Methyl jasmonate-induced ethylene production is responsible for conifer phloem defense responses and reprogramming of stem cambial zone for traumatic resin duct formation

Abstract

Conifer stem pest resistance includes constitutive defenses that discourage invasion and inducible defenses, including phenolic and terpenoid resin synthesis. Recently, methyl jasmonate (MJ) was shown to induce conifer resin and phenolic defenses; however, it is not known if MJ is the direct effector or if there is a downstream signal. Exogenous applications of MJ, methyl salicylate, and ethylene were used to assess inducible defense signaling mechanisms in conifer stems. MJ and ethylene but not methyl salicylate caused enhanced phenolic synthesis in polyphenolic parenchyma cells, early sclereid lignification, and reprogramming of the cambial zone to form traumatic resin ducts in Pseudotsuga menziesii and Sequoiadendron giganteum. Similar responses in internodes above and below treated internodes indicate transport of a signal giving a systemic response. Studies focusing on P. menziesii showed MJ induced ethylene production earlier and 77-fold higher than wounding. Ethylene production was also induced in internodes above the MJ-treated internode. Pretreatment of P. menziesii stems with the ethylene response inhibitor 1-methylcyclopropene inhibited MJ and wound responses. Wounding increased 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase protein, but MJ treatment produced a higher and more rapid ACC oxidase increase. ACC oxidase was most abundant in ray parenchyma cells, followed by cambial zone cells and resin duct epithelia. The data show these MJ-induced defense responses are mediated by ethylene. The cambial zone xylem mother cells are reprogrammed to differentiate into resin-secreting epithelial cells by an MJ-induced ethylene burst, whereas polyphenolic parenchyma cells are activated to increase polyphenol production. The results also indicate a central role of ray parenchyma in ethylene-induced defense.

Figure 1.

Douglas fir stem cross-sections 28 d after application of 0.1% Tween 20 (A), 100 mm MJ (B), ethylene (C), and 100 mm MS (D). Figures include similar region from each treatment with secondary phloem, cambium, and xylem for comparison. Bars = 200 μm. C, Cambial zone; PP, PP cell; PD, phloem resin duct; R, ray parenchyma; S, sieve cells; X, xylem. A, Normal anatomy of Douglas fir (Tween 20 control) with rows of PP cells, normal cambium, and the absence of constitutive axial xylem ducts. B, MJ-induced PP cell swelling, phenolic accumulation, and formation of radial phloem ducts and large axial xylem TDs. C, Ethylene treatment-induced PP cell swelling and phenolic deposition and a single row of TDs in the xylem. D, MS treatment did not induce anatomical changes.

Figure 2.

Giant redwood stem cross-sections 28 d after application of 0.1% Tween 20 (A), 100 mm MJ (B), ethylene (C), and 100 mm MS (D). Figures include similar region from each treatment with secondary phloem, cambium, and xylem for comparison. Bars = 200 μm. C, Cambial zone; F, fiber; PP, PP cell; R, ray parenchyma; S, sieve cells; X, xylem. A, Tween 20 control has normal anatomy of giant redwood, with regular secondary phloem pattern of a row of PP cells followed by sieve cells, fibers, and another row of sieve cells, and the absence of constitutive axial xylem ducts. B, MJ-induced PP cell swelling, phenolic accumulation, lignification of young fibers, and formation of axial xylem TDs. C, Ethylene treatment induced PP cell swelling and phenolic deposition and a row of TDs in the xylem. D, MS treatment did not induce anatomical changes.

Figure 3.

Analysis of change in secondary phloem PP cell phenolics and xylem resin ducts 28 d after application of 0.1% Tween 20 and water (controls), and 100 mm MS, 100 mm MJ, and ethylene in giant redwood and Douglas fir. Shown are total mean polyphenolic body area (A), mean xylem resin duct cross sectional area (B), and mean number of resin ducts per millimeter of xylem (C). Measurements (μm²/mm) represent means from cross-sections from three individual replicate saplings. Means are of three replicates ± se. Forty PP cells and 10 xylem resin ducts were analyzed per section. Different letters indicate significant differences (P < 0.05) for each treatment within a species. Significant differences were found between MJ and ethylene, and MJ and ethylene and Tween 20, water, and MS treatments. Significant differences were not found among Tween 20, water, and MS treatments.

Figure 4.

Response of giant redwood (A and B) and Douglas fir (C and D) following application of 10, 25, 50, and 100 mm MJ. Number of TD per millimeter ± se (A and C) and TD lumen area ± se (B and D), as viewed in cross-section, formed at the treated internode (B), internode below the treated internode (A), internode above treated internode (C), and two internodes above treated internode (D). A dose response is evident with respect to application concentration and distance from treated internode. Refer to text for description of significant differences.

Figure 5.

