Probing the Role of the Chloroplasts in Heavy Metal Tolerance and Accumulation in Euglena gracilis

The E. gracilis Zm-strain lacking chloroplasts, characterized in this study, was compared with the earlier assessed wild type Z-strain to explore the role of chloroplasts in heavy metal accumulation and tolerance. Comparison of the minimum inhibitory concentration (MIC) values indicated that both strains tolerated similar concentrations of mercury (Hg) and lead (Pb), but cadmium (Cd) tolerance of the Z-strain was twice that of the Zm-strain. The ability of the Zm-strain to accumulate Hg was higher compared to the Z-strain, indicating the existence of a Hg transportation and accumulation mechanism not depending on the presence of chloroplasts. Transmission electron microscopy (TEM) showed maximum accumulation of Hg in the cytosol of the Zm-strain and highest accumulation of Cd in the chloroplasts of the Z-strain indicating a difference in the ability of the two strains to deposit heavy metals in the cell. The highly abundant heavy metal transporter MTP2 in the Z-strain may have a role in Cd transportation to the chloroplasts. A multidrug resistance-associated protein highly increased in abundance in the Zm-strain could be a potential Hg transporter to either cytosol or mitochondria. Overall, the chloroplasts appear to have major role in the tolerance and accumulation of Cd in E. gracilis.


Introduction
The use of heavy metals in industrial, medical, household, and agricultural products has led to an increase in the deposition and distribution of these metals in the environment arising serious concerns about their impact on living organisms [1,2]. Different approaches such as application of chemicals have been employed to remove heavy metals from the soil and water but most of these have proven inefficient or expensive. Additional problems are generated by large volumes of contaminated sludge requiring further processing [3]. On the other hand, plants, microorganisms, and microalgae have the ability to bioaccumulate heavy metals sustainably, thus reducing the burden imposed on landfill sites and water resources [4,5].
Phytochelatin-mediated heavy metal detoxification has been previously reported in plants, fungi, and algae [6]. In plants, low molecular weight heavy metals are first complexed with phytochelatins (LMW HM-PC), and transported into vacuoles for the later conversion into high molecular weight

Determination of Heavy Metal Bioaccumulation
The maximum capacity of the E. gracilis Zm-strain to bioaccumulate heavy metals was determined using microwave plasma atomic emission spectroscopy (4100 MP-AES, Agilent, USA). Cells were grown in a GNY medium [29] supplemented with either Cd (200 ppm), Pb (500 ppm) and Hg (50 ppm), selected from the MIC study. Samples were collected at different time points (day 2, 4, 6 and 7) of cultivation and heavy metal accumulation was determined as described previously [28].

Transmission Electron Microscopy (TEM)
Both the Z-and Zm-strains grown in a GNY medium supplemented with a particular heavy metal and controls without heavy metal addition were harvested at the log phase by centrifugation (500× g; 2 min) and washed twice with ddH 2 O. All chemicals used in TEM were purchased from ProSciTech, Australia. Primary fixation of the cells was performed using 2.5% (v/v) glutaraldehyde and 2.5% (v/v) paraformaldehyde for 2 h and secondary fixation was carried out using 2% (v/v) osmium tetroxide (OsO 4 ) for 1.5 h. The cells were dehydrated with graded ethanol solutions (50%, 70%, 90%, 95%, 100% v/v). After this, the cells were infiltrated with increasing concentrations of London resin (LR) white resin medium grade at 50%, 75% and finally 100% (v/v). Infiltration was carried out for 2 h for each concentration of LR white resin except the 100% LR white resin, for which the cells were incubated at RT (room temperature) overnight on a rotator. On the following day, the resin was replaced with fresh 100% LR white resin and the cells were incubated at RT for 3 h. The samples were then allowed to polymerize inside an oven at 60 • C for 48 h.
Ultrathin resin sections were prepared on Ultramicrotome Leica EM UC7 and the sections were stained serially with uranyl acetate and lead citrate. The sections were then studied on a Philips CM10 Transmission Electron Microscope. Control cells without metal treatment were compared against heavy metal treated samples to identify the location of heavy metal accumulation.

Extraction of Proteins and Peptides
Aliquots of the E. gracilis Zm-strain cultures grown with each heavy metal were collected at the maximum heavy metal accumulation time points as determined by MP-AES (Section 3.2) and proteins and peptides were extracted as described earlier for the Z-strain [28]. All experiments were carried out in biological triplicates.

