Isolation and characterisation of a class of carbohydrate oxidases from higher plants, with a role in active defence

In a search for novel plant-derived antimicrobial proteins, we screened extracts from salicylic acid (SA)-treated lettuce and sunflower leaves. These extracts displayed very potent antimicrobial activity against a set of phytopathogens. Characterisation of these extracts revealed that in both extracts, proteins of approximately 60 kDa were responsible for the antimicrobial activity. Further characterisation of these proteins and cloning of the respective cDNAs revealed close homology to a range of (plant) oxidases. Dissection of the enzymatic activity of both proteins revealed them to be carbohydrate oxidases (Helianthus annuus carbohydrate oxidase (Ha-CHOX) and Lactuca sativa carbohydrate oxidase (Ls-CHOX)) with broad substrate specificity and with hydrogen peroxide (H2O2) as one of the reaction products. The sunflower transcript, in addition to being SA inducible, was also inducible by fungal pathogens but not by ethylene and jasmonate. To determine whether Ha-CHOX plays a role in pathogen defence, it was transformed into tobacco and the effect of resistance to Pectobacterium carotovorum ssp. carotovorum was examined. Transgenic plants overexpressing Ha-CHOX displayed enhanced resistance to infection by this pathogen, and the resistance level was proportional to enzyme expression.