Department of Biological and Physical Sciences, University of Southern Queensland, Toowoomba, Queensland 4350, Australia

Background and objectives
The rapid production of reactive oxygen species (ROS) (particularly superoxide anion and its dismutation product hydrogen peroxide) during disease resistance responses has been widely studied [1, 2]. A variety of methods have been used to detect and monitor superoxide radicals during this oxidative burst, in particular the reduction of either the redox protein cytochrome c, or the tetrazolium dye NBT. Both of these methods have potential shortcomings. Cytochrome c is a 12.5-kDa protein and may not diffuse freely across plant cell walls from external solutions to the plasmalemma surface. A large proportion of the radicals may therefore escape reaction with the protein. NBT, although a much smaller molecule and able to cross the cell wall, produces an insoluble formazan when reduced. While this renders this agent useful for localization studies, it does not assist kinetic studies or estimation of the yield of superoxide intercepted by the dye. We have recently developed assays for superoxide based on the tetrazolium dyes XTT and MTS, which reduce to soluble formazans [3]. An XTT-based assay has detected the production of superoxide radicals in tobacco cell cultures challenged with avirulent zoospores of Phytophthora parasitica var. nicotianae [4]. In view of the widespread interest in the mechanisms by which ROS are produced, in particular the possibility that hydrogen peroxide production can occur independently of superoxide generation, our objectives are to compare the yields of superoxide radicals and hydrogen peroxide using a range of assay procedures.

Results and discussion
The stabilities of the pure formazans of XTT and MTS have been assessed at different pHs and in the presence of tobacco cells. While XTT formazan is stable in the presence of tobacco cells in buffer, MTS formazan is slowly degraded. A peroxidase inhibitor, salicylhydroxamic acid, does not prevent this degradation. Spectra indicate that MTS formazan is not re-oxidized to MTS by the plant cells. Neither dye is reduced by hydrogen peroxide. Thus XTT but not MTS appears suitable for the assay of superoxide production by suspension-cultured tobacco cells challenged with an avirulent race of Phytophthora parasitica var. nicotianae.

Superoxide production during the second, major phase of the oxidative burst was then quantified by monitoring the production of formazan in the presence of XTT. The amount of superoxide produced was ca 9x10-9 mol/min/mg protein. Cytochrome c detected only one-tenth this amount of superoxide [4], presumably due to the inability of the protein to cross the cell wall. Two methods are currently being trialled to estimate hydrogen peroxide production. These methods are the reaction of hydrogen peroxide with the fluorescent dye pyranine, and oxygen evolution in the presence of catalase in an oxygen electrode. Initial experiments indicate that the electrode-based method is more reliable than the fluorescence assay, and that significant metabolism of the peroxide in the presence of cells can lead to under-estimation of the rate of production. Early results are consistent with a scheme in which hydrogen peroxide is formed from the dismutation of superoxide radicals.

The authors acknowledge financial support from the Australian Research Council.

1. Sutherland MW, 1991. Physiological and Molecular Plant Pathology 39, 79-93.
2. Wojtaszek P, 1997. Biochemical Journal 322, 681-692.
3. Sutherland MW, Learmonth BA, 1997. Free Radical Research 27, 283-289.
4. Able AJ, Guest DI, Sutherland MW, 1998. Plant Physiology (in press).