Metabolism of Hazardous Phenolic Compounds in Plants

3.3.1. Phenolic Compounds Metabolism and the Green Liver Model

It has generally been accepted that several enzyme systems, not necessarily physiologically connected, form a metabolic cascade for the detoxification, breakdown and final storage of organic xenobiotics (Schröder et al., 2008). Detoxification mechanisms described for phenolic compounds, resemble more the reactions in the animal liver than the bacterial metabolism, following the "green liver" model proposed for the metabolism of other organic pollutants by Sandermann (1994). This network of reactions can be subdivided into three distinct phases: tranformation (phase I), conjugation (phase II) and compartmentation (phase III). Recently, the last phase has been categorized into two independent phases, one confined to transport and storage in the vacuole, and a second one involving final reactions, such as cell wall binding or excretion (Schröder et al., 2007; Abhilash et al., 2009).

3.3.2. Transformation (Phase I)

The first metabolic step is the transformation of the initial substrate and generally includes several enzymatically catalized oxidations. However, this step is not essential for some pollutants. Pollutant transformation increases its solubility and provides an opportunity for conjugation, the next step in the removal process. In many plants, a number of transformations take place simultaneously and different enzymes can be identified. Despite the fact that biochemical processes accompanying phenolic detoxification in plants are not well investigated, there are many examples in the literature indicating that the activities of peroxidase isoenzymes, laccases and other phenol-oxidases are a crucial step in phenolic metabolism (Dec and Bollag, 1994; Agostini et al., 2003; Coniglio et al., 2008). Peroxidases catalyze a reaction known as oxidative coupling, which is involved in the detoxification of phenol in aqueous solutions while in soils this coupling may occur with the humic material (Flocco et al., 2002; Coniglio et al., 2008). For instance, phenol was completely removed from the incubation medium by aseptically grown Vetiveria zizanoides plantlets and this was associated with inherent production of peroxidase and H2O2 (Singh et al., 2008). In addition, a significant increase in peroxidase activity was detected in alfalfa roots exposed to phenol, at 10 and 30 days of exposure (Flocco et al., 2002). In these experiments, as well as in other studies, the products of plant bioconversion of this pollutant remain unidentified (Singh et al., 2008). It is important to note that not only the level but also the isoenzyme pattern of peroxidases could be modified by environmental stress, such as that produced by phenolics at high concentrations (Agostini et al., 2003). These aspects will be discussed with more detail in following sections.

Regarding phenolic full transformation, there are only few reports indicating the degradation of different phenolic compounds to CO2 (mineralization) or regular cell metabolites (Ugrekhelidze et al., 1999). Considering that organic compounds are rarely mineralized in plants (Sandermann, 1992; Schnoor et al., 1995; Schröder and Collins, 2002) and, sometimes, only a small amount of toxicant present in the cell is mineralized while the rest undergoes conjugation, various authors have suggested that this conversion is depreciable or even that does not take place in the metabolic pathway of phenolic compounds (Harvey et al., 2002; Pascal-Lorber et al., 2008). This is in accordance with the knowledge that only few enzymes, present in plants, are able to catalyze ring opening reactions of organic compounds, in contrast to the degradative metabolism of microorganisms. However, few works found in the literature showed oxidation of benzene and phenol by crude enzyme extracts of many plants, which formed muconic acid after ring cleavage, with catechol as an intermediate. Then, further oxidation of muconic acid may lead to the formation of fumaric acid (Durmishidze et al., 1969; Chrikishvili et al., 2005). In addition, Ugrekhelidze et al. (1999) found that a small amount of phenol molecules assimilated through wheat and mung bean roots could be transformed via aromatic ring cleavage and bibasic carbonic acid formation. This process, which involves the mineralization of a pollutant, is usually known as deep oxidation and is one of the most desirable ecological features of plants. However, in nature it rarely occurs. Depending on plant species, the contaminant's nature and its concentration, a relatively small proportion of the environmental contaminant penetrating into the plant cell undergoes deep oxidation. (Kvesitadze et al., 2006).

