Hydrogen peroxide acts as a second messenger for the induction of defense genes in tomato plants in response to wounding, systemin, and methyl jasmonate

Abstract

The systemic accumulation of both hydrogen peroxide (H(2)O(2)) and proteinase inhibitor proteins in tomato leaves in response to wounding was inhibited by the NADPH oxidase inhibitors diphenylene iodonium (DPI), imidazole, and pyridine. The expression of several defense genes in response to wounding, systemin, oligosaccharides, and methyl jasmonate also was inhibited by DPI. These genes, including those of four proteinase inhibitors and polyphenol oxidase, are expressed within 4 to 12 hr after wounding. However, DPI did not inhibit the wound-inducible expression of genes encoding prosystemin, lipoxygenase, and allene oxide synthase, which are associated with the octadecanoid signaling pathway and are expressed 0.5 to 2 hr after wounding. Accordingly, treatment of plants with the H(2)O(2)-generating enzyme glucose oxidase plus glucose resulted in the induction of only the later-expressed defensive genes and not the early-expressed signaling-related genes. H(2)O(2) was cytochemically detected in the cell walls of vascular parenchyma cells and spongy mesophyll cells within 4 hr after wounding of wild-type tomato leaves, but not earlier. The cumulative results suggest that active oxygen species are generated near cell walls of vascular bundle cells by oligogalacturonide fragments produced by wound-inducible polygalacturonase and that the resulting H(2)O(2) acts as a second messenger for the activation of defense genes in mesophyll cells. These data provide a rationale for the sequential, coordinated, and functional roles of systemin, jasmonic acid, oligogalacturonides, and H(2)O(2) signals for systemic signaling in tomato plants in response to wounding.

Figure 1.

A Model for the Differential Regulation of Signal Pathway Genes and Defensive Genes in Leaves of Tomato Plants in Response to Wounding and Systemin. In this model, JA activates the signal pathway genes (early genes) in the vascular bundles, whereas H₂O₂, produced by cell wall–derived oligogalacturonides released by PG is a second messenger that activates defense genes (late genes) in mesophyll cells. DPI, diphenyl iodinium chloride; PG, polygalacturonase; PIs, proteinase inhibitors; PM, plasma membrane; PPO, polyphenol oxidase; R, receptor.

Figure 2.

Inhibition of Wound-Inducible Accumulation of Proteinase Inhibitors I and II Proteins by Different Chemical Inhibitors of NADPH Oxidase. Fourteen-day-old tomato plants were excised at the base of the stems and supplied with solutions of phosphate buffer alone (Control and Wound), 40 mM pyridine, 20 mM imidazole, or 100 μM DPI in phosphate buffer for 30 min. Plants, except controls, then were wounded and incubated in water under light as described in Methods. Proteinase inhibitors I (Inh I) and II (Inh II) were assayed immunologically in leaf juice 24 hr later. Data are means ±sd; n .

Figure 3.

Inhibition of Wound-Inducible Accumulation of Proteinase Inhibitors by the NADPH Oxidase Inhibitor DPI at Different Concentrations. Plants were treated and assayed as described for Figure 1. Inh I, proteinase inhibitor I; Inh II, proteinase inhibitor II. Data are means ±sd; n .

Figure 4.

Inhibition of Elicitor-Induced Accumulation of Inhibitor I by DPI. Plants were supplied with either phosphate buffer alone or 100 μM DPI in phosphate buffer for 30 min and transferred for 30 min to the same buffer solution containing 25 nM systemin, 250 μg/mL OGA, or 125 μg/mL chitosan or exposed to methyl jasmonate (MeJ) vapor as described in Methods. After each treatment, plants were incubated in water for 24 hr and then immunologically assayed for proteinase inhibitor I content in leaf juice. Data are means ±sd; n .

Figure 5.

H₂O₂-Mediated Accumulation of Proteinase Inhibitors I and II Proteins in Tomato Leaves. Fourteen-day-old tomato plants were excised and incubated in phosphate buffer alone (Control and Wound) or in buffer containing 50 μM glucose (Glu), 2.5 units/mL glucose oxidase (Oxidase), glucose plus glucose oxidase (Glu + Oxidase), or 50 μM gluconate (Glco) for 2 hr. Thereafter, plants were incubated in water for 24 hr and assayed immunologically for proteinase inhibitors I (Inh I) and II (Inh II) content in leaf juice. Data are means ±sd; n .

