Iron plaque formation in the roots of Pistia stratiotes L.: importance in phytoremediation of cadmium

Abstract Aquatic macrophytes play an important role in the removal of toxic metals from wastewater. Therefore, the induction of Fe plaque on the roots, and its consequences on Cd tolerance investigated in an aquatic macrophyte Pistia stratiotes L. The presence of Fe2+ ion but not Fe3+ resulted in Fe plaque formation. Induction of Fe plaque decreased Ca and increased K and Fe accumulations in the root. Plaque formed plants had accumulated less Cd until 50.0 µM CdCl2 treatments because plaque acted as a barrier to Cd exposure. However, at higher concentrations (500.0 µM CdCl2), plaque formed plants contained more Cd in the roots. Cadmium inducible ion leakage in the root and lowering of the photosynthetic pigment content were less in plants with a plaque. Stretching of aromatic carbonyl groups and alkyl groups among plaque formed plants upon Cd treatments indicated the putative role of phenolics in Cd detoxification.


Introduction
Mining, industrial operations, and heavy metal contaminated agrochemicals released toxic heavy metals into aquatic environment (Rai 2008;Sebastian and Prasad 2014;Wu et al. 2014). Apart from this, heavy metals get desorbed from the sediment into overlying water when there is a decrease in redox potential and pH (Sebastian and Prasad 2015a;Sebastian et al. 2015). More recently, constructed wetlands used for the cleanup of heavy metal polluted water bodies (Marchand et al. 2010;Hashim et al. 2011). Aquatic macrophytes are constantly used for phytoextraction, rhizofiltration, phytostabilization, and phytotransformation of toxic metals and metalloids in the constructed wetlands (Pilon-Smits 2005;Maleva et al. 2018). These studies highlight the importance of aquatic macrophytes for the phytoremediation of metal contaminated water.
The presence of excess Fe in the rhizosphere resulted in the formation of Fe plaque in plant roots (Longnecker and Welch 1990;Marquel et al. 2014). The oxidation of Fe 2þ to Fe 3þ occurred in the course of Fe plaque formation. The oxidation reactions of Fe occurred due to the release of oxygen or oxidants into the rhizosphere and prevalence of acidic pH, respectively (Sebastian and Prasad 2015a). Acidic pH caused the occurrence of more Fe 2þ in the rhizosphere (Guerinot 2007). Secondly, the submerged conditions preserved the occurrence of Fe as Fe 2þ because of the low oxygen content (Sebastian and Prasad 2015a). Thus, plants, which grow in acidic pH and submerged conditions, accumulated more Fe 2þ in the root. The excess Fe 2þ accumulation triggered Fenton's reactions in plants because of the redox active nature of Fe and resulted in oxidative stress (Guerinot 2007). But the presence of oxygen in the aerenchyma helps conversion of Fe 2þ to Fe 3þ containing oxides, and deposition of these oxides on the root surface as Fe plaque (Marquel et al. 2014;Sebastian and Prasad, 2015a). Thus, it is clear that chance of Fe plaque formation is very high in roots of plants having aerenchyma, and the plaque helps to protect plants from Fe toxicity in the course of submergence.
The formation of plaque on root surface enabled metal tolerance in plants (Yang and Ye 2009). The Fe plaque formation on the root surface of wetland plants prevented accumulation of metal pollutants (e.g., Zn, Cd, Mn, Cu) (Jiang et al. 2009;Sebastian and Prasad 2015a). Cadmium and Al contents in rice (Oryza sativa) root tissues significantly decreased with the formation of Fe plaque (Chen et al. 2006;Sebastian and Prasad 2016). Occasionally, the presence of Fe plaque on the roots served as a reserve of Fe for plant uptake and helps to survive from Fe deficiency (Sebastian and Prasad 2016).
Pistia stratiotes L. is extensively used for phytoremediation (Zayed et al. 1998;Das et al. 2014). This plant helps in the removal of several heavy metals such as Hg, Fe, Cu, Zn, Mn, Cr, and Pb from metal contaminated water (Miretzky et al. 2004). Aerenchyma present in the leaves and roots of Pistia plants helps floating of the plant in water bodies (Sundeep et al. 2015). Aerenchyma also increases the availability of oxygen in the submerged root zone where atmospheric oxygen is seldom available. Thus, the chances of Fe plaque formation are very high in the roots of Pistia plants. In this context, a study was proposed to explore Fe plaque formation in Pistia plants, and its consequences on Cd tolerance. Cadmium draws special attention because the release of Cd-contaminated water very often occurs from mines, smelters, pigment, and alloy industries, and from agriculture fields (Sebastian and Prasad 2014). In this study, an iron plaque was induced in Pistia plants in-vitro with the help of a suitable Fe source. The criteria of iron plaque formation and its impact on the cadmium accumulation in Pistia stratoites have been elaborated. It expedited importance of iron plaque formation in the alleviation of Cd stress, which can be of great advantage for phytoremediation.

