Hydrogen peroxide is generated systemically in plant leaves by wounding and systemin via the octadecanoid pathway

Abstract

Hydrogen peroxide (H2O2) generated in response to wounding can be detected at wound sites and in distal leaf veins within 1 hr after wounding. The response is systemic and maximizes at about 4-6 hr in both wounded and unwounded leaves, and then declines. The timing of the response corresponds with an increase in wound-inducible polygalacturonase (PG) mRNA and enzyme activity previously reported, suggesting that oligogalacturonic acid (OGA) fragments produced by PG are triggering the H2O2 response. Systemin, OGA, chitosan, and methyl jasmonate (MJ) all induce the accumulation of H2O2 in leaves. Tomato plants transformed with an antisense prosystemin gene produce neither PG activity or H2O2 in leaves in response to wounding, implicating systemin as a primary wound signal. The antisense plants do produce both PG activity and H2O2 when supplied with systemin, OGA, chitosan, or MJ. A mutant tomato line compromised in the octadecanoid pathway does not exhibit PG activity or H2O2 in response to wounding, systemin, OGA, or chitosan, but does respond to MJ, indicating that the generation of H2O2 requires a functional octadecanoid signaling pathway. Among 18 plant species from six families that were assayed for wound-inducible PG activity and H2O2 generation, 14 species exhibited both wound-inducible PG activity and the generation of H2O2. Four species, all from the Fabaceae family, exhibited little or no wound-inducible PG activity and did not generate H2O2. The time course of wound-inducible PG activity and H2O2 in Arabidopsis thaliana leaves was similar to that found in tomato. The cumulative data suggest that systemic wound signals that induce PG activity and H2O2 are widespread in the plant kingdom and that the response may be associated with the defense of plants against both herbivores and pathogens.

Figure 1

The generation of H₂O₂ in leaves of tomato plants in response to wounding and wound signals. Fourteen-day-old plants were excised at the base of the stems and supplied with DAB for 8 hr. The plants then were either wounded, supplied with solutions of systemin, OGA, or chitosan in DAB through the cut stems, or exposed to MJ vapors in a closed Plexiglas box as described in Materials and Methods. (A) The time course of H₂O₂ production in tomato leaves in response to wounding. (B) The systemic production of H₂O₂ in leaves of tomato plants wounded only on the lower leaf. (C) The production of H₂O₂ in leaves of tomato plants 4 hr after treatment with systemin (SYS), OGA, chitosan (CHIT), or MJ.

Figure 2

Inhibition of wound-inducible H₂O₂ production by the NADPH oxidase inhibitor DPI. Fourteen-day-old tomato plants were excised and treated as in Fig. 1A, except that at the time of wounding DPI and DAB were supplied to the plants.

Figure 3

The induction of H₂O₂ production by wounding in leaves of (A) tomato plants transformed with a prosystemin antisense prosystemin gene and (B) a mutant tomato line (def1) that is compromised in the octadecanoid signaling pathway. C, control; W, wounded; SYS, systemin; CHIT, chitosan. The plants were treated as in Fig. 1C.

Figure 4

Assays of H₂O₂ levels in leaves of wild-type tomato plants and transformed tomato plants constitutively expressing prosystemin (ProSYS). The upper terminal leaflet of 14-day-old tomato plants and the terminal leaflet of the third leaf from the apex of 3-month-old tomato plants were excised and assayed by using DAB as described in Materials and Methods.

Figure 5

Assay for wound-inducible H₂O₂ production in leaves of selected species from several plant families. Leaves of plants were excised and supplied with a solution of DAB for 8 hr and assayed (0 time) or wounded and assayed after an additional 4-hr incubation with DAB (4H). Details of the experiments are described in Materials and Methods.

Figure 6

Wound-induced PG activity and H₂O₂ production in leaves of A. thaliana plants. (A) Time course of PG activity in leaves from wounded plants (●) and leaves from unwounded plants (■). (B) Time course of H₂O₂ production in the wounded leaves. Details of the experiments are in Materials and Methods.