Mass Spectrometry Analysis
A TripleTOF 5600 mass spectrometer was used to analyze the peptides from the proteins extracted from the Zm-strain treated with heavy metals. All experiments were carried out in biological triplicates ( Figure 1). The SWATH experiment was carried out in two-steps: information-dependent acquisition-mass spectrometry (IDA-MS) was used to generate the ion library and SWATH-MS was applied for label-free quantification ( Figure 1).
Microorganisms 2019, 7, x; doi: FOR PEER REVIEW www.mdpi.com/journal/microorganisms Figure 1. Workflow of SWATH-MS. The empirical ion/spectral library was generated by IDA-MS. SWATH acquisition enabled comparison of the spectra to the spectral library and label-free relative quantification of proteins. The relative abundance of proteins was compared between heavy metal treated and untreated control samples. Statistical analysis was performed as stated in Section 2.9 Figure 1. Workflow of SWATH-MS. The empirical ion/spectral library was generated by IDA-MS. SWATH acquisition enabled comparison of the spectra to the spectral library and label-free relative quantification of proteins. The relative abundance of proteins was compared between heavy metal treated and untreated control samples. Statistical analysis was performed as stated in Section 2.9. Extracted peptides were fractionated with modifications as described [28]. The generated ion library was searched against the Euglena non-redundant protein database available at the John Innes Centre website (http://jicbio.nbi.ac.uk/euglena/), acquired in September 2016. In the search parameter, carbamidomethylation of cysteine residues was used as a fixed modification. Candidate proteins were identified using the following criteria: Unused Score > 2 (signifying 99% confidence level) and global peptide false discovery rate (FDR) < 1%.

SWATH-MS Data Processing and Statistical Analysis
SWATH mass spectrometry and data processing were performed as described previously [28]. A complete SWATH-MS workflow is presented in Figure 1. Peptide extraction, SWATH-MS, and data processing have been carried out with the Z-strain previously [28] and were used here for comparison with the corresponding data obtained from the Zm-strain in this study. Proteins with relative expression fold-changes of ±1.5 and p-value < 0.05 were considered statistically significantly altered in expression between respective conditions. Proteomic survey will reveal the proteins involved in heavy metal tolerance and accumulation and the role of chloroplasts and chloroplast-related proteins in heavy metal sequestration and transportation into chloroplasts.

Functional Annotation
The Blast2GO software was used to annotate the proteins [28]. Sequences obtained from the Euglena non-redundant proteins database were matched with the UniProtKB/Swiss-Prot database with an E-value cut-off of 1e −10 . Gene Ontology (GO) information of proteins was derived using the UniProt database.

Fluorescence Microscopy of the E. gracilis Strains
The loss of chloroplasts in the Zm-strain was confirmed with fluorescence microscopy ( Figure 2) and further by TEM (Section 3.3). The wild type Z-strain had a bright red fluorescence brought about by the chlorophyll in the chloroplasts, however, the Zm-strain lacked the red fluorescence ( Figure 2). Streptomycin treatment can eliminate chloroplasts from Euglena without significantly inhibiting cell division or the viability of the cells [30,31].

Creation of a Peptide Ion Library (IDA-MS)
Extracted peptides were fractionated with modifications as described [28]. The generated ion library was searched against the Euglena non-redundant protein database available at the John Innes Centre website (http://jicbio.nbi.ac.uk/euglena/), acquired in September 2016. In the search parameter, carbamidomethylation of cysteine residues was used as a fixed modification. Candidate proteins were identified using the following criteria: Unused Score > 2 (signifying 99% confidence level) and global peptide false discovery rate (FDR) < 1%.

SWATH-MS Data Processing and Statistical Analysis
SWATH mass spectrometry and data processing were performed as described previously [28]. A complete SWATH-MS workflow is presented in Figure 1. Peptide extraction, SWATH-MS, and data processing have been carried out with the Z-strain previously [28] and were used here for comparison with the corresponding data obtained from the Zm-strain in this study. Proteins with relative expression fold-changes of ±1.5 and p-value < 0.05 were considered statistically significantly altered in expression between respective conditions. Proteomic survey will reveal the proteins involved in heavy metal tolerance and accumulation and the role of chloroplasts and chloroplastrelated proteins in heavy metal sequestration and transportation into chloroplasts.

Functional Annotation
The Blast2GO software was used to annotate the proteins [28]. Sequences obtained from the Euglena non-redundant proteins database were matched with the UniProtKB/Swiss-Prot database with an E-value cut-off of 1e −10 . Gene Ontology (GO) information of proteins was derived using the UniProt database.

Fluorescence Microscopy of the E. gracilis Strains
The loss of chloroplasts in the Zm-strain was confirmed with fluorescence microscopy ( Figure  2) and further by TEM (Section 3.3). The wild type Z-strain had a bright red fluorescence brought about by the chlorophyll in the chloroplasts, however, the Zm-strain lacked the red fluorescence ( Figure 2). Streptomycin treatment can eliminate chloroplasts from Euglena without significantly inhibiting cell division or the viability of the cells [30,31].

