A. A. Calderón

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.

Peroxidase-mediated formation of resveratrol oxidation products during the hypersensitive-like reaction of grapevine cells to an elicitor from Trichoderma viride

Suspension cell cultures of grapevine ( Vitis vinifera cv. Monastrell) treated with the elicitor Onozuka R-10 ccllulase, isolated from Trichoderma viride , showed a specific hypersensitive-like response characterized by the formation of resveratrol (3,5,4′-trihydroxystilbene) oxidation products (ROPs). Elicitor-induced formation of ROPs was partially stimulated by superoxide dismutase and only partially prevented by catalase. The inhibitor of polyphenol oxidase, L-mimosine, had no effect on the formation of ROPs, while tropolone and ascorbic acid inhibited their formation. The effect of tropolone may be due to its properties as a superoxide anion scavenger. Furthermore, tentoxin, a fungal toxin that inhibits the processing of nuclear-encoded chloroplast polyphenol oxidase also had no effect on the elicitor-mediated formation of ROPs. These results, and the previous observation that grapevine peroxidase is capable of oxidizing resveratrol-like stilbene model compounds, suggest an exclusive role for peroxidase in the elicitormediated formation of ROPs. In this context, elicitor treatment produced an increase in the level of extracellular peroxidases and the appearance of a new basic peroxidase isoenzyme, B 3 , which was correlated with the formation of ROPs.

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 %.

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.

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.

A rapid and continuous spectrophotometric method to measureβ-glucosidase activity based on p-nitrophenylβ-O-D-glucopyranoside hydrolysis

An improved p-nitrophenyl-β-O-D-glucopyranoside method to measure β-glucosidase (EC. 3.2.1.21) activity is presented in which a cyclodextrin is added to the enzymatic mixtures. The inclusion complex consisting of p-nitrophenol and α-cyclodextrin has a higher e than p-nitrophenol alone, thus increasing the sensitivity of the original method. The proposed method makes it possible continuously to record the formation of the reaction product and, since it is possible to measure the initial reaction rates, the method is suitable for kinetic studies. Copyright

Differential expression of a cell wall-localized peroxidase isoenzyme capable of oxidizing 4-hydroxystilbenes during the cell culture of grapevine (Vitis vinifera cv. Airen and Monastrell)

Differential expression and localization of peroxidase isoenzymes capable of oxidizing 4-hydroxystilbenes was studied during establishment of callus cultures from Vitis vinifera ‘Airen’ (anthocyanin-non accumulating) and ‘Monastrell’ (anthocyanin-accumulating) berries. Callus formation from mesocarp tissues was accompanied by differential expression of several peroxidase isoenzyemes located in cell walls, among which only peroxidase isoenzyme A1 was capable of oxidizing 4-hydroxystilbene to any great extent. Likewise, grape cell cultures were capable of accumulating the grape stilbene phytoalexin, resveratrol. However, ε-viniferin, the most powerful phytoalexin in grapevines and previously considered as the product of peroxidase-mediated oxidative coupling of two resveratrol moieties, was only detectable in trace amounts. Since grapevine suspension cell cultures were unable to produce H2O2 as revealed by the luminol test, H2O2 production by the cultured cells appears to be one of the main factors which limits resveratrol oxidation in the cell walls of grapevine cells cultured in suspension.

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.

A spectrophotometric assay for quantitative analysis of the oxidation of 4-hydroxystilbene by peroxidase-H2O2 systems

A new and sensitive spectrophotometric assay has been developed to study and to characterize kinetically the oxidation of 4-hydroxystilbene (an analogue of the putative viniferin precursor, trans-resveratrol, directly involved in the resistance mechanism of the grapevine to fungal diseases) by peroxidase-H2O2 systems. The technique measures the overall increase in absorbance at 248 nm in the reaction media, probably due to the formation of a phenoxy radical of the 4-hydroxystilbene (4-HS). This technique was developed by using the purified isoenzyme C of horseradish peroxidase and all the validity criteria (sensitivity and reproducibility) were checked. The results show that it is especially suitable for low activity measurements. It was finally applied to the determination of the oxidation rate of 4-HS by peroxidases isolated from the media of suspension-cultured grapevine cells, at two different developmental stages.