M. López-Serrano

Purification and characterization of α‐3′,4′‐anhydrovinblastine synthase (peroxidase‐like) from Catharanthus roseus (L.) G. Don

An H2O2‐dependent enzyme capable of coupling catharanthine and vindoline into α‐3′,4′‐anhydrovinblastine (AVLB) was purified to apparent homogeneity from Catharanthus roseus leaves. The enzyme shows a specific AVLB synthase activity of 1.8 nkat/mg, and a molecular weight of 45.40 kDa (SDS‐PAGE). In addition to AVLB synthase activity, the purified enzyme shows peroxidase activity, and the VIS spectrum of the protein presents maxima at 404, 501 and 633 nm, indicating that it is a high spin ferric heme protein, belonging to the plant peroxidase superfamily. Kinetic studies revealed that both catharanthine and vindoline were substrates of the enzyme, AVLB being the major coupling product.

Cloning and Molecular Characterization of the Basic Peroxidase Isoenzyme from Zinnia elegans, an Enzyme Involved in Lignin Biosynthesis

The major basic peroxidase from Zinnia elegans (ZePrx) suspension cell cultures was purified and cloned, and its properties and organ expression were characterized. The ZePrx was composed of two isoforms with a Mr (determined by matrix-assisted laser-desorption ionization time of flight) of 34,700 (ZePrx34.70) and a Mr of 33,440 (ZePrx33.44). Both isoforms showed absorption maxima at 403 (Soret band), 500, and 640 nm, suggesting that both are high-spin ferric secretory class III peroxidases. Mr differences between them were due to the glycan moieties, and were confirmed from the total similarity of the N-terminal sequences (LSTTFYDTT) and by the 99.9% similarity of the tryptic fragment fingerprints obtained by reverse-phase nano-liquid chromatography. Four full-length cDNAs coding for these peroxidases were cloned. They only differ in the 5′-untranslated region. These differences probably indicate different ways in mRNA transport, stability, and regulation. According to the kcat and apparent KmRH values shown by both peroxidases for the three monolignols, sinapyl alcohol was the best substrate, the endwise polymerization of sinapyl alcohol by both ZePrxs yielding highly polymerized lignins with polymerization degrees ≥87. Western blots using anti-ZePrx34.70 IgGs showed that ZePrx33.44 was expressed in tracheary elements, roots, and hypocotyls, while ZePrx34.70 was only expressed in roots and young hypocotyls. None of the ZePrx isoforms was significantly expressed in either leaves or cotyledons. A neighbor-joining tree constructed for the four full-length cDNAs suggests that the four putative paralogous genes encoding the four cDNAs result from duplication of a previously duplicated ancestral gene, as may be deduced from the conserved nature and conserved position of the introns.

Peroxidase: a multifunctional enzyme in grapevines

Peroxidases are heme-containing enzymes that catalyse the one-electron oxidation of several substrates at the expense of H2O2. They are probably encoded by a large multigene family in grapevines, and therefore show a high degree of polymorphism. Grapevine peroxidases are glycoproteins of high thermal stability, whose molecular weight usually ranges from 35 to 45 kDa. Their visible spectrum shows absorption bands characteristic of high-spin class III peroxidases. Grapevine peroxidases are capable of accepting a wide range of natural compounds as substrates, such as the cell wall protein extensin, plant growth regulators such as IAA, and phenolics such as benzoic acids, stilbenes, flavonols, cinnamyl alcohols and anthocyanins. They are located in cell walls and vacuoles. These locations are in accordance with their key role in determining the final cell wall architecture, especially regarding lignin deposition and extensin insolubilization, and the turnover of vacuolar phenolic metabolites, a task that also forms part of the molecular program of disease resistance. Although peroxidase is a constitutive enzyme in grapevines, its levels are strongly modulated during plant cell development and in response to both biotic and abiotic environmental factors. To gain an insight into the metabolic regulation of peroxidase, several authors have studied how grapevine peroxidase and H2O2 levels change in response to a changing environment. Nevertheless, the results obtained are not always easy to interpret. Despite such difficulties, the response of the peroxidase-H2O2 system to both UV-C radiation and Trichoderma viride elicitors is worthy of study. Both UV-C and T. viride elicitors induce specific changes in peroxidase isoenzyme / H2O2 levels, which result in specific changes in grapevine physiology and metabolism. In the case of T. viride-elicited grapevine cells, they show a particular mechanism for H2O2 production, in which NADPH oxidase-like activities are apparently not involved. However, they offer a unique system whereby the metabolic regulation of peroxidase by H2O2, with all its cross-talks and downstream signals, may be elegantly dissected.

Spectrophotometric determination of rosmarinic acid in plant cell cultures by complexation with Fe2+ ions

A spectrophotometric method for determining rosmarinic acid (α-O-caffeoyl-3,4-dihydroxyphenyllactic acid) in unpurified methanolic extracts from Lavandula cell cultures is reported. It is based on a complexation reaction of rosmarinic acid with Fe2+ to give a blue-dark product with λmax=572 nm and ɛ573=3.82×103 l mol−1 cm−1. The stoichiometry of the reaction and the optimal conditions for colour development are checked. The sensitivity and accuracy of this spectrophotometric method are compared with UV-spectral and HPLC methods for determining rosmarinic acid in plant cell cultures; similar results are obtained.

Characterization of the last step of lignin biosynthesis in Zinnia elegans suspension cell cultures

The last step of lignin biosynthesis in Zinnia elegans suspension cell cultures (SCCs) catalyzed by peroxidase (ZePrx) has been characterized. The k 3 values shown by ZePrx for the three monolignols revealed that sinapyl alcohol was the best substrate, and were proportional to their oxido/reduction potentials, signifying that these reactions are driven exclusively by redox thermodynamic forces. Feeding experiments demonstrate that cell wall lignification in SCCs is controlled by the rate of supply of H2O2. The results also showed that sites for monolignol β‐O‐4 cross‐coupling in cell walls may be saturated, suggesting that the growth of the lineal lignin macromolecule is not infinite.