Minimum Inhibitory Concentration (MIC)
The MIC of the three heavy metals for the Zm-strain varied considerably. The lowest MIC (90 ppm) was observed with Hg, which, however is higher than the Hg MIC of 20 ppm assigned for the freshwater microalgae Phormidium ambiguum, Pseudochlorococcum typicum and Scenedesmus quadricauda var quadrispina [32]. Hg tolerance of 71 ppm has been recorded for brown algae [33]. Although the E. gracilis Zm-strain lacking chloroplasts had a slightly higher Hg tolerance when compared to that of the Z-strain (80 ppm) [28], the relatively small difference and non-existing information available for other algae do not allow drawing sound conclusions for the possible role of the chloroplasts in Hg tolerance in algae. The MICs of the Z-strain [28] and Zm-strain ( Figure 3) were brought about by the difference in the cellular ability of the strains to withstand the toxicity of each heavy metal since the growth rates between the untreated Z-and Zm-strains were not significantly different (p > 0.05) (Supplementary Figure S1).

Minimum Inhibitory Concentration (MIC)
The MIC of the three heavy metals for the Zm-strain varied considerably. The lowest MIC (90 ppm) was observed with Hg, which, however is higher than the Hg MIC of 20 ppm assigned for the freshwater microalgae Phormidium ambiguum, Pseudochlorococcum typicum and Scenedesmus quadricauda var quadrispina [32]. Hg tolerance of 71 ppm has been recorded for brown algae [33]. Although the E. gracilis Zm-strain lacking chloroplasts had a slightly higher Hg tolerance when compared to that of the Z-strain (80 ppm) [28], the relatively small difference and non-existing information available for other algae do not allow drawing sound conclusions for the possible role of the chloroplasts in Hg tolerance in algae. The MICs of the Z-strain [28] and Zm-strain ( Figure 3) were brought about by the difference in the cellular ability of the strains to withstand the toxicity of each heavy metal since the growth rates between the untreated Z-and Zm-strains were not significantly different (p > 0.05) (Supplementary Figure S1). The MIC for Pb in the Zm-strain was 9000 ppm, which is about the same as that of the Z-strain from our previous study [28]. Thus, similarly to Hg tolerance, chloroplasts may not be involved in the tolerance to Pb. Differently to the about similar Hg and Pb MIC values established for the two strains, the MIC for Cd with the Zm-strain lacking chloroplasts was 300 ppm, which is about half of the 600 ppm determined earlier for the Z-strain containing chloroplasts [28]. A lower tolerance to Cd The MIC for Pb in the Zm-strain was 9000 ppm, which is about the same as that of the Z-strain from our previous study [28]. Thus, similarly to Hg tolerance, chloroplasts may not be involved in the tolerance to Pb. Differently to the about similar Hg and Pb MIC values established for the two strains, the MIC for Cd with the Zm-strain lacking chloroplasts was 300 ppm, which is about half of the 600 ppm determined earlier for the Z-strain containing chloroplasts [28]. A lower tolerance to Cd in the Zm-strain lacking chloroplasts may indicate a role for these organelles in Cd tolerance in Euglena.

Evaluation of the Heavy Metal Accumulation
MP-AES analysis of the Zm-strain showed that it could accumulate Hg in higher amounts, 6.54 mg/g dry weight (DW) at day 2 compared to Cd and Pb. The maximum Cd accumulation was recorded on day 6 (4.14 mg/g DW) while only 1.93 mg/g DW accumulation of Pb was observed on day 4.
Although Hg is more toxic than Cd and Pb, the order of heavy metal bioaccumulation by the Zm-strain was Hg > Cd > Pb ( Figure 4). This suggests that the Zm-strain has an ability to sequester Hg into a non-toxic form even in the absence of chloroplasts. In our previous study, the average Hg accumulation from day 2 to day 7 by the wild type Z-strain was 3.77 mg/g (DW) [28], which is comparatively lower than that of the Zm-strain (5.84 mg/g, DW) under similar conditions. in the Zm-strain lacking chloroplasts may indicate a role for these organelles in Cd tolerance in Euglena.

