Comparative biochemistry of the oxidative burst produced by rose and french bean cells reveals two distinct mechanisms

Abstract

Cultured cells of rose (Rosa damascena) treated with an elicitor derived from Phytophthora spp. and suspension-cultured cells of French bean (Phaseolus vulgaris) treated with an elicitor derived from the cell walls of Colletotrichum lindemuthianum both produced H2O2. It has been hypothesized that in rose cells H2O2 is produced by a plasma membrane NAD(P)H oxidase (superoxide synthase), whereas in bean cells H2O2 is derived directly from cell wall peroxidases following extracellular alkalinization and the appearance of a reductant. In the rose/Phytophthora spp. system treated with N, N-diethyldithiocarbamate, superoxide was detected by a N, N'-dimethyl-9,9'-biacridium dinitrate-dependent chemiluminescence; in contrast, in the bean/C. lindemuthianum system, no superoxide was detected, with or without N,N-diethyldithiocarbamate. When rose cells were washed free of medium (containing cell wall peroxidase) and then treated with Phytophthora spp. elicitor, they accumulated a higher maximum concentration of H2O2 than when treated without the washing procedure. In contrast, a washing treatment reduced the H2O2 accumulated by French bean cells treated with C. lindemuthianum elicitor. Rose cells produced reductant capable of stimulating horseradish (Armoracia lapathifolia) peroxidase to form H2O2 but did not have a peroxidase capable of forming H2O2 in the presence of reductant. Rose and French bean cells thus appear to be responding by different mechanisms to generate the oxidative burst.

Figure 4

A, H₂O₂ accumulation. Inhibition by DPI of the luminol-dependent chemiluminescence of rose cells elicited with cell wall extract from Phytophthora spp. and of French bean cells elicited with cell wall extract from C. lindemuthianum. B, Enzyme activity. Inhibition by DPI of the synthesis of superoxide by rose cell NADH oxidase and of H₂O₂ (luminol-dependent chemiluminescence) by horseradish peroxidase in the presence of 0.5 mm Cys. The points indicate means (bars are ses) from at least three independent experiments.

Figure 1

Accumulation of H₂O₂ (luminol-dependent chemiluminescence) in the medium of rose (A) and French bean (B) cells elicited by cell wall extract from Phytophthora spp. or C. lindemuthianum. Representative plots are shown. For rose cells, the peak H₂O₂ concentrations were 20 ± 8 μm (mean ± se; n = 4) with Phytophthora spp. elicitor and 7 ± 2 μm (n = 4) with C. lindemuthianum elicitor. For French bean cells, the peak H₂O₂ concentrations were 133 ± 39 (n = 5) with C. linedmuthianum elicitor and 16 ± 3 (n = 3) with Phytophthora spp. elicitor.

Figure 2

Effects of washing on the luminol-dependent chemiluminescence of rose cells elicited by cell wall extract from Phytophthora spp. and of French bean cells elicited by cell wall extract from C. lindemuthianum. The data for rose cells were calculated from Qian et al. (1993). Results are means ± se (n = 3). For rose cells, values were normalized to the mean with washed cells; for French bean cells, values were normalized to the mean with unwashed cells.

Figure 3

Effect of KCN on luminol-dependent chemiluminescence of French bean cells, elicited by cell wall extract from C. lindemuthianum and assayed for 10 or 15 min after the addition of elicitor. The points indicate means (bars are ses) from two to three independent experiments.

Figure 5

Induction by cell wall extracts of Phytophthora spp. (Ph. elicitor) and C. lindemuthianum (C. elicitor) of secretion from rose cells of an agent that stimulates H₂O₂ synthesis by horseradish peroxidase. The data represent means from three independent experiments. By analysis of variance, variation among beakers was shown to be significant at the 2% confidence level; variation among sampling times was significant at the 0.2% level. Beaker 3 (C. elicitor) at 30 min was significantly different from all other samples (Student's t test, 5% level).

Figure 6

Reaction of 0.1 mm DDC with 4 mm H₂O₂. The peaks at 258 and 283 nm represent DDC in the absence of H₂O₂; the spectrum for 4 mm H₂O₂ alone is also shown. The curves identified as 10 s, 3 min, and 10 min show the spectrum of the DDC sample at the indicated times after the addition of H₂O₂.