Melina A. Talano

Establishment of transgenic tobacco hairy roots expressing basic peroxidases and its application for phenol removal

Transgenic hairy root (HR) systems constitute an interesting alternative to improve the efficiency of phytoremediation process. Since peroxidases (Px) have been associated with phenolic compounds removal, in the present work, transgenic tobacco HR, which expressed basic Px genes from tomato (tpx1 and tpx2), were established and assayed for phenol removal. Tobacco HR clones were obtained, including those transgenic for TPX1 or TPX2, those double transgenic (DT) for both Px and the corresponding controls. Based on growth index, the presence of rol C sequence, tpx1 and/or tpx2 genes and the coded proteins, as well as Px activity determinations, we selected 10 tobacco HR clones for phenol removal assays. The removal efficiencies were high for all the HR, although, some transgenic HR showed significantly higher removal efficiencies compared with controls. The results demonstrate that TPX1 is involved in phenol removal not only when it was overexpressed in tomato, but also when it was expressed in other plant, such as tobacco. The higher efficiency of TPX2 transgenic HR showed that this Px also participates in the process. The contribution of other mechanisms (adsorption, H2O2 independent enzymatic processes) could be considered depreciable, which establishes the great implication of Px in phenol removal.

Removal of 2,4-diclorophenol from aqueous solutions using tobacco hairy root cultures

2,4-Dichlorophenol (2,4-DCP) is harmful for aquatic life and human health, so many attempts have focused on removing it through innocuous technologies. Hairy roots (HR) represent an interesting plant system to study the process and to remove efficiently this compound. In the present work, tobacco HR clones were obtained and one of them was selected for 2,4-DCP phytoremediation assays. These cultures removed 2,4-DCP in short time and with high efficiency (98%, 88% and 83%) for solutions initially containing 250, 500 and 1000 mg/L, respectively. Removal process was mainly associated with peroxidase activity. The highest efficiency for 2,4-DCP (500 mg/L) removal was reached at 60 min and using 10 mM H(2)O(2). Moreover, HR could be re-used, almost for three consecutive cycles. The diminution of pH and the increase of chloride ions in post-removal solutions suggested that 2,4-DCP dehalogenation was mediated by peroxidases. Moreover, changes in deposition pattern of lignin in HR exposed to 2,4-DCP suggested that cell walls of xylem and phloem elements would be the site of deposition of some products formed and they would be a lignin-type polymer. These findings contribute to understand 2,4-DCP removal process with tobacco HR and it might have implications in the use of this system for decontamination of polluted waters.

Hairy Roots, their Multiple Applications and Recent Patents

In the last years, hairy root (HR) cultures are gaining attention in the biotechnology industry. This particular plant cell culture derives from explants infected with Agrobacterium rhizogenes. They constitute a relatively new approach to in vitro plant biotechnology and modern HR cultures are far away from the valuables findings performed by Philip R. White in the 1930s, who obtained indefinite growth of excised root tips. HR cultures are characterized by genetic and biochemical stability and high growth rate without expensive exogenous hormones source. HR cultures have allowed a deep study of plant metabolic pathways and the production of valuable secondary metabolites and enzymes, with therapeutic or industrial application. Furthermore, the potential of HR cultures is increasing continuously since different biotechnological strategies such as genetic engineering, elicitation and metabolic traps are currently being explored for discovery of new metabolites and pathways, as well as for increasing metabolites biosynthesis and/or secretion. Advances in design of proper bioreactors for HR growth are being of great interest, since scale up of metabolite production will allow the integration of this technology to industrial processes. Another application of HR cultures is related to their capabilities to biotransform and to degrade different xenobiotics. In this context, removal assays using this plant model system are useful tools for phytoremediation assays, previous to the application in the field. This review highlights the more recent application of HRs and those new patents which show their multiple utilities.

Application of hairy roots for phytoremediation: what makes them an interesting tool for this purpose?