Evaluation of the Heavy Metal Accumulation
MP-AES analysis of the Zm-strain showed that it could accumulate Hg in higher amounts, 6.54 mg/g dry weight (DW) at day 2 compared to Cd and Pb. The maximum Cd accumulation was recorded on day 6 (4.14 mg/g DW) while only 1.93 mg/g DW accumulation of Pb was observed on day 4.
Although Hg is more toxic than Cd and Pb, the order of heavy metal bioaccumulation by the Zm-strain was Hg > Cd > Pb ( Figure 4). This suggests that the Zm-strain has an ability to sequester Hg into a non-toxic form even in the absence of chloroplasts. In our previous study, the average Hg accumulation from day 2 to day 7 by the wild type Z-strain was 3.77 mg/g (DW) [28], which is comparatively lower than that of the Zm-strain (5.84 mg/g, DW) under similar conditions. The average Cd accumulation by the Zm-strain was 3.5 mg/g (DW), which is lower than in the Z-strain (5.23 mg/g of DW) [28]. This suggests that chloroplasts may contribute to Cd bioaccumulation. Cd accumulation in the chloroplasts has been reported for Hordeum vulgare, Zea mays [34] and a few other plants [35]. Although some studies are currently underway to understand the role of chloroplasts in Cd accumulation, most studies have been dedicated to reveal the effects of Cd toxicity on inhibition of photosynthesis [36,37].
The least amount of heavy metal accumulation by the Zm-strain was for Pb, which remained almost constant between day 2 and 7. MIC results indicated that Pb was also the least toxic of the three metals tested ( Figure 4). The average Pb bioaccumulation was 1.62 mg/g (DW), which is similar The average Cd accumulation by the Zm-strain was 3.5 mg/g (DW), which is lower than in the Z-strain (5.23 mg/g of DW) [28]. This suggests that chloroplasts may contribute to Cd bioaccumulation. Cd accumulation in the chloroplasts has been reported for Hordeum vulgare, Zea mays [34] and a few other plants [35]. Although some studies are currently underway to understand the role of chloroplasts in Cd accumulation, most studies have been dedicated to reveal the effects of Cd toxicity on inhibition of photosynthesis [36,37].
The least amount of heavy metal accumulation by the Zm-strain was for Pb, which remained almost constant between day 2 and 7. MIC results indicated that Pb was also the least toxic of the three metals tested (Figure 4). The average Pb bioaccumulation was 1.62 mg/g (DW), which is similar to the 1.82 mg/g (DW) by the Z-strain [28]. As the difference between the accumulation of Pb by chloroplast and non-chloroplast-containing strains was not significant, chloroplasts may not greatly contribute to Pb bioaccumulation. Despite its lower toxicity, Pb has been reported to cause inhibition of the photosystem in plants, leading to swollen and deformed chloroplasts [38,39]. In the alga Chara vulgaris, Pb impaired the membrane system of chloroplasts and destroyed the thylakoid structure [40]. However, Pb does not appear to impair the chloroplasts of E. gracilis (Figure 5g). to the 1.82 mg/g (DW) by the Z-strain [28]. As the difference between the accumulation of Pb by chloroplast and non-chloroplast-containing strains was not significant, chloroplasts may not greatly contribute to Pb bioaccumulation. Despite its lower toxicity, Pb has been reported to cause inhibition of the photosystem in plants, leading to swollen and deformed chloroplasts [38,39]. In the alga Chara vulgaris, Pb impaired the membrane system of chloroplasts and destroyed the thylakoid structure [40]. However, Pb does not appear to impair the chloroplasts of E. gracilis (Figure 5g). : Zstrain exposed to Hg; (d): Zm-strain exposed to Hg; (e): Z-strain exposed to Cd; (f): Zm-strain exposed to Cd; (g): Z-strain exposed to Pb; (h): Zm-strain exposed to Pb. P: Paramylon; N; nucleus, M; mitochondria, Ch; chloroplast. Black dots represent heavy metal inclusion and are marked by a red arrow. The 4 µm bar corresponds to all images. : Z-strain exposed to Hg; (d): Zm-strain exposed to Hg; (e): Z-strain exposed to Cd; (f): Zm-strain exposed to Cd; (g): Z-strain exposed to Pb; (h): Zm-strain exposed to Pb. P: Paramylon; N; nucleus, M; mitochondria, Ch; chloroplast. Black dots represent heavy metal inclusion and are marked by a red arrow. The 4 µm bar corresponds to all images.

Visualization of Heavy Metal Bioaccumulation
TEM was used to locate heavy metal bioaccumulation in different cellular organelles in the two E. gracilis strains. Treated cells were compared against control cells without heavy metal treatment. Ultrathin sections revealed the internal structure of the cell more clearly in the control cells, while those treated with heavy metals seemed to have lost some cellular integrity. TEM images in Figure 5 show recognizable cellular structures such as nuclei (N), mitochondria (M) and chloroplasts (Ch), and the "ridges and grooves" pattern of the pellicle (cell wall). As expected, there were no chloroplasts in the Zm-strain. The accumulated heavy metals appeared as small black dots while aggregated paramylon granules appeared as white round and oval structures. Euglena does not have a designated reservoir organelle similar to vacuoles in plants [41].
The chloroplasts of E. gracilis are generally spindle-shaped [42]. In the Z-strain treated with Pb, the long spindle-shaped chloroplasts were clearly visible (Figure 5g) while most of the chloroplast structures were damaged in the Hg-treated Z-strain cells, probably because of its toxicity (Figure 5c). The shape of the chloroplasts in the Cd-treated cells of the Z-strain were affected (not spindle shape), probably due to Cd bioaccumulation and toxicity (Figure 5e). The Zm-strain lacked visible chloroplasts.
In the Z-strain, accumulation of Hg took place in the cytosol and mitochondria ( Figure 5c); a similar deposition pattern was also observed for the Zm-strain (Figure 5d). However, it appears that the number of black dots representing Hg accumulation was higher in the Zm-strain than in the Z-strain. This result correlates with the fact that the Zm-strain was able to accumulate more Hg compared to the Z-strain (Figure 4) [28]. This also suggests that chloroplasts do not play a major role in Hg accumulation or the chloroplasts in the Z-strain were damaged by Hg resulting in a decrease in their ability to take up the metal. Damage to the chloroplast by mercury has been reported previously in plants and the green alga Chara vulgaris [40,43].
Cd accumulation mostly occurred in the chloroplasts in the Z-strain, although Cd accumulation was also found in the cytosol and mitochondria (Figure 5e). In the Zm-strain, Cd deposition was seen mostly in the cytosol (Figure 5f). The Z-strain accumulated more Cd than the Zm-strain which suggests that chloroplasts may have a role in Cd accumulation in the Z-strain. The maximum bioaccumulation was observed during Cd treatment of the Z-strain, suggesting an established mechanism to sequester large amounts of Cd in the chloroplasts, rendering this strain a Cd hyperaccumulator [28].
Pb accumulation was the least among the heavy metals studied here and is consistent with Pb accumulation shown in Figure 4. Pb accumulation was distributed fairly evenly in the chloroplasts and cytosol of the Z-strain (Figure 5g). The Zm-strain was found to accumulate Pb in the cytosol and mitochondria. Pb accumulation may have been assisted by chloroplasts in the Z-strain, as some black dots were observed in these organelles although both the Z-and Zm-strains had an almost similar ability to accumulate Pb. Chloroplasts seem to be intact and not deformed by Pb compared to the negative effect of Hg, which highlights the different effects of these heavy metals on cellular organelles of E. gracilis.
The preliminary data obtained from MIC studies, metal accumulation and heavy metal localization revealed differences in the ability and mode of the two strains to handle Hg, Pb, and Cd.