Plant material
Pistia stratiotes L. (water lettuce) is a perennial monocotyledonous plant with thick, soft leaves that form a rosette. This plant is found in nearly all tropical and subtropical fresh waterways. Weedy plant growth has observed in contaminated water where there is the human loading of sewage or fertilizers. The plant floats on the surface of the water, and the roots are hanging submersed beneath floating leaves.
Pistia stratiotes L. collected from a lake located at Madinaguda, Hyderabad, Telangana, India (17 29 0 11.79 00 N, 78 20 0 39.94 00 E) ( Figure 1a). The pH and conductivity of the lake water were 7.5 and 2000 mS/cm, respectively (Figure 1b). Plant culture maintained at constant temperature (25.0 C) and light intensity (1400.0 mmol photons m À2 s À1 ) in a greenhouse, at University of Hyderabad. Recorded humidity was 45%-60% during the experimental period. The multiplication period of the plant was 25.0 days, and the Hoagland solution (25.0%) changed after two days of interval.

Hydroponics
Experimental plants were maintained in Hoagland nutrient solution (Smith et al. 1983). The nutrient solution contained macronutrients of 6.  experimental condition. All solutions prepared in Milli-Q water (Millipore quantum, Merck, USA). All the experiments were repeated twice within 70 days' time period.

Induction of iron plaque
Plaque formation was checked under varying conditions of Fe salts and pH in this study. The efficacy of different Fe salts to form Fe plaque near neutral pH (6.5) screened with additions of 200.0 mM of each FeSO 4 , Fe 2 (SO 4 ) 3 , FeCl 3 , Fe-EDTA, and C 6 H 5 FeO 7 in nutrient solution. The influence of pH on plaque formation also checked at pH 4.0 and pH 6.5, respectively, with FeSO 4 .

Cadmium treatments
Plants were subjected to Cd stress for studying the influence of Fe plaque on Cd stress tolerance. Plaque-induced in plants during growth in Hoagland solution (25.0%) contain FeSO 4 at pH 4.0. Plaque formed plants transferred to the 25.0% Hoagland media (pH 6.5) containing 5.0, 50.0, and 500.0 mM CdCl 2 , respectively, for Cd treatment. Cadmium treatment continued for 7 days. To visualize that plaque act as a barrier to contaminant uptake, plants having plaque grown in media containing 0.1% safranin. It assumed that if the plaque acts as a barrier to safranin, there will be more absorbance in the media at 540.0 nm, which is the absorbance peak of safranin.

Biomass estimation
Biomass is an indicator of metal tolerance. Therefore, the biomass of the experimental plants was measured with the help of a weight balance (Sartorius, Germany). For biomass analysis, plants were manually cleaned with milliQ water for three times, and the water remaining on plant surface removed with a blotting paper. The increase of biomass expressed as mg fresh weight.
Scanning electron microscope coupled with energy dispersive spectroscopy analysis Surface morphology and elemental composition of the plaque formed roots analyzed with the help of field-emission scanning electron microscope (Zeiss Merlin Compact, Germany) coupled to energy dispersive spectroscopy (Oxford-X-max, UK). The roots were washed in double MilliQ water and dried in a hot air oven. The dried plant material was gold coated for enhancing conductivity (Quorum-Q150T, UK). The SEM-EDS analyses were carried out at operating voltage of 15.0 KeV at a working distance of 10.0 mm with counts per seconds >1000.

