Volatile-mediated killing of Arabidopsis thaliana by bacteria is mainly due to hydrogen cyanide

Abstract

The volatile-mediated impact of bacteria on plant growth is well documented, and contrasting effects have been reported ranging from 6-fold plant promotion to plant killing. However, very little is known about the identity of the compounds responsible for these effects or the mechanisms involved in plant growth alteration. We hypothesized that hydrogen cyanide (HCN) is a major factor accounting for the observed volatile-mediated toxicity of some strains. Using a collection of environmental and clinical strains differing in cyanogenesis, as well as a defined HCN-negative mutant, we demonstrate that bacterial HCN accounts to a significant extent for the deleterious effects observed when growing Arabidopsis thaliana in the presence of certain bacterial volatiles. The environmental strain Pseudomonas aeruginosa PUPa3 was less cyanogenic and less plant growth inhibiting than the clinical strain P. aeruginosa PAO1. Quorum-sensing deficient mutants of C. violaceum CV0, P. aeruginosa PAO1, and P. aeruginosa PUPa3 showed not only diminished HCN production but also strongly reduced volatile-mediated phytotoxicity. The double treatment of providing plants with reactive oxygen species scavenging compounds and overexpressing the alternative oxidase AOX1a led to a significant reduction of volatile-mediated toxicity. This indicates that oxidative stress is a key process in the physiological changes leading to plant death upon exposure to toxic bacterial volatiles.

FIG. 1.

Plant growth inhibition and cyanogenesis by different bacterial strains. White bars indicate plant biomass (fresh weight) after 3 weeks of exposure to bacterial volatiles, expressed as a percentage of control plant biomass (not exposed to volatiles). Black bars indicate total HCN production (μmol) over 3 weeks. P.fluo: Pseudomonas fluorescens CHA0; P.chloro, Pseudomonas chlororaphis subsp. aureofaciens; C.viol, Chromobacterium violaceum CV0; S.plym, Serratia plymuthica IC14; S.marc, Serratia marcescens MG1. Averages of three to four replicates and standard errors are shown. Different lowercase letters (a to g) indicate significant differences (Student t test, P < 0.05).

FIG. 2.

Plant growth inhibition and cyanogenesis by P. fluorescens wild type (CHA0) and an HCN-negative mutant (CHA77). White bars indicate the plant biomass (fresh weight) after 3 weeks of exposure to bacterial volatiles produced by CHA0 or CHA77, expressed as a percentage of control plant biomass (not exposed to volatiles). Black bars indicate the total HCN production (μmol) over 3 weeks. CHA0, P. fluorescens wild type; CHA77, P. fluorescens hcnABC mutant. The averages of three to four replicates and standard errors are shown. The pictures are representative examples of control plants (upper picture), CHA0-treated plants (middle picture), and CHA77-treated plants (lower picture) after 3 weeks of incubation.

FIG. 3.

Plant growth inhibition by chemical addition of HCN. (A) Plant biomass (fresh weight) after 3 weeks of exposure to different amounts of HCN, expressed as a percentage of control plant biomass (not exposed to HCN). Averages of three to four replicates and standard errors are shown. Different lowercase letters (a to c) indicate significant differences (Student t test, P < 0.05). (B) Representative pictures of plants grown for 3 weeks after treatment with different amounts of HCN.

FIG. 4.

Effect of quorum sensing on cyanogenesis and phytotoxicity. White bars indicate the plant biomass (fresh weight) after 3 weeks of exposure to bacterial volatiles, expressed as a percentage of control plant biomass (not exposed to volatiles). Black bars give the total HCN production (μmol) over 3 weeks. (A) Chromobacterium violaceum CV0; (B) Pseudomonas chlororaphis subsp. aureofaciens; (C) Pseudomonas aeruginosa PAO1b; (D) Pseudomonas aeruginosa PUPa3. See Table 1 for more details. sup., supplemented with AHLs. Averages of three to four replicates and standard errors are shown. Different lowercase (a to e) letters indicate significant differences (Student t test, P < 0.05).

FIG. 5.

Effect of ascorbate addition and alternative oxidase (AOX) overexpression on A. thaliana's tolerance to deleterious bacterial volatiles. Black bars, no ascorbate addition; white bars, addition of 0.1 mM ascorbate as sodium ascorbate to the plant's culture medium. ctrl., empty vector plants (N6590); AOX+, plants overexpressing AOX (N6595); AOX−, plants silencing AOX (N6599). See Table 2 for more details. Averages of three to four replicates and standard errors are shown. Within each group (bacterial strain), different letters indicate significant differences (Student t test, P < 0.05).