Ethylene biosynthesis in Douglas fir stems at different times following wounding or 100 mm MJ treatment. Samples were collected at 6, 24, and 48 h. Means of three replicates ± se are indicated. Data are for MJ-treated internode (▪), internode above MJ treatment (▴), and wounded internode (♦). Ethylene was detected at the internode above the wound, although it was below the quantifiable limits of the calibration curve and, thus, is not shown on this scale. Ethylene was not detected after Tween 20 control treatments. Images to the right of each curve are representative of the appearance of the new sapwood 4 weeks after treatment. Different letters indicate significant differences (P < 0.05) between treatments at each time point.

Figure 6.

Anatomical changes in Douglas fir stems following 100 mm MJ application or wounding in the presence or absence of the ethylene response inhibitor 1-MCP 3 weeks after treatment. Bars = 200 μm. PB, Phenolic band; PP, PP cell. A, Typical response of Douglas fir following exogenous application of MJ. PP cell swelling and a single tangential band of xylem TDs were formed. B, Pretreatment with 1-MCP substantially reduced the MJ effect with only a rare TD and some PP cell swelling. C and D, Cork borer wounding to the cambium (position indicated by cylinder) results in PP cell swelling and development of a band of phenolic cells and undifferentiated TDs (*) in the xylem. The responses extend well beyond the wound. D, Enlarged area showing response of secondary phloem and xylem. E and F, Cork borer wounding of saplings exposed to 1-MCP. E, Image of stem section showing that the wound response is restricted to the xylem immediately adjacent to the wound in the presence of 1-MCP. F, Enlarged area showing limited response of current year phloem and xylem tissue to wounding after 1-MCP treatment, indicating that ethylene is involved in the wound response.

Figure 7.

ACC oxidase protein in Douglas fir bark 6, 48, and 96 h after wounding, MJ, or control treatment. A, Western-blot analysis of ACC oxidase following Tween 20 control treatment (lanes 2 and 3), cork borer wounding (lanes 4–6) and MJ treatment (lanes 7–9). A total of 18 μg of total protein was loaded per lane, separated by SDS-PAGE, blotted to a PVDF membrane, and probed with an anti-ACC oxidase antibody. Prestained M_r (MW) markers were loaded in lane 1. B, Complementary SDS-PAGE stained with Coomassie Blue to verify equal protein loading. Lane 1 was loaded with MW standards.

Figure 8.

Immunocytochemical localization of ACC oxidase in resin-embedded Douglas fir stem sections. MJ treatments were 100 mm as described in “Materials and Methods.” Reflected/transmitted images of silver-enhanced gold labeling (yellow particles). CZ, Cambial zone; E, resin duct epithelial cell; L, resin duct lumen; N, nucleus; PP, PP cell; R, ray parenchyma cell; S, sieve cell; V, vacuole. A, Secondary phloem of MJ-treated stem. Label is abundant in ray parenchyma, and some label is in young PP cells. Bar = 40 μm. B, Cambial zone from MJ-treated stem. Label is associated with the thin cytoplasm of the cambial zone cells and the cytoplasm and nucleus of ray parenchyma. Arrow points to where cytoplasm has pulled away from the wall, demonstrating label is in cytoplasm and not in the wall or vacuole. Bar = 10 μm. C, Section of a ray parenchyma cell from MJ-treated stem treated with nonimmune serum. Label is absent. Bar = 20 μm. D, Ray parenchyma cell from MJ-treated stem section treated with ACC oxidase antibody showing abundant label in cytoplasm and nucleus. Bar = 20 μm. E, Section of a ray parenchyma cell from control stem treated with ACC oxidase antibody. Bar = 10 μm. F, Section from MJ-treated stem showing labeling of the epithelial cells, including nuclei, surrounding a radial resin duct lumen. Bar = 10 μm. G, Section from wounded stem showing labeling of the epithelial cells of a constitutive cortical resin duct. Bar = 30 μm. H, Enlargement of an epithelial cell from (G) showing label in the cytoplasm and nucleus but not in the vacuole or wall. Bar = 15 μm.

Figure 9.

Model of potential interaction of jasmonates and ethylene with secondary phloem and cambium resulting in anatomically based defense responses in conifer stems. Bark invasion generates jasmonates that induce ACC oxidase (ACCO) and ethylene synthesis in ray parenchyma, cambium, and resin duct epithelia. Ethylene is transported radially by rays and to adjacent cells tangentially, enhancing ethylene levels in PP cells, fibers, cambial cells, and constitutive resin ducts. Ethylene promotes phenolic and resin synthesis and early lignification of fibers. A high-level ethylene burst induces reprogramming of xylem mother cells to form TDs and PP cells to form stone cells. Transport of ethylene or jasmonates by secondary phloem and xylem results in similar responses at some distance from attack, giving rise to a form of systemic resistance.