Ion leakage in the roots
Membrane damage leads to release of ions from the cells and results in an increase of conductivity of the nutrient solution. Ion leakage was monitored by measuring the conductivity of nutrient solution using a digital conductivity meter after the harvest of plants (Digisun, Hyderabad). The conductivity of the nutrient solution expressed in micro Siemens per centimeter (mS/cm).

Estimation of malondialdehyde (MDA) for lipid peroxidation analysis
Membrane damage was analyzed by estimating the production of MDA from both Plaque and non-plaque formed tissue according to the method followed by Huang et al. 2005 with slight modifications. Briefly, 100.0 mg tissue from both leaves and roots homogenized with 1.0 mL 0.1% (w/v) Trichloroacetic acid (TCA) followed by centrifugation at 10,000.0 rpm for 10 min. The supernatant mixed with a solution containing 0.5% Trichlorobutyric acid in 10.0% TCA. The mixture boiled, and the supernatant collected after centrifugation at 10,000.0 rpm for 10 min. The absorbance of the supernatant measured at 532.0 and 600.0 nm, respectively, using a UV-visible spectrophotometer (Cintra5-GBC scientific, Australia). MDA present in the reaction mixture calculated using the extinction coefficient of 155.0 mM À1 cm À1 .

Estimation of phenolics
The total phenolic content determined as per Kaur and Kapoor, 2002. The dried root and the leaf (100.0 mg) were extracted in 2.0 mL of 80.0% methanol. The extract mixed with the Folin-Ciocalteau reagent at 1:2 ratio, and few drops of 1.0 M sodium carbonate added to the resultant mixture. The absorbance of the mixture measured at 650.0 nm using a UV-visible spectrophotometer after 1.0 h (Cintra 5 -GBC scientific, Australia). Amount of phenolics calculated from the gallic acid standard graph.

Fourier-transform infrared (FTIR) spectroscopy
Air-dried powder of plant tissues (10.0 mg) made into a pellet with potassium bromide, and the pellet loaded into FTIR spectrometer (Nicolet 380 FTIR, Thermo scientific, USA) at room temperature to find out the changes in chemical forms of compounds in plant material. Spectral wave numbers with ranges of 400-4000 cm À1 were recorded. The transmittance peak and stretches in transmittance peak in the infrared absorbance spectra correspond to the abundance of functional group and chemical changes, respectively.

Photosynthetic pigment analysis
Photosynthetic pigment analysis was performed on leaf discs (2 cm diameter). The discs were incubated overnight in DMSO-Acetone mixture. The absorbance of the extracts recorded at 663.0, 645.0, and 480.0 nm in a UV-visible spectrophotometer (Shimadzu UV-1800, Japan). The concentrations of pigments were calculated using Arnon's Equations (Arnon, 1949)

The metal content analysis in plant tissues
Plants manually cleaned with 0.1 M HCl and 0.5 M EDTA, and thereafter dried at 80.0 C in a hot air oven. Dried samples were acid digested using HNO 3 -HClO 4 (3:1) mixture, and the reaction continued until the acid mixture completely evaporates out. The white colored ash deposited on the bottom of the flask dissolved in 0.1 M HCl and subjected to poplar leaf NCSDC 73550 reference calibrated atomic absorption spectrophotometer (Sebastian and Prasad 2013) (GBC 932, Australia).

Statistical analysis
The results of various treatments analyzed with Duncan's multiple range test to understand statistical significance of plant growth responses under various treatments. The alphabets a, b, c, d, and e represent first, second, third, fourth, and fifth levels of statistical significance. All analyses were considered significant at P < .05.