3.3.3. Conjugation (Phase II)

Once transformation occurs, conjugation with endogenous compounds (mono-, oligo-and polysaccharides, proteins, peptides, amino acids, organic acids, lignin, etc.) is predominantly the next step in the detoxification or metabolism of pollutants (Phase II). However, some pollutants are conjugated without being preceded by transformation. The formation of conjugates leads to an enhancement of the hydrophilicity of organic contaminants, and consequently to an increase in their mobility. Such characteristics simplify further compartmentation of the transformed toxic compounds.

The process of conjugation is usually carried out by enzymes, such as O-glucosyl transferases (OGT; EC 2.4.1.7), N-glucosyltransferases (EC 2.4.1.71), N-malonyltransferases (EC 2.3.1.114), glutathione S-transferase (EC 2.8.1.18), etc. This process leads to the formation of peptide, ether, ester, thioether or other bonds of a covalent nature. Hydroxyl, NH2-, SH- and COOH functions on a molecule usually trigger glycosyl-transfer mediated by glycosyltransferases, whereas the presence of conjugated double bonds or halogen-functions proceeds to glutathione conjugation (catalyzed by glutathione S-transferase). In this context,

Schröder and Collins (2002) have pointed out that to know the type of primary conjugation is crucial, because this will determine the final fate of the compound in phytoremediation.

Regarding phenolics, they can be conjugated with carbohydrates such as glucose and glucuronic acid, in different proportions depending on the plant species. In fact, in vitro glycosilation of simple phenols was demonstrated several times. For example, this typical detoxification mechanism has been reported for the metabolism of phenol, 2,4-DCP and 2,4,5-TCP in duckweed (Lemna gibba). In this work, ß-glucoside conjugates were detected as final products of phenolic metabolism, which were progressively dehalogenated (Ensley et al., 1997). However, when the metabolic fate of 2,4-DCP was investigated in six macrophytes, the 2,4-DCP-glucoside conjugate was described as an intermediate metabolite (Pascal-Lorber et al., 2004). Once this intermediate metabolite is formed, more complex monoglucoside esters, either malonyl or acetyl, are detected in these macrophytes. These authors also described an unusual glucosyl-pentose conjugate as the 2,4-DCP major metabolite in Lemna minor and Glyceria maxima. Conjugation with pentose would prevent further saccharide chain elongation. Moreover, soluble ß-D glucoside and O-malonyl-ß-D-glucoside conjugates were detected after metabolism of PCP by wheat and soybean plants, which translocate and accumulate them in vacuoles (Schmitt et al., 1985). Similarly, Pascal-Lorber et al. (2003) reported that both plant species shared a common metabolism for [14C]-2,4-DCP since the malonylated glucoside conjugates were found as the final major metabolites. Conjugation with malonic acid is a common process, specific to plants, and may represent a signal for sequestration of glycoside conjugates in vacuoles. In addition, Day and Saunders (2004) have characterized more complex 2,4-DCP and 2,4,5-TCP glycosides such as a glucose-apiose, a hydroxymethyl-3-tetrose conjugate, in Lemna minor. Lately, the presence of complex glycosides has also been described in edible plants, such as spinach, radish and lettuce (Table 3) and glucuronide conjugates have only been characterized in spinach treated with 2,4-DCP and 2,4,5-TCP (Pascal-Lorber et al., 2008). It is important to point out that these glucuronide-conjugates have rarely been described in plants, and there are only few reports in the literature (Bokern, et al., 1996; Laurent et al., 2007). More examples of phenolic-conjugates described in different plant species are presented in Table 3.

