Horacio A. Tigier

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.

Tomato root peroxidase isoenzymes: kinetic studies of the coniferyl alcohol peroxidase activity, immunological properties and role in response to salt stress

Summary More than eleven soluble and ionically-bound cell wall peroxidase (EC 1.11.1.7; donor: hydrogen-peroxidase oxido-reductase) isoenzymes were found in tomato ( Lycopersicon esculentum (L.) Mill. cv. Pera) roots. Isoperoxidases with isoelectric points 9.6, 8.2, 7.5, 6.5, and 3.6, were partially purified by ammonium sulfate precipitation, gel filtration on Sephacryl-S200 and ion exchange chromatography on DEAE Sephacel and/or SP-Sephadex C50 columns. Isoenzymes of pI 9.6, 3.6, and 6.5 showed the highest catalytic efficiencies with coniferyl alcohol, a true monolignol, used as substrate marker of peroxidases presumably involved in lignification. In addition, they showed cross-reactivity in Western blots of crude extracts when antibodies anti pI 3.6 isoenzyme were used for the immunodetection, despite their different pI values. It was demonstrated that the levels of pI 3.6 isoenzyme increased with the time of growth but did not respond to salt stress, unlike the isoenzyme with pI 9.6, which did. Thus, the expression of peroxidase isoenzymes involved in lignification may be differentially controlled by either developemental or stress signals.

Changes in indole-3-acetic acid, indole-3-acetic acid oxidase, and peroxidase isoenzymes in the seeds of developing peach fruits

Changes in indole-3-acetic acid (IAA) content of peach (Prunus persica L. Batsch cv. Merry) seeds were followed during fruit development. The highest concentration of IAA, 2.7 μg/g fresh weight, was found at the beginning of Stage III of fruit development, approximately 50–60 days after anthesis. The IAA-decarboxylating capacity of crude extracts of seeds was also greatest at 55–60 days after anthesis. Four soluble peroxidase isoenzymes were found on anionic electrophoresis. There were no marked changes in two isoenzymes (Rf 0.23 and 0.51), which were present in all three stages of fruit growth. There was a marked increase in a band atRf 0.59 between Stages II and III, and a decrease in a band atRf 0.68 from Stages II to III. Neither band (Rf 0.59 and 0.68) was present at Stage I.

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.

Purification of an anionic isoperoxidase from peach seeds and its immunological comparison with other anionic isoperoxidases

A soluble anionic isoperoxidase (EC 1,11,1,7) was purified from peach (Prunus persica L. Batsch cv. Merry) seeds. Purification was achieved by DEAE-Sephacel, Sephacryl S-300 and CM-cellulose chromatography. The purified isoperoxidase de-carboxylated indole-3-acetic acid (S(0.5) 0.13 mM, Hill coefficient 1.7). Molecular mass, determined by gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis, was ca 60 kDa. Polyclonal antibodies were raised in rabbit against this isoperoxidase. Using immunoprecipitation this isoenzyme was found to be immunologically different from other soluble anionic isoperoxidases isolated from peach seeds.