Results and discussion
Iron plaque formation and nutrient accumulation in the roots of Pistia stratiotes Phytoremediation with aquatic macrophytes is attractive because of the rapid multiplication and easy harvest of the plants. Pistia stratiotes accumulate toxic heavy metals, and therefore strategies that enhance phytoremediation capacity of this plant are a matter of interest in the remediation of metal contaminated water bodies. Rapid multiplication of Pistia results in the formation of mats over the water bodies ( Figure 1c). The formation of mat blocks the entry of atmospheric oxygen to water and create anoxygenic conditions in the rhizosphere of Pistia plants. But the aerenchyma tissue allows the passage of atmospheric oxygen into the roots, and hence the rhizosphere of Pistia mat is oxygenic (Denisele et al. 2016) (Figure 1d). The dissolution of metals from sediments into water is a typical phenomenon in submerged fields such as habitat of Pistia plants (Crowe et al. 2007). Secondly, Fe 2þ is very common in water bodies during anoxia because of low redox potential. The release of oxygen into the rhizosphere or lowering of pH caused Fe plaque formation in the roots of wetland plants (Sebastian and Prasad 2015a). All the above studies pointed out that the chance of Fe plaque formation in the roots of Pistia stratiotes is very high.
Iron plaque formation in Pistia roots was tested with different Fe salts at a pH of 6.5 because of the close similarity to pH of natural water bodies. Addition of FeSO 4 resulted in black coloration on the root surface at pH 6.5 (Figure 2a, Supplementary data S1). A dark color change of the root indicated Fe plaque formation in aquatic plants (Louise and Peter 1996). The supply of ferric Fe salts such as ferric sulfate(Fe 2 (SO 4 ) 3 ), ferric chloride(FeCl 3 ), a ferric chelate of ethylenediaminetetraacetic acid (Fe-EDTA), and ferric citrate (C 6 H 5 FeO 7 ) did not induce plaque at pH 6.5 in 6.0 days (Supplementary data S2). The abundance of oxygen caused conversion of Fe 2þ to Fe 3þ oxides in the root, and the oxide got deposited on root surface (Sebastian and Prasad 2016). The similar result obtained in this study pointed out that oxidation of Fe 2þ caused induction of Fe plaque in Pistia. Secondly, it confirmed that rather than pH, the availability of Fe 2þ is the critical factor involved in Fe plaque formation in roots. There was no test conducted with ferric salts at lower pH because of the conversion of Fe 3þ into Fe 2þ under acidic pH. The extent of plaque deposition in rice roots increased with lowering of pH (Sebastian and Prasad 2016). However, it is contradictory with reports of Chen et al. 2006 where the pH had no significant effect on the formation of Fe plaque. Therefore, the study continued to reveal the influence of pH on Fe plaque formation with FeSO 4 . It was found that both the pH 4.0 and 6.5 induced Fe plaque in Pistia plants (Figure 2a, Supplementary data S1). However,  there existed two days delay to form Fe plaque at pH 6.5 compared with pH 4.0 (Supplementary data S3). This delay occurred because of the increase in reduction potential at high pH, which prevents oxidation of Fe 2þ .
The morphology of Fe plaque formed in the root was compared with that of roots without plaque (Figure 2b). The analysis found that plaque formed roots had a rough surface with small spikes of Fe deposits. This type of morphology is well known to increase surface area for adsorption of metal ions, which hinder uptake of nutrients. The EDS analysis showed that plants with plaque accumulate less Si, Cl, and Ca in the root (Figure 2c, Supplementary data S4-S6). This effect arises because plaque acted as a barrier, which hinders exposure of roots to nutrients. However, the induction of Fe plaque promoted accumulation of K and Fe (Figure 2c, Supplementary data S4-S6). More K accumulation occurred as a result of higher H þ -ATPase activity for counteracting phosphate deficiency due to plaque formation (Palmgren 2001;Yan et al. 2002). Iron plaque acted as a source of Fe, and this effect accounts for the increase in Fe content after the induction of Fe plaque. Thus, the Fe plaque formation in Pistia depends on the availability of Fe 2þ ion, and the plaque affects nutrient accumulation in plants.

