Detection of several enzymatic activities in leaf prints of cucumber plants

Abstract

Leaf printing directly onto nitrocellulose membranes under high pressure was adapted for screening activities of defence-related enzymes after inoculation of cucumber plants with Colletotrichum lagenarium, tobacco necrosis virus (TNV) or treatment with dipotassium phosphate and after inoculation of tobacco plants with tobacco mosaic virus (TMV). Increased activities of peroxidase, polyphenol oxidase, lipoxygenase, chitinase and α-glucosidase were detected in cucumber leaves in the vicinity of lesions caused by pathogens or phosphate application. The activity of β-glucosidases was increased in cucumber leaves by TNV, to a lesser extent with C. lagenarium and was not altered by phosphate treatment. Galactosidase activity was not induced in cucumber leaves. Low activity of β-1,3-glucanase in cucumber precluded the detection of this enzyme by the tissue printing technique. However, an increase of β-1,3-glucanase activity was detected around lesions caused by TMV in tobacco. Peroxidase, polyphenol oxidase, α- and β-glucosidases and galactosidase were detected with specific substrates on the membranes after leaf printing. Activities of chitinase and β-1,3-glucanase were determined as lytic zones in substrate-containing overlaying gels incubated with leaf prints on the membranes and lipoxygenase was detected on the membranes by the reaction with specific antibodies. The leaf printing technique is applicable to the determination of the spatial distribution of various defence-related enzymatic activities and their relevance to disease resistance in plants.