The oxidative burst, the generation of
reactive oxygen species (ROS) in response to microbial pathogen attack, is a ubiquitous early part of the resistance mechanisms of plant cells. It has also become apparent from the study of a number of plant-pathogen interactions and those modelled by elicitor treatment of cultured cells that there may be more than one mechanism operating. However, one mechanism may be dominant in any given species.
NADPH oxidases have been implicated in a number of systems and have been cloned and characterized. However, the
enzyme system which is the major source of ROS in French bean (Phaseolus vulgaris) cells treated with a cell wall elicitor from Colletotrichum lindemuthianum, appears to be dependent on an exocellular
peroxidase. The second component, the extracellular alkalinization, occurs as a result of the Ca(2+) and
proton influxes and the K(+) efflux common to most elicitation systems as one of the earliest responses. The third component, the actual
reductant/substrate, has remained elusive. The low molecular weight compound composition of apoplastic fluid was compared before and after elicitation. The substrate only becomes available some min after elicitation and can be extracted, so that by comparing the profiles by LC-MS it has been possible to identify possible substrates. The mechanism has proved to be complex and may involve a number of low molecular weight components. Stimulation of H(2)O(2) production was observed with
saturated fatty acids such as
palmitate and
stearate without concomitant
oxylipin production. This biochemical evidence is supported by immunolocalization studies on papillae forming at
bacterial infection sites that show the
peroxidase isoform present at sites of H(2)O(2) production revealed by
cerium chloride staining together with the cross-linked wall
proteins and
callose and
callose synthase. The
peroxidase has been cloned and expressed in Pichia pastoris and has been shown to catalyse the oxidation reaction with the same kinetics as the purified
enzyme. Furthermore, Arabidopsis plants transformed heterologously using the French bean
peroxidase in antisense orientation have proved to be highly susceptible to bacterial and fungal pathogens. Thus it is possible that Arabidopsis is another species with the potential to mount an apoplastic oxidative burst and these transformed plant lines may be useful to identify the
peroxidase that is responsible.