Iron plaque conferred cadmium tolerance in Pistia stratiotes
Cadmium accumulation in plants retarded activity of photosynthesis and nitrogen fixation respectively (Sebastian and Prasad 2015b). These effects were the results of Cd-induced Fe deficiency related to oxidative stress. Plants exposed to Cd also had low photosynthetic pigments and biomass (Singh and Tuteja 2011). The recovery of plants from Cd stress reported in the course of Fe supplement (Sebastian and Prasad 2015b). Pistia plants with Fe plaque had more Cd tolerance. These plants had the healthy appearance and more gain in biomass (Figure 3a,b). The reasons for enhanced Cd tolerance with induction of plaque were both increase of Fe and decrease of Cd accumulation in plants (Figure 4a-d). The increase of Fe accumulation in plaque formed plants was $3-7 times in the roots and 16%-38.0% in the leaves, respectively (Figure 4a,b). The iron plaque has been reported to acting as a source of Fe and barrier of Cd in rice plants (Ye et al. 1998;Xu and Yu 2013;Sebastian and Prasad 2016). Hence, it concluded that more Fe accumulation in plaque-induced plants was the outcome of immediate access of Fe from the plaque. Cadmium accumulation decreased in the roots of plaque formed plants at 5.0 and 50.0 mM CdCl 2 treatments. However, the accumulation of Cd increased 14.0% in the roots of plaque formed plants at 500 mM CdCl 2 treatment (Figure 4c). However, Cd accumulation decreased (12.0%-21.0%) in the leaves of plaque formed plants (Figure 4d). The decrease in Cd accumulation was the result of blockage of contact of roots with Cd due to Fe plaque. By growing the plants in a media containing dye, it can visualize the evidence of plaque acting as a barrier on the root surfaces. The barrier activity of plaque in the root blocking entry of contaminants visualized with a decrease in plant uptake of safranin in this study (Supplementary data S7). For instance, the color of the safranin in the media where plaque formed plants grown was more than that of the non-plaque formed plants, indicating less adsorption of safranin. This result confers the possible role of plaque as a barrier on the plant root surfaces. However, comparatively more increase in Cd accumulation in root at 500.0 mM treatment could be the result of more vigor in the growth of plants compared with those plants without plaque. This finding supported with a higher rate of biomass production observed in plants with a plaque at 500. 0 mM CdCl 2 treatments (Figure 3b).
Cadmium toxicity caused membrane damage in plants (Sanita di Toppi and Gabbrielli 1999). Exposure to Cd caused accumulation of reactive oxygen species (ROS), and membrane damage in wheat (Ekmekci et al. 2008). Membrane damage resulted in the release of cellular ions and thereby increased the conductivity of a nutrient solution . However, plants with Fe plaque had less ion leakage compared with plants without a plaque in the course of Cd treatment (Figure 3c). Breakdown of the cell membrane leads to the production of MDA (Chaoui et al. 1997). In Pistia, Cd accumulation leads to increase in antioxidant production and MDA level (Li et al. 2013). A similar result was also observed in this study where MDA level increased with Cd treatments (Figure 5a,b). However, the MDA level in Fe plaque formed plants was significantly less compared with plants without Fe plaque. Thus, it confirmed that Fe plaque prevents Cd exposure and membrane damage in roots, and this account for lowering of MDA accumulation and ion leakage among plaque formed plants during Cd treatments.
