M. A. Pedreño

Effect of dimethyl-β-cyclodextrins on resveratrol metabolism in Gamay grapevine cell cultures before and after inoculation with shape Xylophilus ampelinus

Gamay cell cultures were treated with dimethyl-β-cyclodextrins in order to ascertain their effect on resveratrol metabolism before and after inoculation with Xylophilus ampelinus. The results showed that, in grapevine cell suspensions, dimethyl-β-cyclodextrins themselves do not need the co-cultivation with bacteria to act as elicitors of the cells producing trans-resveratrol, a phytoalexin of grapevines. Dimethyl-β-cyclodextrins protected cell suspensions against bacteria by maintaining a high level of peroxidase activity.

Bioinformatic and functional characterization of the basic peroxidase 72 from Arabidopsis thaliana involved in lignin biosynthesis.

Lignins result from the oxidative polymerization of three hydroxycinnamyl (p-coumaryl, coniferyl, and sinapyl) alcohols in a reaction mediated by peroxidases. The most important of these is the cationic peroxidase from Zinnia elegans (ZePrx), an enzyme considered to be responsible for the last step of lignification in this plant. Bibliographical evidence indicates that the arabidopsis peroxidase 72 (AtPrx72), which is homolog to ZePrx, could have an important role in lignification. For this reason, we performed a bioinformatic, histochemical, photosynthetic, and phenotypical and lignin composition analysis of an arabidopsis knock-out mutant of AtPrx72 with the aim of characterizing the effects that occurred due to the absence of expression of this peroxidase from the aspects of plant physiology such as vascular development, lignification, and photosynthesis. In silico analyses indicated a high homology between AtPrx72 and ZePrx, cell wall localization and probably optimal levels of translation of AtPrx72. The histochemical study revealed a low content in syringyl units and a decrease in the amount of lignin in the atprx72 mutant plants compared to WT. The atprx72 mutant plants grew more slowly than WT plants, with both smaller rosette and principal stem, and with fewer branches and siliques than the WT plants. Lastly, chlorophyll a fluorescence revealed a significant decrease in ΦPSII and qL in atprx72 mutant plants that could be related to changes in carbon partitioning and/or utilization of redox equivalents in arabidopsis metabolism. The results suggest an important role of AtPrx72 in lignin biosynthesis. In addition, knock-out plants were able to respond and adapt to an insufficiency of lignification.

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.

The Cell Wall Localization of two Strongly Basic Isoperoxidases in Etiolated Lupinus albus Hypocotyls and its Significance in Coniferyl Alcohol Oxidation and Indole-3-Acetic Acid Catabolism

Summary Two strongly basic peroxidase (EC 1.11.1.7) isoenzymes, B3 and B4, of isoelectric points 9.5 and 9.7, are localized in cell walls of young Lupinus albus hypocotyls. The binding of B3 and B4 isoenzymes to cell walls is specific and, unlike that observed for acidic isoperoxidases, is maximal at neutral pH values. By comparison with A1 and A2 acidic isoperoxidases, cell wall-bound basic B3 and B4 isoperoxidases are little involved in coniferyl alcohol oxidation and indole-3-acetic acid catabolism. However, due to the localization of these isoenzymes in vacuoles also, they are probably involved in some type of H2O2-dependent oxidation reaction common to both lytic compartments. These results are discussed on the basis of a possible participation of these isoenzymes, as indicated by other authors, in the oxidative transformation of tetracyclic bisquinolizidine-type alkaloids in lupin.

Two distinct cell sources of H2O2 in the lignifying Zinnia elegans cell culture system

Summary.The use of transdifferentiating Zinnia elegans mesophyll cells has proved useful in investigations of the process of xylem differentiation from cambial derivatives. Cultured mesophyll cells can be induced by external stimuli to proceed through temporally controlled developmental programs which conclude in the formation of single-cell-derived dead vascular tracheids and parenchyma-like elements. However, there is a gap in our knowledge concerning the role played by reactive oxygen species (O2− and H2O2) in the development of these vascular elements. In this study, we show by the following four independent and highly selective methods that transdifferentiating Z. elegans mesophyll cells are capable of producing reactive oxygen species: the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay, which monitors O2− production, and the xylenol orange, 2,7-dichlorofluorescein diacetate, and CeCl3 assays, which monitor H2O2 production and localization. The joint use of these biochemical (XTT and xylenol orange) assays and cytochemical (2,7-dichlorofluorescein diacetate and CeCl3) probes revealed that transdifferentiating Z. elegans mesophyll cells do not show an oxidative burst but live in a strongly oxidative state during the entire culture period. In this state, H2O2 is produced by both tracheary and parenchyma-like elements, the nonlignifying parenchyma-like cells acting quantitatively as the main source. The existence of these two sources of H2O2 in this in vitro cell culture system may be especially relevant during the later stages of tracheary cell wall lignification, in which lignifying tracheary elements become hollow. In the case of differentiating tracheary elements, H2O2 was located in the same place and at the same time as the onset of tracheary element lignification, i.e., at the primary cell wall during secondary thickening, supporting the view that the H2O2 produced by this in vitro culture system is destined for use during lignin biosynthesis.

Indole-3-methanol is the main product of the oxidation of indole-3-acetic acid catalyzed by two cytosolic basic isoperoxidases from Lupinus.

