Defense Responses in Aspen with Altered Pectin Methylesterase Activity Reveal the Hormonal Inducers of Tyloses

Abstract

Tyloses are ingrowths of parenchyma cells into the lumen of embolized xylem vessels, thereby protecting the remaining xylem from pathogens. They are found in heartwood, sapwood, and in abscission zones and can be induced by various stresses, but their molecular triggers are unknown. Here, we report that down-regulation of PECTIN METHYLESTERASE1 (PtxtPME1) in aspen (Populus tremula × tremuloides) triggers the formation of tyloses and activation of oxidative stress. We tested whether any of the oxidative stress-related hormones could induce tyloses in intact plantlets grown in sterile culture. Jasmonates, including jasmonic acid (JA) and methyl jasmonate, induced the formation of tyloses, whereas treatments with salicylic acid (SA) and 1-aminocyclopropane-1-carboxylic acid (ACC) were ineffective. SA abolished the induction of tyloses by JA, whereas ACC was synergistic with JA. The ability of ACC to stimulate tyloses formation when combined with JA depended on ethylene (ET) signaling, as shown by a decrease in the response in ET-insensitive plants. Measurements of internal ACC and JA concentrations in wild-type and ET-insensitive plants treated simultaneously with these two compounds indicated that ACC and JA regulate each other's concentration in an ET-dependent manner. The findings indicate that jasmonates acting synergistically with ethylene are the key molecular triggers of tyloses.

Figure 1.

Tyloses (arrows) found in developing wood of aspen trees with suppressed expression of PtxtPME1. Shown are a longitudinal (A) and a transverse (B) section of the developing wood of line 5 (Siedlecka et al., 2008) approximately 0.5 mm from the cambium, stained with safranin and alcian blue. Bar = 50 μm.

Figure 2.

Hydrogen peroxide levels and peroxidase activity are increased in the leaves and the wood of PME up- and down-regulated lines (PME+ and PME−, respectively). A and B, Visualization of endogenous peroxidase activity (brown), after reaction with H₂O₂ and DAB, in the wood (A) and the leaves (B). C, H₂O₂ (brown) distribution in the leaves after staining with DAB. A to C, Representative reaction among two transgenic lines per construct, each represented by six trees. Bar = 100 μm. Densitometry of images of all trees is shown on the right. D, H₂O₂ levels in the leaves of PME+ and PME− lines as compared to the wild type. In A to D, Means ± se; two independent lines were analyzed per genotype with two trees each. Means accompanied by the same letters are not statistically different (Tukey test, P ≤ 5%).

Figure 3.

Treatment of intact aspen plants with jasmonates induces formation of tyloses (arrows) and gels (arrowheads) and inhibits plant growth. The panels show transverse (A–C) and radial longitudinal (D–F) sections through the secondary xylem of aspen plants treated with either JA in DMSO, MeJA in DMSO, or DMSO (control) for 2 weeks. Bar (same for all micrographs) = 100 μm. G and H, Inhibition of plant (G) and xylem (H) growth by jasmonate treatment. Data are means for two plants per treatment; asterisk indicates significant difference from the control according to a Tukey post ANOVA test at P ≤ 5%.

Figure 4.

ACC combined with JA, or JA alone, can induce the formation of tyloses (arrows) and gels (arrowheads) in aspen cuttings. Transverse (A–D) and radial longitudinal (E–H) sections of aspen stems 3 weeks after treatment with hormones or DMSO (control). Bar = 100 µm.

Figure 5.

Internal levels of JA (A) and ACC (B) after ACC and JA treatments in wild-type aspen and in transgenic aspen lines 1E and 3E, which have reduced sensitivity to ethylene. Means ± se; n = 5 biological replicates. Asterisk indicates a significant difference between control and hormonal treatment for each genotype (post ANOVA Tukey test, P ≤ 5%).

Figure 6.

Proposed pathways leading to tyloses induction based on experiments presented in this study. Solid arrows refer to interactions shown by specific figures in this article or by Love et al. (2009). Stippled arrows indicate different possible interactions suggested by our data or by the literature.