Photosynthetic pigments considered as a biomarker of Cd toxicity (Ferhad et al. 2015). The decrease in chlorophyll during Cd treatment occurred due to oxidative stress (Guo et al. 2016). It was noticed that more chlorophyll and carotenoid present in the leaves of the plaque formed plants (Figure 6a-d). The increase in chlorophyll a and b was in the rage of 10.0%-100.0% (Figure 6a,b). Chlorophyll a is part of the reaction center of the photosystems, and therefore this pigment is critical for the harvest of light energy (Fromme and Grotjohann 2008). Therefore, it is predicted that plants with Fe plaque had better photosynthetic light harvest efficiency under Cd stress. Chlorophyll b and carotenoids are known as accessory pigments that help in the channeling of light energy to reaction center with the dissipation of excess light energy. The increase in carotenoid content was in the range of 14.0%-58.0% among the Fe plaque formed plants during Cd treatments (Figure 6d). More light harvest resulted in an increase of accessory pigments in plants (Trees et al. 2000). Therefore, the increase in accessory pigments observed in the study considered as a response to increase in light harvest due to more chlorophyll among plaque-induced plants. Total chlorophyll content in the plant was used as an indicator of Fe nutrition status of the plants (Terry and Low 1982;Rafael et al. 2013). Therefore, the higher total chlorophyll content among plaque formed plants indicates that Cd-induced Fe deficiency did not occur in plants with plaque ( Figure 6c). This finding also confirmed with more Fe accumulation observed in the leaves of plaque formed plants (Figure 4b).
Heavy metal stress-induced changes in metabolite profile of plants (Sebastian and Prasad 2014). Influences of plaque formation and Cd stress on metabolites profile were revealed through FTIR analysis in this study. Stretching of -CO and -C-H group were the noticeable changes during Cd stress in the leaves of plaque formed plants compared with control plants (Table 1, Supplementary data S8). This indicated putative role of phenolics containing an aromatic carbonyl group in Cd tolerance among plaque formed plants (Holser 2012). Accumulation of phenols and polyphenols are an indication of antioxidant activities to deduce the effect of Cd toxicity in both aquatic and terrestrial plants (Lavid et al. 2001;M arquez-Garc ıa et al. 2012). Cadmium exposure to plants involves the production of ROS, and phenolics acts as a scavenger ROS under Cd stress (Sytar et al. 2013). A significant difference in phenolics was noticed only at 500. 0 mM CdCl 2 treatment where plaque formed plants had 40. 0% and 36.0% increase of phenolics in roots and leaves respectively for defense against Cd-induced ROS (Supplementary data S9). This result confirmed the role of antioxidant activity of phenolics in Cd tolerance. At lower The alphabets a, b, c, d, and e represent first, second, third, fourth, and fifth levels of statistical significance. All analysis considered significant at P < 0.05. Cd concentration the phenolics helped to mitigate Cd stress by chelation because there occurred only stretching of the -OH functional group. The C-Cl stretch was observed only in the leaves of plaque formed plants. This effect was due to the counteraction of more Fe accumulated in the leaf (Wenrong et al. 2010). Similar findings noticed in the marine green macroalga Caulerpa lentillifera too (Pavasant et al. 2006 ). Stretching of ester group H-C ¼ O:C-H detected in the leaves of all plants during Cd stress. This change was the outcome of the interaction of Cd with ester-containing compounds in the cells.

Conclusions
Iron plaque formation in Pistia stratiotes was dependent on the presence of Fe 2þ . The acidic pH decreased the time duration required for plaque formation. Plaque formation decreased the accumulation of plant nutrients such as Ca and increased accumulation of Fe and K, respectively. Plants with plaque had Cd tolerance, and it helped to accumulate more Cd in the roots at higher concentration of Cd in the media. Both increases in Fe accumulation and a decrease in Cd accumulation resulted in Cd tolerance among plants with a plaque. Metabolites flux varies in response to Fe plaque formation especially those with aromatic carbonyl group and esters in the leaf. The study summarized that Pistia can accumulate high concentrations of Cd with the formation of iron plaque on its root surface. Therefore, Pistia plants with Fe plaque will help phytoremediation of heavily Cd-polluted water bodies such as mine leachate. This study also helps to elucidate the importance of aquatic macrophytes in facilitating phytoremediation for global sustainability and environmental pollution.