Purification and characterization of a basic peroxidase isoenzyme from strawberries

Abstract Canned syrup strawberries (Fragaria ananassa var. Oso Grande, Tudla and Chandler) after appertization showed significant signs of red pigment decay and the appearance of visible browning reactions. Even after this industrial processing, residual peroxidase activity may be measured in drained (syrup-free) canned strawberries, suggesting that a partially thermostable peroxidase activity may be involved in these browning reactions. Strawberries contain a peroxidase isoenzyme of basic pI, which is the only component of peroxidase polymorphism in the whole fruit. For this reason, this isoenzyme was purified by preparative isoelectric focusing in glycerol-stabilized 3.5–10.0 pH gradients and by liquid chromatography on CM-cellulose, and characterized as regards its catalytic properties against several phenols. The results showed that this isoenzyme is capable of oxidizing phenols only in the presence of H2O2, lacking oxidase (catecholase, cresolase, and laccase) activities. These results, and the previous observation that its homologous isoenzyme in other plant species may play a role in anthocyanin turnover and degradation, suggest that a participation of this peroxidase isoenzyme in browning reactions in canned syrup strawberries should be taken into account.

Reversed-phase and size-exclusion chromatography as useful tools in the resolution of peroxidase-mediated (+)-catechin oxidation products

The peroxidase-catalysed oxidation of plant phenolics involves one-electron oxidation reactions, and yields unstable mono-radical species, which couple to generate heterogeneous product mixtures of different degrees of polymerisation. One such phenolic susceptible to oxidation by peroxidase is (+)-catechin. Low-pressure chromatography on Sephadex LH-20, using methanol as mobile phase, resolves the main peroxidase-mediated (+)-catechin oxidation products into a dimeric compound (dehydrodicatechin A) and an oligomeric fraction with a polymerisation degree equal or greater than 5. These pure fractions were used to develop rapid high-performance liquid chromatographic methods, both reversed-phase and size-exclusion chromatography for the direct analysis of the peroxidase-mediated (+)-catechin oxidation products. The joint use of both chromatographic systems permitted the qualitative and quantitative identification of the peroxidase-mediated (+)-catechin oxidation products, and can thus be considered as a useful tool for analysing the complex mixtures of natural bioactive plant products synthesized in reactions catalyzed by plant peroxidases.

Peroxidase in unripe and processing-ripe strawberries

Abstract The activity, enzymatic polymorphism and tissue localization of peroxidase in unripe and processing-ripe strawberries ( Fragaria ananassa var. Oso Grande) was studied. The results showed that both unripe and processing-ripe strawberries contain a peroxidase isoenzyme of basic pI, which is the only component of peroxidase polymorphism in the fruits and which is mainly localized in the concentric array of the vascular bundles and in the vascular connections with the seeds. This isoenzyme is also located in the epidermal cells. The activity of this basic peroxidase isoenzyme, which is apparently bound to acidic polysaccharides during extraction and from which it may be solubilized by treatment with Caylase 345L, did not vary with ripening. From these results, it may be concluded that this basic peroxidase isoenzyme, similar to its homologous isoenzyme in grape berries, is involved in the lignification of the vascular tissues during fruit development.

Ca2+ and Mg2+ ions counteract the reduction by fosetyl-Al (aluminum tris[ethyl phosphonate]) of peroxidase activity from suspension-cultured grapevine cells

Grapevine (Vitis vinifera cv. Monastrell) cell suspension cultures were treated with 1.5 mM fosetyl-Al, a frequently used systemic fungicide for grapevine diseases caused by oomycetes. These cells showed a reduction in the level of peroxidase activity secreted into the culture media when compared to non-treated cells, the effect being mainly related to a decrease in the level of the basic B1 peroxidase isozyme. The effect of fosetyl-Al on peroxidase was analogous to that observed with the Ca2+-channel blockers Co2+, Cd2+ and La3+, and was counteracted by Ca2+ ions, but was not reversed when the Ca2+-ionophore A23187 was added to the culture media. Moreover, the effect of fosetyl-Al on peroxidase activity and peroxidase isozymes was also partially reversed by Mg2+ ions but not by Sr2+, and was accentuated by Ba2+ ions. These results suggested that Ca2+ and Mg2+ ions specifically overcome the inhibitory effect of fosetyl-Al on peroxidase. In this context, an apoplastic Ca2+/Mg2+-displacement hypothesis is proposed for the mechanism of action of fosetyl-Al on peroxidase from grapevine cells.

A Blue Pigment Derived from Rosmarinic Acid and FeSO4 is Responsible for the Blue Pigment Secreted into the Medium by Lavandula x intermedia Cell Cultures Grown in the Dark

Abstract Rosmarinic acid (α-O-caffeoyl-3,4-dihydroxy phenyllactic acid) is the main phenolic constituent (1.0–7.0 μmol/kg) present in methanolic extracts of the medium from Lavandula × intermedia cell cultures which have been grown on agar gel containing Murashige & Skoog medium. In this medium, the concentration of FeSO4 is 100 μmol/kg. Since rosmarinic acid yields a blue pigment upon reaction with FeSO4 (λmax = 572 nm and ϵ572 = 3,820 M−1 cm−1 at pH 6.0), this this pigment appears to be the main compound responsible for the blue colour which is secreted into the medium by Lavandula × intermedia cell cultures grown in the dark.