Proteome Profiling of Zm-Strain in Response to Heavy Metals Using SWATH-MS
Differentially abundant proteins during heavy metal treatment provide a basis to understand the molecular mechanism of heavy metal tolerance and bioaccumulation. The differentially abundant proteins in the Zm-strain exposed to heavy metals were compared to the control without heavy metal exposure. The differentially abundant proteins of the previously assessed Z-strain [28] and the Zmstrain identified in this study were also compared. Through a SWATH-MS analysis, a total of 5325 proteins with FDR < 1% were identified across the strains and heavy metal treatments, and 4493 proteins were quantified.
Proteome profile of the E. gracilis Zm-strain was significantly changed after exposure to the heavy metals Cd, Pb and Hg when compared to the untreated control of the same strain (Table 1). The number of differentially abundant proteins in the Hg-treated Zm-strain was 141, which is higher than the Z-strain (76 proteins) [1]. Despite the high level of toxicity of Hg compared to the other two heavy metals, the Zm-strain was able to accumulate large quantities of Hg (5.84 mg/g, DW). A larger number of differentially abundant proteins resulting from Hg exposure indicated a substantial involvement of proteins to either defend the Hg toxicity or regulate the cellular machinery to accumulate it for further detoxification. During the Cd treatment, 960 proteins were increased in abundance (FC > 1.5) in the Z-strain [28], compared to just 100 proteins in the Zm-strain. This vast difference in the number of differentially abundant proteins indicates that the chloroplasts in the Z-strain may play a major role in Cd accumulation and detoxification. Cd accumulation by the Z-strain was the highest among the heavy metals studied here and was also higher than that of the Zm-strain. Pb exposure returned a similar number of differentially abundant proteins for both strains, which is reflective of the similar MIC and accumulation patterns ( Figure 6).  The number of differentially abundant proteins in the Hg-treated Zm-strain was 141, which is higher than the Z-strain (76 proteins) [1]. Despite the high level of toxicity of Hg compared to the other two heavy metals, the Zm-strain was able to accumulate large quantities of Hg (5.84 mg/g, DW). A larger number of differentially abundant proteins resulting from Hg exposure indicated a substantial involvement of proteins to either defend the Hg toxicity or regulate the cellular machinery to accumulate it for further detoxification. During the Cd treatment, 960 proteins were increased in abundance (FC > 1.5) in the Z-strain [28], compared to just 100 proteins in the Zm-strain. This vast difference in the number of differentially abundant proteins indicates that the chloroplasts in the Zstrain may play a major role in Cd accumulation and detoxification. Cd accumulation by the Z-strain was the highest among the heavy metals studied here and was also higher than that of the Zm-strain. Pb exposure returned a similar number of differentially abundant proteins for both strains, which is reflective of the similar MIC and accumulation patterns ( Figure 6).
The differentially abundant proteins between the two strains were compared to identify whether common or unique proteins were involved in heavy metal tolerance and accumulation ( Figure 6). Among the 213 differentially abundant proteins during Hg treatment, only 4 proteins (1.87%) were common to both strains, while for the Cd and Pb treatments 26 proteins (2.51%) and 11 proteins (3.38 %) out of a total of 1034 and 325 differentially abundant proteins were common, respectively. This small percentage of common differentially abundant proteins between the strains suggests that strains with and without chloroplasts have different mechanisms for heavy metal tolerance and accumulation. The complete list of differentially abundant proteins common to both strains is shown in Supplementary Table S1.

