José Miguel Zapata

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.

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.

Oxidation of hydroquinone by both cellular and extracellular grapevine peroxidase fractions

The oxidation of hydroquinone by two peroxidase (EC 1.11.1.7) fractions obtained from the cells and spent medium of cell cultures of grapevine (Vitis vinifera cv Monastrell) has been studied, and their comparative efficacy (kcat/KM ratio) studied in both the H2O2-consuming and hydroquinone-consuming reactions. While the efficacy in the H2O2-consuming reaction is practically identical for both enzyme fractions, the cellular peroxidase has five-fold more efficacy in the hydroquinone-consuming reaction than the peroxidase located in the spent medium. Screening of cellular peroxidases capable of oxidizing hydroquinone on polyacrylamide gels, by means of a staining reaction based on the nucleophilic attack of 4-aminoantipyrine on p-benzoquinone in acidic media, reveals that all the cellular peroxidase isoenzymes are capable of oxidizing hydroquinone, probably yielding a quinone-diimine as a product of the staining reaction. Since isoperoxidases found in cellular fractions are also present in the spent medium, the values found for the different efficacies in the hydroquinone-consuming reaction must be considered as the results of the different proportions in which each peroxidase isoenzyme was found in the two fractions. The localization of a benzoquinone-generating system of high efficacy inside the plant cell, and probably located in vacuoles, is discussed with respect to the harmful role which the quinone/semiquinone pair might play in cell death, as part of the hypersensitive response expressed within the mechanism of plant disease resistance.

Inactivation of the peroxidase isoenzyme groups, APrx and Lpl BPrx, markers of the in vitro culture of grapevine, by fosetyl-Al (aluminum tris[ethyl phosphonate])

Summary Grapevine ( Vitis vinifera cv. Monastrell) fruits contain, as the only component of peroxidase polymorphism, the peroxidase isoenzyme B 5 , which is the sole component of the peroxidase isoenzyme group HpI BPrx in grapevines. Establishment of suspension cell cultures from grapevine fruits is accompanied by the de novo expression of one acidic (APrx) and one basic (LpI BPrx) peroxidase isoenzyme group in the culture medium, which indicates that the in vitro culture is associated with the new expression of two groups of cell wall-located peroxidase isoenzymes. Treatment of suspension cultured cells with aluminum tris [ethyl phosphonate] (fosetyl-Al) reduces the level of peroxidase activity found in the culture medium, this effect being specific to those isoenzymes that are expressed de novo during cell culture, the isoenzyme groups APrx and LpI BPrx. Thus, fosetyl-Al selectively inactivates the peroxidase isoenzyme groups, APrx and LpI BPrx, which are markers of the process that accompanies the establishment of grapevine cell cultures.

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.

Constitutive expression of extracellular peroxidase isoenzymes capable of oxidizing 4-hydroxystilbenes during the growth cycle of grapevine suspension cell cultures

The constitutive expression of peroxidase isoenzymes which are capable of oxidizing 4-hydroxystilbenes was studied during the growth cycle of suspension cell cultures from grapevine (Vitis vinifera L. cv. Monastrell) berries. the results showed that the growth of suspension cell cultures is accompanied by the constitutive expression of the peroxidase isoenzymes HSPrx 1 and HSPrx 2, previously characterised by their properties for oxidizing 4-hydroxystilbene, the expression of these isoenzymes being characteristic of Monastrell vines brought to a resistant state. However, although the differential catalytic properties of these grapevine isoenzymes are also shown by the horseradish peroxidase, no immunological relationships have been found between the peroxidases from either source.