In recent years, hairy roots (HRs) have been successfully used as research tools for screening the potentialities of different plant species to tolerate, accumulate, and/or remove environmental pollutants, such as PCBs, TNT, pharmaceuticals, textile dyes, phenolics, heavy metals, and radionuclides. This is in part due to several advantages of this plant model system and the fact that roots have evolved specific mechanisms to deal with pollutants because they are the first organs to have contact with them. In addition, by using HRs some metabolic pathways and enzymatic catalyzed reactions involved in pollutants detoxification can be elucidated as well as the mechanisms of uptake, transformation, conjugation, and compartmentation of pollutants in vacuoles and/or cell walls, which are important detoxification sites in plants. Plant roots also stimulate the degradation of contaminants by the release of root exudates and oxido-reductive enzymes, such as peroxidases (Px) and laccases, that are associated with the removal of some organic pollutants. HRs are also considered good alternatives as enzyme sources for remediation purposes. Furthermore, application of genetic engineering methods and development of microbe-assisted phytoremediation are feasible strategies to enhance plant capabilities to tolerate, accumulate, and/or metabolize pollutants and, hence, to create or find an appropriate plant system for environmental cleanup. The present review highlights current knowledge, recent progress, areas which need to be explored, and future perspectives related to the application and improvement of the efficiency of HRs for phytoremediation research.

Brassica napus hairy roots and rhizobacteria for phenolic compounds removal.

Phenolic compounds are contaminants frequently found in water and soils. In the last years, some technologies such as phytoremediation have emerged to remediate contaminated sites. Plants alone are unable to completely degrade some pollutants; therefore, their association with rhizospheric bacteria has been proposed to increase phytoremediation potential, an approach called rhizoremediation. In this work, the ability of two rhizobacteria, Burkholderia kururiensis KP 23 and Agrobacterium rhizogenes LBA 9402, to tolerate and degrade phenolic compounds was evaluated. Both microorganisms were capable of tolerating high concentrations of phenol, 2,4-dichlorophenol (2,4-DCP), guaiacol, or pentachlorophenol (PCP), and degrading different concentrations of phenol and 2,4-DCP. Association of these bacterial strains with B. napus hairy roots, as model plant system, showed that the presence of both rhizospheric microorganisms, along with B. napus hairy roots, enhanced phenol degradation compared to B. napus hairy roots alone. These findings are interesting for future applications of these strains in phenol rhizoremediation processes, with whole plants, providing an efficient, economic, and sustainable remediation technology.

Transgenic plants and hairy roots: exploiting the potential of plant species to remediate contaminants.

Phytoremediation has emerged as an attractive methodology to deal with environmental pollution, which is a serious worldwide problem. Although important advances have been made in this research field, there are still some drawbacks to become a widely used practice, such as the limited plants metabolic rate and their difficulty to break down several organic compounds or to tolerate/accumulate heavy metals. However, biotechnology has opened new gateways in phytoremediation research by offering the opportunity for direct gene transfer to enhance plant capabilities for environmental cleanup. In this context, hairy roots (HRs) have emerged as an interesting model system to explore the potential of plants to remove inorganic and organic pollutants. Besides, their use in rhizoremediation studies has also been explored. In this minireview we will discuss the most recent advances using genetic engineering for enhancing phytoremediation capabilities of plants and HRs.

Tomato (Lycopersicon esculentum cv. pera) hairy root cultures: Characterization and changes in peroxidase activity under NaCl treatment

SummaryHairy root cultures of Lycopersicon esculentum L. Mill ev. Pera were established by infection of leaf explants with Agrobacterium rhizogenes LBA 9402. The pattern of peroxidase isoenzymes in these tissues was similar to that of roots excised from tomato plants grown in hydroponic cultures. Hairy root cultures may be an appropriate system to analyze the peroxidase involvement in the response of isolated roots to salt stress, avoiding the problem of wounding or changes in hormone levels observed in roots excised from plants. The cultures of hairy roots allowed the evaluation of changes in peroxidase patterns not only in the tissue but also in the culture medium. Hairy roots were subcultured in Murashige and Skoog liquid medium with or without 100 mM NaCl to investigate the evolution of growth, total peroxidase activity of the tissue and culture medium, and changes in the peroxidase isoenzyme patterns under each condition of growth. Control cultures showed a growth index higher than those reported for other hairy root cultures, and it was even higher in the presence of 100 mM NaCl. The total peroxidase activity in the tissue was similar for control and salt-treated roots. Even when the total peroxidase activity of the medium decreased under salt treatment, NaCl induced secretion of a highly basic peroxidase and inhibition of the secretion of some acidic isoenzymes. These changes may explain the physiological role of these enzymes in the response to salt stress that we will possibly establish through a future study of the biochemical properties of those peroxidases.

