Elicitor-stimulated ion fluxes and O2- from the oxidative burst are essential components in triggering defense gene activation and phytoalexin synthesis in parsley

Abstract

Fungal elicitor stimulates a multicomponent defense response in cultured parsley cells (Petroselinum crispum). Early elements of this receptor-mediated response are ion fluxes across the plasma membrane and the production of reactive oxygen species (ROS), sequentially followed by defense gene activation and phytoalexin accumulation. Omission of Ca2+ from the culture medium or inhibition of elicitor-stimulated ion fluxes by ion channel blockers prevented the latter three reactions, all of which were triggered in the absence of elicitor by amphotericin B-induced ion fluxes. Inhibition of elicitor-stimulated ROS production using diphenylene iodonium blocked defense gene activation and phytoalexin accumulation. O2- but not H2O2 stimulated phytoalexin accumulation, without inducing proton fluxes. These results demonstrate a causal relationship between early and late reactions of parsley cells to the elicitor and indicate a sequence of signaling events from receptor-mediated activation of ion channels via ROS production and defense gene activation to phytoalexin synthesis. Within this sequence, O2- rather than H2O2 appears to trigger the subsequent reactions.

Figure 1

Time courses of changes in Ca²⁺ (A), H⁺ (B), K⁺ (C) and Cl⁻ (D) concentrations stimulated in suspension-cultured parsley cells by treatment with crude cell-wall elicitor (50 μg/ml; •), amphotericin B (50 μM, ▪), nystatin (50 μM, □), digitonin (50 μM, ▵), or sterile water (○).

Figure 2

Time courses of H₂O₂ accumulation in the medium of cultured parsley cells treated with crude cell wall-elicitor (50 μg/ml; •), amphotericin B (50 μM, ▪), nystatin (50 μM, □), digitonin (50 μM, ▵), or water (○).

Figure 3

Run-on transcription of defense-related genes in nuclei isolated from cultured parsley cells 3 or 6 hr after treatment with water (“untreated”), crude cell-wall elicitor (50 μg/ml), amphotericin B (50 μM), nystatin (50 μM), or digitonin (50 μM). The homologous cDNA probes used for hybridization encode phenylalanine ammonia-lyase (pal), 4-coumarate:CoA ligase (4cl), chalcone synthase (chs), S-adenosyl-l-methionine:bergaptol O-methyltransferase (bmt), tyrosine decarboxylase (tyrdc), hydroxyproline-rich glycoprotein (hrgp), anionic peroxidase (per), S-adenosyl-l-homocystein hydrolase (shh), S-adenosyl-l-methionine synthetase 1 (sms1), intracellular pathogenesis-related proteins 1 and 2 (pr1 and pr2), proteins of unknown function (eli3, eli6, eli7, eli8, eli10, eli12, eli16, eli17, rep), polyubiquitin (ubi4), and ribosomal 18S RNA (r18s).

Figure 4

Induction of phytoalexin production in cultured parsley cells by O₂⁻. KO₂ (250 μM, suspended in dimethyl sulfoxide [0.1%]) was added to the culture medium. Equivalent amounts of dimethyl sulfoxide were added to the incubations without KO₂. For comparison, parsley cells were treated with the peptide elicitor, Pep-13 (400 nM). The inhibitors DDC and DPI were applied at 0.5 mM and 10 μM, respectively. Error bars indicate standard deviations (9–28 independent experiments) within a 95% confidence level.

Figure 5

Hypothetical scheme for elicitor-induced signal transduction in parsley.