Elizabeth Agostini

Genetically transformed roots: from plant disease to biotechnological resource

Hairy root syndrome is a disease that is induced by Agrobacterium rhizogenes infection and characterized by a proliferation of excessively branching roots. However, in the past 30 years A. rhizogenes-mediated transformation has also provided a valuable platform for studying biosynthesis pathways in plants. Furthermore, the genetically transformed root cultures are becoming increasingly attractive, cost-effective options for mass-producing desired plant metabolites and expressing foreign proteins. Numerous proof-of-concept studies have demonstrated the feasibility of scaling up hairy-root-based processes while maintaining their biosynthetic potential. Recently, hairy roots have also shown immense potential for applications in phytoremediation, that is, plant-based decontamination of polluted environments. This review highlights recent progress and limitations in the field, and outlines future perspectives for the industrial exploitation of hairy roots.

Phytoremediation of 2,4-dichlorophenol by Brassica napus hairy root cultures

We have obtained hairy root cultures of Brassica napus with high biomass and genetic stability which produce peroxidases, enzymes involved in biodegradation processes. In this work, these hairy root cultures were used to study the removal of 2,4‐dichlorophenol (2,4‐DCP), a common contaminant in industrial effluents that is highly toxic for human and aquatic life. The optimum conditions to obtain high efficiency in the removal process were established. Roots were able to remove 2,4‐DCP from aqueous solutions containing 100–1000 mg/l, in the presence of H2O2 concentrations ranging from 5 to 10 mM. After a short period of incubation (15 min), high removal efficiencies were achieved (91–94%) and maximal removal, of approx. 97–98%, was obtained with 1 h of reaction. High removal efficiencies (93–95%) were observed in a broad pH range (pH 3–9), reaching 98–99% in the range pH 4–8. Moreover, roots could be re‐used, almost for six consecutive cycles, to remove 2,4‐DCP. The oxidation catalysed by peroxidases would be the main mechanism involved in this process. The results suggest that these cultures could be useful tools for phytoremediation.

A peroxidase isoenzyme secreted by turnip (Brassica napus) hairy-root cultures: inactivation by hydrogen peroxide and application in diagnostic kits

We have purified various peroxidase isoenzymes from roots and hairy‐root cultures of turnip (Brassica napus) which could potentially be used for commercial applications such as an enzyme immunoassays, diagnostic test kits, wastewater treatment and soil remediation. One of them, a basic peroxidase called HR2, was secreted into the medium of turnip hairy‐root cultures. HR2 had a pI of 9.6, a molecular mass of 39.3 kDa and showed great thermostability. The inactivation of HR2 by H2O2 in the absence of reductant substrates was studied. Under these conditions H2O2 acted as a suicide substrate. The kinetic constants calculated have been compared with those of a basic isoperoxidase from horseradish (Armoracia sp.) roots (HRP‐C), which is commonly used in commercial kits. The results for HR2 indicated that it was more resistant to inactivation because it presented a lower inactivation efficiency and a higher value for the partition ratio (r=1250) than those described for HRP‐C. These results make turnip peroxidase HR2 suitable for use in systems in which high H2O2 concentrations are found. Such an application is demonstrated, namely an enzymic diagnostic kit for determination of uric acid in which HR2 was found to be as efficient as the enzyme originally included in standard kits.

Application of Brassica napus hairy root cultures for phenol removal from aqueous solutions

Phenolic compounds present in the drainage from several industries are harmful pollutants and represent a potential danger to human health. In this work we have studied the removal of phenol from water using Brassica napus hairy roots as a source of enzymes, such as peroxidases, which were able to oxidise phenol. These hairy roots were investigated for their tolerance to highly toxic concentrations of phenol and for the involvement of their peroxidase isoenzymes in the removal of phenol. Roots grew normally in medium containing phenol in concentrations not exceeding 100 mg l(-1), without the addition of H(2)O(2). However, roots were able to remove phenol concentrations up to 500 mg l(-1), in the presence of H(2)O(2), reaching high removal efficiency, within 1h of treatment and over a wide range of pH (4-9). Hairy roots could be re-used, at least, for three to four consecutive cycles. Peroxidase activity gradually decreased to approximately 20% of the control, at the fifth cycle. Basic and near neutral isoenzymes (BNP) decreased along time of recycling while acidic isoenzymes (AP) remained without changes. Although both group of isoenzymes would be involved in phenol removal, AP showed higher affinity and catalytic efficiency for phenol as substrate than BNP. In addition, AP retained more activity than BNP after phenol treatment. Thus, AP appears to be a promising isoenzyme for phenol removal and for application in continuous treatments. Furthermore, enzyme isolation might not be necessary and the entire hairy roots, might constitute less expensive enzymatic systems for decontamination processes.

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.

Scale up of 2,4-dichlorophenol removal from aqueous solutions using Brassica napus hairy roots.

Chlorophenols are harmful pollutants, frequently found in the effluents of several industries. For this reason, many environmental friendly technologies are being explored for their removal from industrial wastewaters. The aim of the present work was to study the scale up of 2,4-dichlorophenol (2,4-DCP) removal from synthetic wastewater, using Brassica napus hairy roots and H(2)O(2) in a discontinuous stirred tank reactor. We have analyzed some operational conditions, because the scale up of such process was poorly studied. High removal efficiencies were obtained (98%) in a short time (30 min). When roots were re-used for six consecutive cycles, 2,4-DCP removal efficiency decreased from 98 to 86%, in the last cycle. After the removal process, the solutions obtained from the reactor were assessed for their toxicity using an acute test with Lactuca sativa L. seeds. Results suggested that the treated solution was less toxic than the parent solution, because neither inhibition of lettuce germination nor effects in root and hypocotyl lengths were observed. Therefore, we provide evidence that Brassica napus hairy roots could be effectively used to detoxify solutions containing 2,4-DCP and they have considerable potential for a large scale removal of this pollutant. Thus, this study could help to design a method for continuous and safe treatment of effluents containing chlorophenols.

Lethal and teratogenic effects of phenol on Bufo arenarum embryos.

Phenol and their derivatives are used in several industries and they have a high potential toxicity for animal and plant species. They were found in variable concentrations, as high as 1000 mg/L, in industrial wastewater and, they are often discharged into the environment. Amphibian embryos are useful indicators of environmental pollution. However, to our knowledge, there are not studies focussed on the toxic effects of phenol on Bufo arenarum, which is an anuran widely distributed in South America. Therefore, the effect of phenol on the survival and morphogenesis of these amphibian embryos was evaluated by means of AMPHITOX test. Embryos at 25 stage of development (acute test) and embryos at 2-4 blastomers stage (early life stage test), were exposed to phenol solutions in concentrations ranging from 25 to 250 mg/L, which were frequently found in the environment. Mortality and malformations were registered each 24h. LC(50), LC(99), NOEC, TC(50) and TI(50) values were 183.70, 250, 60, 113 mg/L and 1.62, respectively, at 96 h of treatment. Mortality and the percentage of malformations increased with increasing phenol concentrations. Teratogenic effects more frequently produced by phenol were: axial flexure, persistent yolk plug and different abnormalities which caused death of blastulae. Moreover, other malformations were registered, such as irregular form, acephalism, edema, axial shortening and underdevelopment of gills, among others. Larvae of B. arenarum, at early embryonic stages (blastulae), showed higher sensitivity to phenol than tadpoles at stage 25. Results confirm high susceptibility of amphibians to phenol and that environmental concentrations of this pollutant might be harmful to these populations.

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.