Characterization of a phenol-degrading bacterium isolated from an industrial effluent and its potential application for bioremediation

The use of native microorganisms is a useful strategy for phenol bioremediation. In the present work, a bacterial strain, named RTE1.4, was isolated from effluents of a chemical industry. The strain was able to grow at high concentrations of phenol and its derivatives, such as guaiacol, 2,4-dichlorophenol and pentachlorophenol, as well as in a medium containing industrial effluents. This bacterium was identified as Acinetobacter sp. using morphological, physiological, biochemical and 16S rRNA gene analysis. Acinetobacter sp. RTE1.4 degraded phenol (200 to 600 mg/L) at wide pH range and temperature (5–9 and 25–37°C, respectively) demonstrating high adaptation ability to different conditions. The strain would metabolize phenol by the ortho-pathway since catechol 1,2-dioxygenase activity was detected. When bacteria were grown in medium containing phenol, an altered whole-cell protein pattern was observed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), with the lack of some low-molecular mass polypeptides and an increase in the relative abundance of high-molecular mass proteins after treatment. Considering that the use of native strains in bioremediation studies shows several ecological advantages and that the studied bacterium showed high tolerance and biodegradation capabilities, Acinetobacter sp. RTE1.4 could be an appropriate microorganism for improving bioremediation and biotreatment of areas polluted with phenol and/or some of its derivatives. Moreover, the establishment of the optimal growth conditions (pH, temperature, concentration of the pollutant) would provide baseline data for bulk production of the strain and its use in bioremediation processes.

Application of two bacterial strains for wastewater bioremediation and assessment of phenolics biodegradation

The use of native bacteria is a useful strategy to decontaminate industrial effluents. In this work, two bacterial strains isolated from polluted environments constitutes a promising alternative since they were able to remove several phenolic compounds not only from synthetic solutions but also from effluents derived from a chemical industry and a tannery which are complex matrices. Acinetobacter sp. RTE1.4 showed ability to completely remove 2-methoxyphenol (1000 mg/L) while Rhodococcus sp. CS1 not only degrade the same concentration of this compound but also removed 4- chlorophenol, 2,4-dichlorophenol and pentachlorophenol with high efficiency. Moreover, both bacteria degraded phenols naturally present or even exogenously added at high concentrations in effluents from the chemical industry and a tannery in short time (up to 5 d). In addition, a significant reduction of biological oxygen demand and chemical oxygen demand values was achieved after 7 d of treatment for both effluents using Acinetobacter sp. RTE1.4 and Rhodococcus sp. CS1, respectively. These results showed that Acinetobacter sp. RTE1.4 and Rhodococcus sp. CS1 might be considered as useful biotechnological tools for an efficient treatment of different effluents, since they showed wide versatility to detoxify these complex matrices, even supplemented with high phenol concentrations.

Effect of arsenic on tolerance mechanisms of two plant growth-promoting bacteria used as biological inoculants

Bacterial ability to colonize the rhizosphere of plants in arsenic (As) contaminated soils is highly important for symbiotic and free-living plant growth-promoting rhizobacteria (PGPR) used as inoculants, since they can contribute to enhance plant As tolerance and limit metalloid uptake by plants. The aim of this work was to study the effect of As on growth, exopolysaccharide (EPS) production, biofilm formation and motility of two strains used as soybean inoculants, Bradyrhizobium japonicum E109 and Azospirillum brasilense Az39. The metabolism of arsenate (As(V)) and arsenite (As(III)) and their removal and/or possible accumulation were also evaluated. The behavior of both bacteria under As treatment was compared and discussed in relation to their potential for colonizing plant rhizosphere with high content of the metalloid. B. japonicum E109 growth was reduced with As(III) concentration from 10 μM while A. brasilense Az39 showed a reduction of growth with As(III) from 500 μM. EPS and biofilm production increased significantly under 25 μM As(III) for both strains. Moreover, this was more notorious for Azospirillum under 500 μM As(III), where motility was seriously affected. Both bacterial strains showed a similar ability to reduce As(V). However, Azospirillum was able to oxidize more As(III) (around 53%) than Bradyrhizobium (17%). In addition, both strains accumulated As in cell biomass. The behavior of Azospirillum under As treatments suggests that this strain would be able to colonize efficiently As contaminated soils. In this way, inoculation with A. brasilense Az39 would positively contribute to promoting growth of different plant species under As treatment.