cd4P80, increased proline contents by 17.2 % and 26.3 %, compared to Cd0.8P0 and Cd4P0 treatments, respectively. 7

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5. Conclusions and recommendations

Antioxidant enzymes SOD, CAT and POD were all found to be notably higher in ryegrass plants under Cd stress. This is attributed to the plant’s natural defense mechanism against Cd toxicity. The increased SOD activity relates to increased O2%– levels (Daud et al., 2013), as SOD is involved in the conversion of this species to H2O (Gill and Tuteja, 2010). CAT is a critical enzyme for detoxification of H2O2 to H2O and O2 (Daud et al., 2013), while POD is involved in minimizing Cd- induced H2O2 generation. Under increased P levels, SOD, CAT and POD activities in Cd-stressed plants decreased, signifying decreased oxidative stress. However, the overall enzyme activity was found to be insufficient to fully eliminate the oxidative stress caused by Cd exposure. This was evidenced by elevated MDA contents signifying lipid peroxidation. The effect of reduced Cd stress following exogenous application was shown in Fig. 3, which illustrated a clear trend between plant biomass and the MDA content of the plants (R2 > 0.95). Importantly, the MDA content significantly decreased and plant biomass increased in proportion to the amount of exogenous P applied. P is an important element in plant amino acid synthesis, and exogenous P application boosted the production of the amino acid proline in the ryegrass root tips. These amino acids important for maintaining water balance and cytoplasmic pH under Cd stress, as well as enhancing plant ROS scavenging ability (Zouari et al., 2016). This finding is notable because Dai et al. (2017) reported that Cd stress can lead to low osmotic potential and a water loss effect in plant cells. Hence, exogenous P may play an important role in ryegrass plants in stabilizing protein structures and regulating protein metabolism when under Cd stress. Moreover, proline acts as a molecular chaperone to protect protein structures by producing non-toxic Cd-proline chelates (Sharma et al., 1998). Chelation reactions play an important role in mitigating Cd stress (Sghayar et al., 2015). This mostly encompasses detoxification processes involving NPTs, GSH and PCs, owing to the high affinity between thiols and Cd. This study found that exogenous P significantly increased the plant synthesis of these substances (NPTs, GSH and PCs) (Fig. 4). Increased GSH and PCs levels are known to be favorable for the formation of Cd-PCs chelates (Gupta et al., 2010; Ye et al., 2016; Lou et al., 2017) in plants under Cd stress (Mendoza-Cózatl et al., 2010; Dai et al., 2017). In particular, it is understood that GSH participates in ascorbateGSH reactions that extend the capacity to synthesize PCs (May et al., 1998). Exogenous P input significantly increased the levels of Cd accumulated in the ryegrass roots and shoots. This is thought to be because Cd will mainly translocate in the form of Cd complexes (Vatamaniuk et al., 2000; Mendoza-Cozatl et al., 2005), which play important roles in ensuring the normal eco-physiological activities of the plant tissues under Cd toxicity (Dai et al., 2017; Hazrat et al., 2013). Links between Cd-induced stress, Cd transport, and exogenous P input, are suggested to relate to physiological responses (amino acid and antioxidant activity) that enhance toxicity defense and antioxidant capacity (Fig. 7). Among potential Cd complexes formed in the root, PCs-Cd chelates are considered most stable, being associated with a higher dissociation constants (Kd) (7.9 × 10−17 M ) than GSH-Cd chelates (3.16 × 10-11 M), Cys -Cd chelates (1.28 × 10-10 M), and phosphate-Cd compounds (2.51 × 10-6 M) (Mendoza-Cózatl et al., 2010; Sillen and Martell, 1964; Erk and Raspor, 1998). It is suggested that PCs-Cd and GSH-Cd play important roles in ensuring the normal physiological activities of ryegrass plant tissues under Cd stress (Dai et al., 2017; Hazrat et al., 2013). Importantly, notable relationships were determined between the amount of Cd uptaken in the root and the amount of nonprotein thiols (R2 = 0.914), glutathione (R2 = 0.805) and Phytochelatins (R2 = 0.904) in proportion to the amount of exogenous P applied (Fig. 6). In other words, exogenous P is associated with enhanced Cd chelation and translocation from root to shoot. This effect is suggested to enhance Cd defense, with NPTs in its major form as GSH and GSHderived PCs playing the key roles.