There are findings which suggested that different plant species have distinct OGTs to detoxify different xenobiotics, rather than utilize the same enzyme in each case (Brazier et al., 2003). For instance, an OGT has been partially purified from wheat shoots (Brazier et al., 2003). This enzyme was characterised as a monomeric 53 kDa protein and was distinct from other OGTs previously identified in wheat (Schmitt et al., 1985). Among the xenobiotic phenols tested, the purified enzyme preparation showed at least a 10-fold preference for 2,4,5-TCP instead of 4-nitrophenol, PCP and 2,4,6-TCP. Interestingly, the OGT from soybean had a similar substrate preference for synthetic phenols as the OGT described by Brazier et al. (2003). Contrarily, the 43 kDa OGT described by Schmitt et al. (1985) was more active in conjugating PCP. However, low substrate specificity and cross reactivity of OGT isoenzymes (i.e. use of xenobiotic as well as natural substrates) were also reported. The metabolism of 2,4,5-TCP in Arabidopsis is a remarkable example. This compound is used by at least six members of the glucosyltransferase superfamily (Sandermann, 2004 and references therein).

Table 3. Examples of several phenolics conjugates from different plant and cell cultures species

Phenolic compound

Plant/Tissue Culture

Plant species

Conjugate type

References

PCP

Cell suspensions

Wheat (Triticum aestivum) Soybean (Glycine max)

P-D-glucosides o-malonyl- P-D glucosides

Schmitt et al., 1985

Phenol

Nitrophenol

Hydroxiphenols

Cell suspensions

Gardenia jasminoides Ellis

P-D-monoglucosides

Misukami et al., 1987

4 n-nonylphenol

Cell suspensions

Wheat (Triticum aestivum)

Glucuronide-conjugates

Bokern et al., 1996

Phenol

2,4-DCP

2,4,5-TCP

Aquatic plant

Duckweed (Lemna gibba)

P-D-glucosides

Ensley et al., 1997

Phenol

Sterile seedlings

Mung bean (Phaseolus aureus) Wheat (Triticum vulgare)

Peptide conjugates

Ugrekhelidze et al., 1999

4 n-nonylphenol

Plant

Lemna minor

Deoxipentose conjugates

Thibaut et al., 2000

2,4-DCP

Cell suspension

Wheat (Triticum aestivum) Soybean (Glycine max)

DCP-(malonyl)-glucosides

Pascal-Lorber, et al., 2003

2,4-DCP

Plants

Lemna minor

2,4-dichorophenyl-P-D-glucopyranoside

2,4-dichorophenyl-P-D-(6-O-malonyl)-

glucopyranoside

2,4-dichorophenyl-P-D-glucopyranosyl-(6,1)-P-D-apiofuranoside

Day and Saunders, 2004

2,4-DCP

Aquatic plant

Myriophyllum spicatum Hippuris vulgaris Mentha aquatica Glyceria maxima

DCP-(malonyl)-glucoside

Pascal-Lorber et al., 2004

2,4-DCP

Aquatic plant

Salvinia natans

DCP-(acetyl)-glucoside

Pascal-Lorber et al., 2004

2,4-DCP

Aquatic plant

Lemna minor

DCP-(pentosyl)-glucoside

Pascal-Lorber et al., 2004

Phenol

Sterile seedlings

Ryegrass (Loliumperenne L.)

Phenol-peptide conjugates

Chrikishvili et al., 2005

Phenol

Chlorophenols

Hairy roots

Daucus carota Ipomoea batatas Solanum aviculare

Polar conjugates (possible with sugars or proteins)

Santos de Araujo et al., 2006

Phenolic compound

Plant/Tissue Culture

Plant species

Conjugate type

References

2,4-DCP

(Nicotiana tabacum)

DCP-glucoside conjugates DCP-(6-O-malonyl)-glucoside DCP-(6-O-acetyl)-glucoside DCP-a1,6-glucosyl-pentose DCP-triglycoside-glucuronic acid

Laurent et al., 2007

4-CP

Plant

Radish (Raphanus sativus)

4-CP-(acetyl)-hexose (major) 4-CP-(malony l)-hexose

Pascal-Lorber et al., 2008

2,4-DCP

Plant

Radish (Raphanus sativus)

2,4-DCP-(acetyl)-hexose (major)

Pascal-Lorber et al., 2008

2,4,5-TCP

Plant

Radish (Raphanus sativus)

2,4,5-TCP-hexo se- sulfate 2,4,5-TCP-(malonyl)-hexose-sulfate

Pascal-Lorber et al., 2008

2,4,5-TCP

Plant

Lettuce (Lactuca sativa L.)

