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Total soluble phenols, soluble flavanols, (+)-catechin, ferulic acid and 1-O-feruloyl-beta-d-glucose were analyzed during the development of a strawberry (Fragariaxananassa, cv. Chandler) callus culture. The time-course changes of the different phenols assayed were well correlated with callus growth and morphology. The changes in polyphenol oxidase (EC 1.10.3.1-2) and beta-glucosidase (EC 3.2.1.21) activities in the callus were also examined. The total phenol, soluble flavanols and (+)-catechin contents were high during the preexponential and exponential phases of growth. The subsequent decrease in (+)-catechin concentration coincided with high levels of polyphenol oxidase activity. The 1-O-feruloyl-beta-d-glucose content was highest as callus growth ceased, and its subsequent decrease was accompanied by the increased production of ferulic acid. This increase in ferulic acid was accompanied by an increase in beta-glucosidase activity. The ferulic acid content decreased at the end of culture, when callus growth had stopped and showed clear symptoms of senescence. This decrease in the ferulic acid concentration was accompanied by an increase in the levels of ferulic acid bound to cell wall components.

SUMMARYSuspension cell cultures of grapevine {Vitis vinifera, cv. Monastrell) treated with an elicitor (cellulase, Onozuka R-10) from Trichoderma viride showed a hypersensitive-like response. This was characterized by cell plasmolysis and was accompanied by localized cell death, which was concomitant with cell culture browning, itself probably due to an activation of oxidative phenolic metabolism driven by a large increase in endogenous levels of H.^O2. In addition to these responses, the treatment of cell cultures with the elicitor produced an increase in amounts of benzoic acid and of resveratrol, the latter a potent phytoalexin of grapevines. This hypersensitive-like response was specific since none of the above responses was obtained with other cell wall-degrading enzymes from several sources, or with inocula of either mycelial extracts or culture filtrates of Botrytis cinerea. These results are discussed in the light of a disease-resistance reaction induced in grapevine cells by a product of T. viride, a fungal agent characterized by its effective biocontrol of Botrytis cinerea, the causal agent of grey mould in grapevines.

A new and sensitive chromogenic assay has been developed to characterize peroxidative activities in plant tissues, applicable to kinetic, zymographic and cytochemical studies on plant peroxidases. This technique is based on the H,O,-dependent oxidation of 4-methoxy-a-naphthol (4-MN) to yield a non-diffusable deep-blue product, the appearance of which is kinetically correlated with the disappearance of 4-MN. Oxidation conditions have been optimized. Since 4-MN is a specific substrate for peroxidase against other heme proteins (catalase and cytochrome c) and, because of the high sensitivity and low detection limit of peroxidase activity in the 4-MN assay, this compound is a powerful reagent in the study of peroxidase activity from plant tissues. The assay has been applied to the kinetic, zymographic and cytochemical characterization of peroxidase activity in grape cell cultures.

Two species of Brassica were used to study their acclimation to heat and high illumination during the first stages of development. One, Brassica fruticulosa, is a wild species from south-east Spain and is adapted to both heat and high light intensity in its natural habitat, while the other, Brassica oleracea, is an agricultural species that is widely cultivated throughout the world. Growing Brassica plants under high irradiance and moderate heat was seen to affect the growth parameters and the functioning of the photosynthetic apparatus. The photosystem II (PSII) quantum yields and the capacity of photosynthetic electron transport, which were lower in B. fruticulosa than in B. oleracea, decreased in B. oleracea plants when grown under stress conditions, indicating inhibition of PSII. However, in B. fruticulosa, the values of these parameters were similar to the values of control plants. Photosystem I (PSI) activity was higher in B. fruticulosa than in B. oleracea, and in both species this activity increased in plants exposed to heat and high illumination. Immunoblot analysis of thylakoid membranes using specific antibodies raised against the NDH-K subunit of the thylakoidal NADH dehydrogenase complex (NADH DH) and against plastid terminal oxidase (PTOX) revealed a higher amount of both proteins in B. fruticulosa than in B. oleracea. In addition, PTOX activity in plastoquinone oxidation, and NADH DH activity in thylakoid membranes were higher in the wild species (B. fruticulosa) than in the agricultural species (B. oleracea). The results indicate that tolerance to high illumination and heat of the photosynthetic activity was higher in the wild species than in the agricultural species, suggesting that plant adaptation to these stresses in natural conditions favours subsequent acclimation, and that the chlororespiration process is involved in adaptation to heat and high illumination in Brassica.

Cell wall fragments, isolated from dark‐grown Lupinus albus L. (cv. multolupa) hypocotyls, and purified by washing with Triton X‐100, did not show detectable contamination by enzyme markers of cytosol or endoplasmic reticulum in which peroxidase activity was also located by electron microscopy. Peroxidase (EC 1.11.1.7) isoenzymes, solubilized from these cell wall fractions by high saline forces, showed a high affinity towards guaiacyl type substrates, whereas syringyl type substrates were not oxidized. On the other hand, both the isoenzyme patterns and the substrate specificity of these cell wall isoperoxidases could be altered by chromatographic and denaturating/renaturating procedures, which break the protein‐phenol interactions, and this suggests the presence of phenol‐induced conformers. These results draw attention to the origin of the enzymatic polymorphism, and catalytic properties, of the peroxidase activity located in the cell wall.

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