The present study explored Cd detoxification mechanisms in perennial ryegrass (Lolium perenne L.) using hydroponic experiments. The findings suggest that exogenous P may exert a protective effect in plants under Cd stress, being associated with reduced lipid peroxidation as evidenced by decreased malondialdehyde (MDA) levels. Antioxidant enzyme activities were also found to decrease with exogenous P input, suggesting enhanced protection from oxidative stress. Importantly, exogenously applied P was found to increase root Cd concentrations and the amount of PCs, GSH and NPTs was in proportion the amount of exogenous P. This finding is associated with enhanced Cd chelation, therefore, exogenous P may help mitigate Cd stress in ryegrass, with PCs-Cd and GSH-Cd chelates playing important roles. Further studies on Cd transport from root phloem to the xylem, and whether P contributes to transport of Cd between apoplast and the symplast, is warranted. Different soil types are known to alter P availability (Nobile et al., 2018), an evaluation exogenous P dynamics, lability, desorption kinetics and bioavailability (Almeida et al., 2018) to ryegrass under Cd stress in different soils is needed. Moreover, other soil constituents and amendments that affect Cd bioavailability (Shen et al., 2019a; O’Connor et al., 2018) should also be considered in the context of environmental remediation (O’Connor et al., 2019b; Shen et al., 2019b; Zhang et al., 2020; Shen et al., 2019c). Author contributions The original idea was conceived by DH and HJ; the manuscript was drafted by HJ, DO’C, and DH; figures, tables and draft discussions were prepared by HJ, SP, JZ; and the manuscript was revised by DH, DO’C, NB and JM. All authors have given approval to the manuscript. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgement This work was supported by the National Key Research and Development Program of China (Grant No. 2018YFC1801300). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.jhazmat.2019.121849. References Aebi, H., 1984. Catalase in vitro. Methods Enzymol. 105, 121–126. Ali, S., Chaudhary, A., Rizwan, M., Anwar, H.T., Adrees, M., Farid, M., Irshad, M.K., Hayat, T., Anjum, S.A., 2015. Alleviation of chromium toxicity by glycinebetaine is related to elevated antioxidant enzymes and suppressed chromium uptake and oxidative stress in wheat (Triticum aestivum L.). Environ. Sci. Pollut. Res. Int. 22, 10669–10678. Almeida, D.S., Menezes-Blackburn, D., Rocha, K.F., de Souza, M., Zhang, H., Haygarth, P.M., Rosolem, C.A., 2018. Can tropical grasses grown as cover crops improve soil phosphorus availability? Soil Use Manag. 34, 316–325. Bates, L.S., Waldren, R.P., Teare, I.D., 1973. Rapid determination of free proline for water-stress studies. Plant Soil 39, 205–207. Bhargava, P., Srivastava, A.K., Urmil, S., Rai, L.C., 2005. Phytochelatin plays a role in UVB tolerance in N2-fixing cyanobacterium Anabaena doliolum. J. Plant Physiol. 162, 1220–1225. Bolan, N.S., Adriano, D.C., Duraisamy, P., Mani, A., Arulmozhiselvan, K., 2003a. Immobilization and phytoavailability of cadmium in variable charge soils. I. Effect of phosphate addition. Plant Soil 250, 83–94. Bolan, N.S., Adriano, D.C., Naidu, R., 2003b. Role of phosphorus in (Im)mobilization and bioavailability of heavy metals in the soil-plant system. Rev. Environ. Contam. Toxicol. 177, 1–44.

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