2,4,5-TCP-(acetyl)-hexose (major) 2,4,5-TCP-deoxypentose-(malonyl) hexose

Pascal-Lorber et al., 2008

2,4-DCP

Plant

Spinach (Spinacea oleracea)

2,4-DCP-(malonyl) hexose-hexuronic acid (major)

Pascal-Lorber et al., 2008

Moreover, a glucosyltransferase isoenzyme mixture from tobacco leaves can be efficiently used to glucosylate 2,4,5-TCP, PCP and 4-nitrophenol (Harvey et al., 2002).

Thus, different plant species can differ in the degree of cross-reactivity of their detoxifying enzymes (Sandermann, 2004). In some cases when phenols are glycosylated, the existence of di- and triglycosides has been demonstrated. For instance, diglycoside (gentiobioside) and triglycosides were formed from exogenous hydroquinone in wheat embryos (Harborne, 1977). In addition, disaccharide conjugates, formed by glycosil extension, were also described in the metabolic pathway of 2,4,5-TCP in radish (Pascal-Lorber et al., 2008).

Contrarily, phenol was not glycosylated in intact plants of maize (Zea mays), pea (Pisum sativum L.) and pumpkin (Cucurbita pepo) (Arziani et al., 2002). In some annual plant seedlings, phenol was not glycosylated either, but was conjugated with low-molecular-weight peptides, forming phenol-peptide conjugates. A study of [l-6-14C] phenol metabolism in sterile seedlings of mung bean (Phaseolus aureus) and wheat (Triticum vulgare) demonstrated that phenol formed conjugates with low-molecular-mass peptides (Ugrekhelidze et al., 1999). However, among the peptides participating in conjugation, glutatione and homoglutatione were not found. Other monophenols also formed conjugates with peptides in plants, namely a-naphthol in maize, pea, and pumpkin seedlings (Ugrekhelidze et al., 1980; Ugrekhelidze et al., 1983); o-nitrophenol in pea seedlings (Ugrekhelidze et al., 1980; Ugrekhelidze et al., 1983); and a hydroxyl derivative of 2,4-D in maize, pumpkin, and pea seedlings (Arziani et al., 1983; 2002). It was observed that in some plants treated with phenol, the low molecular-mass peptides concentration increases (Ugrekhelidze et al., 1983). Besides, phenols are covalently bound to peptides via hydroxyl groups. It is important to note that peptides participating in the conjugation of phenols considerably differ in their aminoacid composition. According to the existing information, in some plants, conjugation with low-molecular-mass peptides seems to be an important detoxification pathway for monophenols (Arziani et al., 2002).

Furthermore, direct conjugation to lignin can occur. Lignin is a phenolic, structurally nonrepeating macromolecule, which is active in conjugation reactions, and often plays the role of a carrier of xenobiotics and their primary transformants (Sandermann, 1994). Such compounds are incorporated into the lignin structure by being covalently coupled with the biopolymer. It has been shown that tautomeric forms of the lignin monomer coniferyl alcohol (quinone-methyl) couple xenobiotics with amino and hydroxyl groups. In this sense, it is interesting to mention that if PCP is hydroxylated, upon which it acquires a second hydroxyl group, this intermediate can be conjugated with lignin, forming an insoluble compound, which is removed from the cell and stored in the cell wall (Sandermann, 1994). Also, coniferyl alcohol is easily conjugated with 1,2-dihydroxy-3,4,5,6-tetrachlorobenzene, which is an intermediate of PCP hydroxylation (Sandermann, 1987).

In addition, Bokern and Harms (1997), investigating the metabolism of [14C] 4-nonylphenol in suspension cultures of 12 plant species, found that in 7 of the cultures, the xenobiotic is conjugated with lignin. However, lignin is not the only biopolymer involved in binding with xenobiotics. In the leaves, xylan and lignin are the preferred compounds, whereas in the stems pectin and lignin are the main components which bind xenobiotics.

