R. Muñoz

Characterization of the antiradical activity of betalains from Beta vulgaris L. roots

Betalains (betacyanins and betaxanthins), the main pigments of red beet (Beta vulgaris) roots, showed an antiradical effect when measured by the destruction of the 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid (ABTS) free radical generated by the horse-radish peroxidase/hydrogen peroxide-mediated oxidation of ABTS. The antiradical activity of betacyanins was greater than that of the betaxanthins and increased with the pH of the reaction medium. The different antiradical properties shown by both types of betalain is discussed in light of the respective ease with which it is possible to withdraw one electron from their molecules and the stability of their corresponding radicals.

Chlororespiration and tolerance to drought, heat and high illumination

Sun (Chrysanthemum morifolium) and shade (Spathiphyllum wallisii) plants were used to study the effects of drought, heat and high illumination. The stress conditions caused a greater accumulation of hydrogen peroxide in Chrysanthemum morifolium than in Spathiphyllum wallisii leaves. They also resulted in down-regulation of linear electron transport in the leaves of both species, as indicated by a gradual reduction in the photochemistry efficiency of PS II, which was associated with an increase in the non-photochemical quenching of fluorescence. Only a slight decrease in F(v)/F(m) was observed under stress conditions in either plant species, suggesting that the chloroplast is protected by mechanisms that dissipate excess excitation energy to prevent damage to the photosynthetic apparatus. In addition to the effects on photosynthetic activity, changes were also observed by immunoblot analysis in the plastidial NADH DH complex, PTOX and PGR5. The quantities of the PTOX and NDH-H subunit of the thylakoidal NADH DH complex, and the NADH DH activity in the thylakoid membranes were similar in control plants of both species and increased in stressed plants, particularly in Spathiphyllum wallisii. The level of PGR5 polypeptide was higher in Chrysanthemum morifolium than in Spathiphyllum wallisii control plants, while after stress, the quantity of PGR5 increased significantly in Chrysanthemum morifolium and remained constant in Spathiphyllum wallisii. These results indicate that the relative importance of chlororespiration and the cyclic electron pathways in the tolerance to drought, heat and high illumination differs in sun and shade plants, indicating different adaptive mechanisms to the environment. In the conditions studied, the PGR5-dependent cyclic pathway is more active in Chrysanthemum morifolium, a sun species, whereas in Spathiphyllum wallisii, a shade species, other ways involving the NADH DH complex and PTOX are stimulated in response to stress, which results in lower levels of ROS accumulation in the leaves.

Oxidation of coniferyl alcohol by cell wall peroxidases at the expense of indole-3-acetic acid and O2: A model for the lignification of plant cell walls in the absence of H2O2

The oxidation of coniferyl alcohol (CA), a lignin precursor, by cell wall peroxidases may take place at the expense of indole‐3‐acetic acid (IAA) and O2, and in the absence of H2O2. The peroxidase‐catalyzed oxidation of CA shows an optimum at an IAA concentration of 0.33 mM, while higher IAA concentrations are inhibitory. The observation that the oxidation of CA by cell wall peroxidase at the expense of IAA and O2 is inhibited by genistein, a putative endogenous inhibitor of lignification in lupin hypocotyls, supports the view that the H2O2‐generating system coexists with cell wall peroxidase activities involved in lignification, and that it takes place at the expense of IAA and O2.

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.

Reduction of 2,3,5-triphenyltetrazolium chloride by the KCN-insensitive, salicylhydroxamic acid-sensitive alternative respiratory pathway of mitochondria from cultured grapevine cells.

The reduction of 2,3,5-triphenyltetrazolium chloride (TTC) by grapevine cells cultured in suspension was studied in order to assess the reliability of using TTC reduction as a measure of cell viability. Similar to the reduction observed in animals cells, TTC can be reduced in grapevine cells by the cytochrome respiratory path of the mitochondria, although it is mostly reduced (about 72 %) by the alternative respiratory path sensitive to salicylhydroxamic acid. Engagement of the alternative path in TTC reduction was calculated through the √Valt plot, and was established to be 89 %.

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.

Subcellular location of basic and acidic soluble isoperoxidases in lupinus

Abstract The subcellular location of non-particulate (soluble) peroxidases (EC 1.11.1.7) was studied in lignifying lupin ( Lupinus albus L.) hypocotyls. Supernatants of cellular homogenates contain two acidic (A 1 and A 2 ) and two basic (B 1 and B 2 ) isoperoxidases. Studies of centrifugation on isopycnic sucrose gradients of cellular homogenates of plasmolyzed tissues, vacuum infiltration of hypocotyl sections, and binding probes to membranes and cell walls, suggest that while basic soluble isoperoxidases possess a cytoplasmic location, acidic isoperoxidases possess an extracellular location, as freely soluble forms in the intercellular spaces. These results are discussed on the basis of the subcellular location of the auxin catabolism, mediated mainly by the two basic isoperoxidases, which in lupin cells at least, seems to be restricted to the cytosolic compartment.

Isoperoxidase Involvement in the Resistance of Capsicum annuum to Infection by Cucumber Mosaic Virus

Summary Cucumber mosaic virus (CMV) infection of Capsicum annuum leaves provokes a strong increase in soluble peroxidase activity. Isoelectrofocusing of the peroxidase activity in polyacrylamide gel patterns, after staining the soluble protein fractions from both healthy and infected leaves with benzidine/H 2 O 2 , revealed the presence of two acidic and one basic isoperoxidases, the increase in peroxidase activity being mainly due to the former. Staining of peroxidase activity in the gels with coniferyl alcohol/H 2 O 2 showed that acidic isoenzymes were more capable of catalyzing coniferyl alcohol oxidation than basic isoenzymes. Native polyacrylamide gel electrophoresis of the soluble fraction showed that the increase in peroxidase isoenzymes. CMV elicitation of the acidic isoperoxidase through its involvement in the lignification process might be considered as a part of the defense mechanism of Capsicum annuum against viral infection, lignin deposition on plasmodesmata could act as a physical barrier against the cell-to-cell advance of viral particles.

The histochemical localization of anthocyanins in seeded and seedless grapes (Vitis vinifera)

Abstract A technique has been developed for the study of the histochemical localization of anthocyanins in both seeded and seedless grapes ( Vitis vinifera ) through the blotting of freeze/thawed tissue sections on nitrocellulose membranes. After blotting, anthocyanins can be seen principally at the level of the hypodermal tissue before and after exposure to NH 3 vapours. This last treatment provokes the transition of the red anthocyanin flavylium ion to the corresponding blue anthocyanin anhydrobase, this being characteristic of anthocyanin pigments. The reliability of this technique in the histochemical localization of anthocyanins in grapes was confirmed by fractionation and determination of the anthocyanin content in the different 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.