Figure 6.

Differential Inhibition of Systemic Wound Response Genes by DPI. Young excised tomato plants with two expanding leaves and a young growing leaf were supplied with phosphate buffer alone or with 100 μM DPI for 0.5 hr, wounded on the lower leaf at time 0, transferred to water, and assayed by RNA gel blotting at the times indicated (see Methods). 18S-rRNA, rRNA (loading control); AOS, allene oxide oxidase; CDI, aspartic proteinase inhibitor; CPI, metallocarboxypeptidase inhibitor; Inh I, proteinase inhibitor I; Inh II, proteinase inhibitor II; LOX, lipoxygenase; PGcat, leaf polygalacturonase catalytic subunit; PPO, polyphenol oxidase; ProSYS, prosystemin.

Figure 7.

Differential Induction of mRNAs of Systemic Wound Response Proteins by H₂O₂ Generated with Glucose Oxidase in Leaves of Tomato Plants. At time 0, plants were excised and supplied with phosphate buffer alone or with buffer containing 50 μM glucose (Glu), 2.5 units/mL glucose oxidase (Oxidase), or both (Glu + Oxidase). Total leaf RNA was extracted and examined by RNA gel blotting at the times indicated. For the independent (control) treatments with glucose or glucose oxidase alone, only the results obtained after 1 hr (for the signaling genes) and 8 hr (for the defense genes) are shown. Abbreviations are as in Figure 5.

Figure 8.

Cytochemical Localization of Wound-Inducible H₂O₂ in Vascular Bundles of Tomato Leaves. (A) Electron-dense deposits of CeCl₃ indicative of the presence of H₂O₂ in developing secondary cell walls of xylem vessels (XV) of control unwounded leaves (arrows show typical deposits). Note that the cell walls of an adjacent vascular parenchyma cell (VP) show little CeCl₃ staining. (B) Absence of CeCl₃ staining in the cell walls of vascular parenchyma (VP) and neighboring spongy mesophyll (SM) cells associated with the phloem in control unwounded leaves. (C) H₂O₂ generation in the vascular bundle of a wounded tomato leaf 4 hr after wounding. H₂O₂ accumulates strongly in the cell walls of vascular parenchyma cells bordering spongy mesophyll cells and at the intercellular spaces (IS). (D) Systemic accumulation of H₂O₂ in vascular bundles of upper unwounded leaves of young tomato plants 4 hr after wounding of the lower leaf. CC, companion cell; CW, cell wall; SE, sieve element. .

Figure 9.

Cytochemical Localization of Wound-Inducible H₂O₂ in Mesophyll Cells of Tomato Leaves. (A) Spongy mesophyll cells of a control leaf from an unwounded plant does not exhibit H₂O₂ in the cell walls or intercellular spaces (IS). (B) Accumulation of H₂O₂ in the bordering cell walls of a vascular parenchyma and a spongy mesophyll cell of a wounded leaf 4 hr after wounding. (C) Accumulation of H₂O₂ in the cell walls of two spongy mesophyll cells facing an intercellular space 4 hr after wounding of the leaf. (D) Systemic H₂O₂ accumulation in the cell walls of two spongy mesophyll cells facing an intercellular space 4 hr after wounding of the lower leaf. (E) Constitutive accumulation of H₂O₂ in the cell wall of a spongy mesophyll cell of a leaf from a transgenic tomato plant overexpressing prosystemin. The proteinaceous material (P) within the central vacuole corresponds to aggregates containing proteinase inhibitor proteins that constitutively accumulate in transgenic tissue. (F) Inhibition of wound-induced H₂O₂ accumulation by DPI. Leaf samples were obtained 4 hr after wounding. C, chloroplast; CW, cell wall; M, mitochondrion; N, nucleus; P, protein aggregates of defensive inhibitor proteins; V, central vacuole. Bar in (A), (C), (D), and (F) = 2 μm; bar in (B) and (E) = 1 μm.