Regarding PCP metabolism, in the aquatic plant Eichhornia crassipes the major by products were identified as ortho- and para- substituted chlorohydroxyphenols (chlorocatechols and hydroquinones), -anisoles, and -veratroles. Partially dechlorinated products of PCP were also detected. A major portion of the absorbed PCP and metabolites was found in bound/conjugated form. A significant increase was also observed in the activity of glutathione S-transferase, a major conjugating enzyme, and in the activities of superoxide dismutase and ascorbate peroxidase in PCP exposed plants (Roy and Haenninen, 1994). On the other hand, Schäfer and Sandermann (1988) identified tetrachlorocatechol as a primary metabolite of PCP in cell suspension cultures of wheat.

Unlike deep oxidation, conjugation does not lead to complete detoxification of the xenobiotic, which preserves its basic molecular structure and hence reduces only partially its toxicity. So, conjugation is not the most successful pathway of xenobiotic detoxification from an ecological point of view. Conjugates of toxic compounds are especially hazardous upon entering the food chain, because enzymes of the digestive tract of warm-blooded animals can hydrolyze conjugates and release the xenobiotics or products of their partial transformation, which in some cases, are more toxic than the parent xenobiotic. So, it is important to perform an adequate characterization of the structure of different conjugates in order to evaluate their bioavailability and the risk that they represent for human and animal health.

3.3.4. Compartmentation (Phase III)

Once conjugates are formed, they can be sequestered or compartmentalized, which is known as phase III of pollutant metabolism. Soluble conjugates (coupled with peptides, sugars, amino acids, etc.) are accumulated in vacuoles. This process takes place with the participation of ATP-binding cassette (ABC) transporters (Schröder et al., 2007). Metabolites stored in the vacuoles could be further processed before exportation to cell wall. However, very little is known about these processes (Pascal-Lorber et al., 2008). Insoluble conjugates (coupled with protein, lignin, starch, pectin, cellulose, xylan and other polysaccharides), are moved out of the cell via exocytosis and are accumulated in the apoplast or cell wall. This may lead to the formation of so-called 'bound residues' because of their inability to be extracted by chemical methods. These conjugates may be covalently bound to stable tissues in the plant (Trapp and Karlson, 2001). Hence, the main objective of compartmentation is essentially to remove toxic compounds from metabolic tissues. In this sense, plants have a greater ability to compartmentalize the products of metabolism and detoxification within internal structures and between organs, producing different localization between in vitro and in vivo systems.

When xenobiotic chemicals are applied to plant cells, the original compound, the primary products of its metabolism and product conjugates, are distributed between extractable and nonextractable fractions of the biomass. Generally, these nonextractable or 'bound residues' of plant cells cannot be released from the plant matrix by extraction with solvents, probably because of covalent association with lignin, hemicellulose or pectin in the plant cell (Harvey et al., 2002). In fact, incorporation of metabolites in covalent and noncovalent linkage with proteins, lignin, pectin, polysaccharides, cellulose, hemicellulose, starch, and cutin has been reported (Sapp et al., 2003). Bound residues seem to be found in those species that are most tolerant to organic pollutants and the pattern of binding depends on the plant species and the physico-chemical properties of the compound (Harvey et al., 2002). These bound residues have attracted considerable interest and concern in recent years because persistence of chemical residues in edible plants may allow toxic components to enter the food chain (Sandermann, 2004; Trapp et al., 2001). However, there is increasing evidence to suggest that the formation of the bound residue fraction is one of the most important detoxification pathways in plant cells (Harvey et al., 2002).