The nature of the products of the auxin catabolism mediated by both basic and acidic isoperoxidases has been studied. While indole-3-methanol is only a minor product of the oxidation of indole-3-acetic acid catalyzed by extracellular acidic isoperoxidases, it is the only product of the oxidation of indole-3-acetic acid catalyzed by two cytosolic basic isoperoxidases (EC 1.11.1.7) from lupin (Lupinus albus L.) hypocotyls. The putative indole-3-methanol formed by these latter isoperoxidases was isolated and then characterized by mass spectrometry and 1H-nuclear magnetic resonance spectrometry. These results are discussed with respect to the diversity and compartmentation of the catabolism of indole-3-acetic acid in plant tissues.

Purification and kinetic characterization of a basic peroxidase isoenzyme responsible for lignification in Gamay rouge grape (Vitis vinifera) berries

Abstract Gamay rouge grape ( Vitis vinifera ) berries contain a peroxidase isoenzyme of basic pI, the peroxidase isoenzyme B 5 , which is the major component of peroxidase polymorphism in the whole fruit, and is localized in xylem vessels of grape berries at pigmentation. This isoenzyme has been purified by preparative isoelectric focusing in glycerol-stabilized 3.0–10.0 pH gradients, and characterized as regards its catalytic properties against coniferyl alcohol. The results showed that this isoenzyme is capable of oxidizing coniferyl alcohol with an optimal pH in the range 3.0–6.0. K m values were 0.149 m m for coniferyl alcohol and 0.206 m m for H 2 O 2 . These results suggest that, although the affinity of Gamay rouge peroxidase B 5 towards the lignification substrates is low compared with that shown by other peroxidases involved in lignin biosynthesis, participation of this isoenzyme in the lignification of xylem vessels of Gamay rouge grape berries should be taken into account.

Soluble peroxidase gradients in lupin hypocotyls and the control of the level of polarly transported indole-3yl-acetic acid

The distribution of basic soluble isoperoxidases along the growth gradient of lupin hypocotyl was studied in order to establish the role of these isoenzymes in controlling polarly transported indole-3yl-acetic acid (IAA) levels. The observation that the levels of basic isoperoxidases, which diminish from the young (vascular differentiating) to the older (vascular differentiated) tissues, are related with previously reported IAA oxidation rates in decapitated plants, suggests that these isoenzymes can play a role in the oxidation of IAA during polar transport. The fact that the level of basic isoperoxidases is controlled by IAA in hypocotyl sections harvested from different growth zones is in accordance with the previously described adaptative activation of basic isoperoxidases to IAA content. This adaptative activation of basic isoperoxidases might constitute the basic characteristic of a system of subcellular oscillators, coupled at the cellular level, necessary to generate the supracellular auxinwave associated with auxin transport.

Purification of a basic peroxidase isoenzyme fromCapsicum fruits and the immunoinhibition of its capsaicin oxidation capacity by antibodies raised against horseradish peroxidase

ZusammenfassungPfeffer enthält das basische Peroxidase-Isoenzym B6 in den Vacuolen der Fruchtzellen. Es wurde durch präparative isoelektrische Fokussierung gereinigt und charakterisiert. Die Oxidation von Capsaicin mit dem Enzym erwies sich als H2O2-abhängig und wurde vollständig durch Antikörper gegen Meerrettich-Peroxidase gehemmt. Das Enzym ist ein Schlüsselenzym beim Abbau von Capsaicin.AbstractPepper fruits contain a peroxidase isoenzyme of basic pI, the peroxidase isoenzyme B6, located in vacuoles and the principal component of peroxidase polymorphism in the whole fruit. This isoenzyme was purified by preparative isoelectric focusing in glycerol-stabilized 3.0–10.0 pH gradients and characterized for its ability to oxidize capsaicin (8-methyl-N-vanillyl-6-nonenamide). Spectrophotometric studies illustrated that the capsaicin oxidation by pepper peroxidase isoenzyme B6 was H2O2-dependent and was totally abolished by antibodies raised against horseradish peroxidase. From these studies, it can be concluded that capsaicin is oxidized by pepper peroxidase isoenzyme B6, thus confirming a role for this peroxidase isoenzyme in capsaicin turnover and degradation.

Effect of UV-C on peroxidase isoenzymes in axillary bud cultures ofVitis species differing in fungal resistance toPlasmopara viticola

The effect of shortwave (250 nm) UV radiation (UV-C) on the level of peroxidase activity and peroxidase isoenzyme patterns in leaves of resistant ([Vitis vinifera x Viris riparia] x Vitis rupestris andVitis rupestris) and susceptible (Vitis vinifera) grapevine species toPlasmopara viticola (downy mildew) was studied. The results show that although UV-C did not produce significant changes in peroxidase activity in susceptible species, and only minor changes in resistant species, treatment with UV-light induces an acidic isoperoxidase (isoperoxidase A1), capable of oxidising 4-hydroxystilbenes in resistant species. It was named HSPrx 2. Since peroxidase is apparently the enzyme responsible for ε-viniferin synthesis from resveratrol in grapevines, a close relationship between this peroxidase isoenzyme and ε-viniferin synthesis which occurs in grapevine leaves after UV-C treatment must be expected.