Gene Ontology (GO) Annotation
The differentially abundant proteins from the Hg, Cd and Pb treatments were categorized by their GO annotations using the UniProt database and plotted collectively (Figure 7). The differentially abundant proteins between the two strains were compared to identify whether common or unique proteins were involved in heavy metal tolerance and accumulation ( Figure 6).
Among the 213 differentially abundant proteins during Hg treatment, only 4 proteins (1.87%) were common to both strains, while for the Cd and Pb treatments 26 proteins (2.51%) and 11 proteins (3.38 %) out of a total of 1034 and 325 differentially abundant proteins were common, respectively. This small percentage of common differentially abundant proteins between the strains suggests that strains with and without chloroplasts have different mechanisms for heavy metal tolerance and accumulation. The complete list of differentially abundant proteins common to both strains is shown in Supplementary Table S1.

Gene Ontology (GO) Annotation
The differentially abundant proteins from the Hg, Cd and Pb treatments were categorized by their GO annotations using the UniProt database and plotted collectively (Figure 7). The number of differentially abundant proteins in the categories of "metal ion binding" and "transporter activity" were about three times higher in the Z-strain compared to the Zm-strain. The results also revealed that in all subcategories of the set "Cellular components", the Z-strain showed an extensive response towards heavy metal exposure. The Z-strain appeared metabolically very active, most probably because of the presence of chloroplasts and performing photosynthesis. The number of differentially abundant proteins in the Z-strain were higher in both the cytosol and The number of differentially abundant proteins in the categories of "metal ion binding" and "transporter activity" were about three times higher in the Z-strain compared to the Zm-strain. The results also revealed that in all subcategories of the set "Cellular components", the Z-strain showed an extensive response towards heavy metal exposure. The Z-strain appeared metabolically very active, most probably because of the presence of chloroplasts and performing photosynthesis. The number of differentially abundant proteins in the Z-strain were higher in both the cytosol and intracellular organelles. As expected, no chloroplast-related proteins were found in the Zm-strain. In the set "Biological process", the Z-strain was more responsive in the subcategories of "cellular" and "metabolic process" during exposure to heavy metals. This suggests that the Z-strain was more reactive towards external stimuli, such as heavy metal toxicity, producing stress responses during exposure. To further understand the ability of the strains to cope with metal toxicity, the proteins increased in abundance in each type of metal treatment were compared (Figure 8).
Microorganisms 2019, 7, x FOR PEER REVIEW 9 of 18 intracellular organelles. As expected, no chloroplast-related proteins were found in the Zm-strain. In the set "Biological process", the Z-strain was more responsive in the subcategories of "cellular" and "metabolic process" during exposure to heavy metals. This suggests that the Z-strain was more reactive towards external stimuli, such as heavy metal toxicity, producing stress responses during exposure. To further understand the ability of the strains to cope with metal toxicity, the proteins increased in abundance in each type of metal treatment were compared (Figure 8). The proteins increased in abundance, which may have a role in heavy metal bioaccumulation were categorized into different sets. The proteins increased in abundance in the Zm-strain are listed in Supplementary Table S2 and the data of proteins increased in abundance in the Z-strain was sourced from our previous study [28]. The analysis showed that during the Hg treatment, the number of proteins involved in the primary metabolic processes was lower in the Z-strain compared to the Zm-strain. Heavy metal ions can disrupt the primary metabolic function in several ways such as by damaging organelles or by replacing the essential metals in metalloproteins, particularly those that carry out enzymatic reactions. A drastic change in the proteome profile of the Z-strain treated with Cd compared to the Zm-strain was observed ( Figure 8). This provides further evidence that the chloroplasts in E. gracilis assist to develop high tolerance against Cd and may have a proper mechanism to accumulate Cd.
The induction of metal ion binding proteins occurs in conjunction with various metals and the active inducers are Cd, Zn, Pb, silver (Ag) and antimony (Sb) [44]. In our study, the metal-binding proteins were highly abundant in the Z-strain during Cd exposure. Hg and Pb exposure resulted in very few changes in the abundance of metal-binding proteins in both strains. Metal-binding proteins have specific binding abilities to different metals depending on the organism and strain. For instance, the Zm-strain lacks chloroplasts and its ability to bind and accumulate Cd was lower compared to the Z-strain. In addition, the Zm-strain had more specificity for Hg than Cd. Metal-binding proteins also play an important role in the intracellular trafficking of metal ions. Metal-binding proteins such as glutathione synthetase, second enzyme in the glutathione (GSH) biosynthesis pathway, was increased by 14-fold in the Cd-treated Z-strain but not in the Hg and Pb-treated cells (Supplementary  Table S3). Glutathione is crucial for heavy metal chelation and subsequent bioaccumulation in intracellular organelles. The number of several transition metal ion binding proteins was significantly enhanced during Cd treatment of the Z-strain. The increase in the GSH during Cd treatment in the The proteins increased in abundance, which may have a role in heavy metal bioaccumulation were categorized into different sets. The proteins increased in abundance in the Zm-strain are listed in Supplementary Table S2 and the data of proteins increased in abundance in the Z-strain was sourced from our previous study [28]. The analysis showed that during the Hg treatment, the number of proteins involved in the primary metabolic processes was lower in the Z-strain compared to the Zm-strain. Heavy metal ions can disrupt the primary metabolic function in several ways such as by damaging organelles or by replacing the essential metals in metalloproteins, particularly those that carry out enzymatic reactions. A drastic change in the proteome profile of the Z-strain treated with Cd compared to the Zm-strain was observed ( Figure 8). This provides further evidence that the chloroplasts in E. gracilis assist to develop high tolerance against Cd and may have a proper mechanism to accumulate Cd.
The induction of metal ion binding proteins occurs in conjunction with various metals and the active inducers are Cd, Zn, Pb, silver (Ag) and antimony (Sb) [44]. In our study, the metal-binding proteins were highly abundant in the Z-strain during Cd exposure. Hg and Pb exposure resulted in very few changes in the abundance of metal-binding proteins in both strains. Metal-binding proteins have specific binding abilities to different metals depending on the organism and strain. For instance, the Zm-strain lacks chloroplasts and its ability to bind and accumulate Cd was lower compared to the Z-strain. In addition, the Zm-strain had more specificity for Hg than Cd. Metal-binding proteins also play an important role in the intracellular trafficking of metal ions. Metal-binding proteins such as glutathione synthetase, second enzyme in the glutathione (GSH) biosynthesis pathway, was increased by 14-fold in the Cd-treated Z-strain but not in the Hg and Pb-treated cells (Supplementary Table S3). Glutathione is crucial for heavy metal chelation and subsequent bioaccumulation in intracellular organelles. The number of several transition metal ion binding proteins was significantly enhanced during Cd treatment of the Z-strain. The increase in the GSH during Cd treatment in the chloroplast containing strain indicates the cellular ability to accumulate them in the chloroplast as shown by TEM studies (Figure 5e) and sequester them to non-toxic form.
Damage on DNA caused by heavy metals includes breaks in the double-stranded molecules as well as leads to inhibition of some of the critical proteins of the DNA repair pathway [45]. The level of DNA damage depends on the toxicity of the respective heavy metal and its concentration. Most importantly, the cellular ability to initiate the response towards DNA repair and the choice of construction or repair pathway determine the cellular ability to accumulate heavy metals [45]. In our study, five proteins responsible for initiating DNA repair were highly abundant in only the chloroplast containing Z-strain during Cd treatment compared to none in the non-chloroplast Zm-strain (Figure 8). This may indicate the ability of the chloroplast containing strain to bioaccumulate and sequester Cd to protect against DNA damage.