Plant cell suspensions have been used to investigate the properties of bound residues (Sandermann et al., 1983; Sapp et al., 2003). The advantages of in vitro cultures for these studies include elimination of microbial effects and measurement artefacts due to photosynthetic refixation of 14C into natural nonextractable cell components (Sandermann, 2004). However, the question is whether plant cells in culture incorporate metabolites into bound residues in the same way or to the same extent as in plants. Several experiments suggest that measurements of bound components in plant tissue cultures may underestimate the characteristic levels of whole plants, while in other studies cultured plant cells have been found to generate similar levels of bound residues to those found in whole plants (Langebartels and Harms, 1986; Schmidt et al., 1993). In any case, plant tissue cultures have been recommended for initial experiments on bound residues to minimize the expense of greenhouse or field trials (Sandermann, 2004). In this way, Harms et al. (2003), studied the formation of bound residues using 14 different cell cultures derived from different plant species and exposed to 4-nonylphenol. They concluded that the formation of bound residue would be species-specific and the capacity to form such residues may be associated with higher tolerance to the pollutant. Talano et al. (2010) using tobacco hairy roots capable to remove 2,4-DCP with high efficiency, showed the possible fate of a lignin-like polymer in the xylem of roots as a result of this pollutant transformation. Although the results obtained were an indirect evidence of 2,4-DCP final product, it is one of the few reports which showed an in vivo localization of these bound residues and, moreover the possible chemical nature of them.

In plants, due to their lack of an efficient excretory system, xenobiotic conjugates finally are sequestered in plant storage compartments, mainly vacuoles, or are integrated as bound residues in cell walls. However, there are few reports which described the existence of a possible excretory system in many plants. In this sense, environmental pollutants, such as phenolics, absorbed by the roots can also be excreted via leaves, although this excretion is uncommon as compared to root excretion. (Korte et al., 2000). Seidel and Kickuth (1967) described that plants kept on phenol solution excreted this pollutant by the leaves of bulrush (Scirpus lacustris L.). In this case, the excretion occured so rapidly that after 90 min phenol could be measured in the air near the leaves and after several hours it could be detected even by smell. The inference from this and other analogous studies (Schröder et al., 2007) is that some plants can excrete derivatives of the pollutants absorbed from the soil or groundwater and gradually dilute them into the air or into the soil. This could mean that some plants possess an excretion system for unwanted compounds. Moreover, in plant cell cultures, substantial proportions of specific metabolites are often found in the culture medium, suggesting a possible excretion of xenobiotics (Canivenc et al.,1989; Groeger and Fletcher, 1988; Laurent and Scalla, 1999).

In summary, there are several potential mechanisms for uptake, metabolism and degradation of phenolics in plants. They are represented in Figure 2. Possible mechanisms include sorption and uptake of the pollutant and/or its metabolites into the roots, microbial transformation performed by rhizospheric and endophytic microorganisms, several transformations catalized by extra and/or intracellular enzymes, xylem transfer of the compounds to the leaves, foliar uptake from the air, probably phloem transfer and bound residue formation, among other processes. All these mechanisms contribute to the phytoremediation of phenolics from the environment.

Phyto Degradation

Figure 2. Schematic representation of proposed mechanisms involved in phenolic transformations in plant-soil-air-environments. Phenols from different sources can be stabilized or degraded in the rhizosphere and phyllosphere, sorbed and/or absorbed by roots and/or leaves, translocated and metabolized inside the plant cells.

Figure 2. Schematic representation of proposed mechanisms involved in phenolic transformations in plant-soil-air-environments. Phenols from different sources can be stabilized or degraded in the rhizosphere and phyllosphere, sorbed and/or absorbed by roots and/or leaves, translocated and metabolized inside the plant cells.

The degradation pathways of hazardous phenolic compounds seem to be significantly complicated and, at present, they are still unknown in several plant species. In part, the complicated appearance is undoubtedly due to the present lack of essential information. However, it is absolutely clear that in plants as well as in microbes, intracellular enzymatic degradation of contaminants is mainly carried out by oxidative enzymes. Thus, in the remediation process, the knowledge of the variety of enzymes and levels of their activities are the main basis of any kind of phyto- or bioremediation technology. In this sense, an overview of the use of oxidative plant enzymes, as an alternative, in phenol remediation process will be described in the next section.

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