Cell wall damage-induced lignin biosynthesis is regulated by a reactive oxygen species- and jasmonic acid-dependent process in Arabidopsis

Abstract

The plant cell wall is a dynamic and complex structure whose functional integrity is constantly being monitored and maintained during development and interactions with the environment. In response to cell wall damage (CWD), putatively compensatory responses, such as lignin production, are initiated. In this context, lignin deposition could reinforce the cell wall to maintain functional integrity. Lignin is important for the plant's response to environmental stress, for reinforcement during secondary cell wall formation, and for long-distance water transport. Here, we identify two stages and several components of a genetic network that regulate CWD-induced lignin production in Arabidopsis (Arabidopsis thaliana). During the early stage, calcium and diphenyleneiodonium-sensitive reactive oxygen species (ROS) production are required to induce a secondary ROS burst and jasmonic acid (JA) accumulation. During the second stage, ROS derived from the NADPH oxidase RESPIRATORY BURST OXIDASE HOMOLOG D and JA-isoleucine generated by JASMONIC ACID RESISTANT1, form a negative feedback loop that can repress each other's production. This feedback loop in turn seems to influence lignin accumulation. Our results characterize a genetic network enabling plants to regulate lignin biosynthesis in response to CWD through dynamic interactions between JA and ROS.

Figure 1.

Lignin deposition in Col-0 and mutant Arabidopsis seedlings. Dark-field images of primary root tips stained with phloroglucinol for lignin deposition after 12 h. Genotypes and treatments of seedlings are shown in the pictures. Bar = 200 μm.

Figure 2.

JA production in mock- or isoxaben-treated Col-0 and mutant seedlings. A, JA concentration in mock-treated (green) or isoxaben-treated (orange) Col-0 seedlings. B, JA concentration in different mutant seedlings (blue) after 7 h of isoxaben treatment normalized to isoxaben-treated Col-0 seedlings (Col). Significance: *P < 0.05. The others are not significantly different from Col-0 isoxaben-treated seedlings.

Figure 3.

ROS generation in mock- or isoxaben-treated Col-0 and mutant seedlings. A, ROS production in Col-0 seedlings either mock treated (light gray) or isoxaben treated (dark gray). B, ROS production in wild-type and mutant seedlings either isoxaben-treated (dark gray) or mock treated (light gray). Significance: *P < 0.05; **P < 0.001. The others are not significantly different from Col-0 isoxaben. [See online article for color version of this figure.]

Figure 4.

Effects of Ca²⁺ inhibitors, MeJA and DPI, on CWD-induced lignin production in Col-0 seedlings. Dark-field images of phloroglucinol stained primary root tips from Col-0 seedlings treated for 12 h as described on the pictures. Bar = 200 μm.

Figure 5.

Effects of MeJA and LaCl₃ on ROS production in Col-0 seedlings. Results of ROS measurements in Col-0 seedlings mock treated, treated with MeJA, with the calcium antagonist LaCl₃ individually (light gray) or in conjunction with isoxaben (dark gray). Significance: ***P < 0.0001. The others are not significantly different from Col-0 mock. [See online article for color version of this figure.]

Figure 6.

JA production in Col-0 seedlings treated with isoxaben and DPI or Ca²⁺ inhibitors. JA concentration in Col-0 seedlings treated with isoxaben and different ROS and Ca²⁺ signaling antagonists for 7 h normalized to Col-0 isoxaben treated for 7 h (beige), EGTA (violet), LaCl₃ (orange), and DPI (blue). Significance: *P < 0.05. The others are not significantly different from Col-0 isoxaben-treated seedlings.

Figure 7.

Phenotypic effects of delayed addition of different inhibitors. A, Effect of delaying addition of DPI (10 μM; light blue) or LaCl₃ (10 mM; orange) on JA accumulation in Col-0 seedlings treated with isoxaben. The x axis shows length of delay relative to start of isoxaben treatment in hours. Data for isoxaben/DPI- or LaCL₃-treated Col-0 seedlings are shown as fold change after normalization to JA concentrations observed in Col-0 seedlings after 7 h of isoxaben treatment. B, Dark-field images of primary root tips from Col-0 seedlings stained for lignin deposition after 12 h of mock or isoxaben treatment combined with DPI (10 μM), LaCl₃ (10 mM), or EGTA (7.5 mM). Hours on pictures show time points when inhibitors were added. Isoxaben was added at 0 h unless otherwise stated. Bar = 200 μm.

Figure 8.

Quantitative RT-PCR characterization of selected genes in Col-0 and rbohD seedlings. Expression data for OPCL1 (blue), AOS (orange), and RBOHD (purple) were standardized using UBIQUITIN10 and normalized to the corresponding mock treatment (without isoxaben). Values and error bars (STDEV) are based on three biological replicates. ISO, Isoxaben; DPI + 2h, addition of DPI was delayed by 2 h relative to addition of isoxaben.

Figure 9.

Model depicting the two-stage process mediating the response to CWD. Orange, Initial stimulus generated by CWD; blue, putative calcium signaling genes; purple, genes involved in ROS signaling in purple; green, JA-related genes; red,lignin production.