Transporters
Transporters have a significant role in metal homeostasis. There were approximately five times more transporter proteins of which relative abundance was increased during heavy metal treatments in the Z-strain than in the Zm-strain ( Table 2) and most of the changes in their abundance in the Z-strain were caused by Cd treatment. Various heavy metal transporters have been characterized in plants [46,47] and yeast [48,49] and green alga Chlamydomonas [14,50]. The highly abundant transporters in the chloroplast and non-chloroplast containing strains were different except the ABC transporter. Transporters in the Zm-strain may have a role in the intracellular trafficking of heavy metals and in the absence of chloroplasts, the most likely destination of heavy metal deposition would be either the mitochondria or cytosol. Two transporters were higher in abundance during Hg treatment of the Zm-strain (Table 2), including the copper-transporting ATPase RAN1. Copper-ATPases are crucial for maintaining metal homeostasis [51]. A transmembrane transporter multidrug resistance-associated protein member 2 (MRP2) was increased by 11.82-fold in the Zm-strain. MRP2 is involved in the transport of heavy metal conjugates into cellular organelles from the cytosol via ATP-dependent export pumps [52]. This transporter may be involved in Hg transport from the cytosol to mitochondria in the Zm-strain. Moreover, the ABC transporter G family member 36, which has been characterized as a probable efflux pump of heavy metal ions [53], was also higher in abundance in the Cd treated Zm-strain. Overexpression of this transporter has made plants more resistant to heavy metals [53]. The Zm-strain may thus have also used this transporter to efflux the Cd and Pb out of the cells. In the Z-strain, the abundance of transporter proteins was most increased after Cd-treatment compared to other heavy metals whereas in the Zm-strain Hg brought the highest increase in the abundance of transporter proteins ( Figure 8; Table 2). The increase in abundance of different types and numbers of transporter proteins in the two strains highlights the difference in the molecular mechanism of chloroplast and non-chloroplast strains to tolerate or accumulate various heavy metals. Transporters that play a role in intracellular metal transportation were found to be highly abundant in the Z-strain. The cation diffusion facilitator (CDF) family of transporters are well known to chelate the heavy metals and sequester them to cellular organelles like the vacuole [54]. In chloroplast-containing plants, CDFs are known as Metal Tolerance Proteins (MTPs) because of their role in the sequestration of excessive Zn in the vacuoles [55]. MTP2, which was previously characterized as a transporter that has a role in Zn hyperaccumulation in Arabidopsis halleri and Noccaea caerulescens [55], was found to be highly abundant, by 3.61-fold increase, in the Z-strain (Table 2). Since E. gracilis lacks a plant-like proper vacuolar structure, the MTP2 transporter in the Z-strain may be involved in Cd sequestration into the chloroplasts, although the functional characterization of this transporter has not been performed yet. The increase in abundance of a P-type ATPase was 3.6-fold in the Z-strain. P-type ATPases are also known as heavy metal ATPases (HMAs) that are associated with the transport of heavy metals in plants [56] and yeast [57]. P-type ATPase are localized to the vacuolar membrane and helps in detoxification of Zn and Cd through vacuolar sequestration in yeast [58]. In Arabidopsis thaliana P-type ATPase are localized to the chloroplasts and assist in Zn detoxification [59]. The increase in abundance of the ABC transporter G family member 36 was 4.41-fold (Table 2) in the Z-strain treated with Hg; the Z-strain may have used this transporter to efflux the toxic Hg out of the cell similar to the probable efflux of Cd used by the Zm-strain above. Overall, the transporter study indicated that chloroplasts have a role in bioaccumulation of Cd and efflux of Hg in the Z-strain. Although the Zm-strain lacks chloroplasts it showed an excellent capacity to bioaccumulate Hg suggesting a role for the transporters in Hg accumulation and efflux of Cd.

Chloroplast Proteins
A few chloroplast proteins were high in abundance in the Z-strain treated with Cd, while Hg and Pb did not bring about any significant changes in the chloroplast proteins. The Light-harvesting chlorophyll a/b binding protein (LHCB) was increased by 15.35-fold in the Z-strain (Table 3). LHCB expression operates a special mechanism in plants to modulate chloroplast stomatal movement against cellular stress caused by external stimuli such as exposure to heavy metals and drought; overall, LHCB functions to maintain plant fitness [60,61]. Cd is a strong inhibitor of photosynthesis and interrupts the electron transport chain in photosystem I (PSI) and photosystem II (PSII) [62]. The increase in expression of LHCB may help to better deal with the Cd toxicity and cope with the cellular stress brought by it. The level of Cd toxicity and cellular ability to resist it varies among different organisms. In our study, major changes in the abundance of chloroplast related proteins were seen in the Cd-treated Z-strain. As none of these proteins showed similar levels of changes during the other metal exposures, chloroplast related proteins may be specifically helping the Z-strain to withstand the toxicity of Cd.

Conclusions
E. gracilis chloroplasts were found to have a role in assisting the accumulation of the heavy metal Cd, whereas the other two metals tested, Hg and Pb, were mostly deposited in the cytosol of both strains studied. Some deposition of Hg was also seen in the mitochondria. This, together with the proteomic data, indicates that the strains with (Z) and without chloroplasts (Zm) have different abilities and mechanisms to respond to the heavy metal exposure, especially concerning Cd and Hg. Major changes in the abundance of chloroplast related proteins were seen in the Cd-treated Z-strain including light-harvesting chlorophyll a /b binding protein of PSII, indicating that the chloroplast related proteins may be specifically helping the Z-strain to withstand the toxicity of Cd. Exposure to Hg caused disturbance in the cell integrity, especially in the chloroplasts; however, Cd and Pb exposure did not bring about significant changes in cellular structure. While we focused on protein transporters and chloroplast-associated proteins, further analysis of all identified proteins will add to the current knowledge on the molecular mechanism operating in the tolerance and accumulation of heavy metals in E. gracilis. Considering the current data and the fact that E. gracilis has an innate tolerance to relatively high concentrations of heavy metals as demonstrated by the MICs obtained, the organism has the potential for the management of particular heavy metal contaminations of the environment.
Supplementary Materials: The following are available online at http://www.mdpi.com/2076-2607/8/1/115/s1, Figure S1: E. gracilis Z-and Zm-strains cultured in the GNY-medium only. Cells were collected each day until day 7. Error bars represent the standard deviation calculated from biological triplicates. The difference in the growth rate observed between the strains were not significantly different (p > 0.05). Table S1: Common differentially abundant proteins in the E. gracilis Z-and Zm-strains treated with heavy metals Cd, Hg and Pb. Table S2: The high abundance proteins in E. gracilis Zm-strain during exposure to the heavy metals Cd, Pb and Hg. Table S3: The high abundance metal binding proteins (FC >1.5) in the E. gracilis Z-and Zm-strains treated with heavy metals.