N-acylethanolamine signaling in tobacco is mediated by a membrane-associated, high-affinity binding protein

Abstract

N-Acylethanolamines (NAEs) are fatty acid derivatives found as minor constituents of animal and plant tissues, and their levels increase 10- to 50-fold in tobacco (Nicotiana tabacum) leaves treated with fungal elicitors. Infiltration of tobacco leaves with submicromolar to micromolar concentrations of N-myristoylethanolamine (NAE 14:0) resulted in an increase in relative phenylalanine ammonia-lyase (PAL) transcript abundance within 8 h after infiltration, and this PAL activation was reduced after co-infiltration with cannabinoid receptor antagonists (AM 281 and SR 144528). A saturable, high-affinity specific binding activity for [(3)H]NAE 14:0 was identified in suspension-cultured tobacco cells and in microsomes from tobacco leaves (apparent K(d) of 74 and 35 nM, respectively); cannabinoid receptor antagonists reduced or eliminated specific [(3)H]NAE 14:0 binding, consistent with the physiological response. N-Oleoylethanolamine activated PAL2 expression in leaves and diminished [(3)H]NAE 14:0 binding in microsomes, whereas N-linoleoylethanolamine did not activate PAL2 expression in leaves, and did not affect [(3)H]NAE 14:0 binding in microsomes. The nonionic detergent dodecylmaltoside solubilized functional [(3)H]NAE 14:0-binding activity from tobacco microsomal membranes. The dodecylmaltoside-solubilized NAE-binding activity retained similar, but not identical, binding properties to the NAE-binding protein(s) in intact tobacco microsomes. Additionally, high-affinity saturable NAE-binding proteins were identified in microsomes isolated from Arabidopsis and Medicago truncatula tissues, indicating the general prevalence of these binding proteins in plant membranes. We propose that plants possess an NAE-signaling pathway with functional similarities to the "endocannabinoid" pathway of animal systems and that this pathway, in part, participates in xylanase elicitor perception in tobacco.

Figure 1

A and B, Effect of mammalian CB receptor antagonists AM 281 and SR 144528 on NAE 14:0-mediated inhibition of xylanase-induced alkalinization of tobacco cell culture medium. Medium alkalinization was recorded in 15 mL of cultured cells that were pre-equilibrated to a constant pH for 20 to 30 min at room temperature. Xylanase (1.0 μg mL⁻¹), NAE 14:0 (10.0 μm), AM 281 (10.0 μm), and/or SR 144528 (10.0 μm) were added alone or in combinations as preparations in water, spent culture medium, and 0.1% (v/v) DMSO, respectively, as described in “Materials and Methods.” Results shown are from a typical experiment using a single population of cells for all treatments in A or B. Replicate experiments with different batches of cell suspensions showed similar results, although the magnitude of the pH change varied between culture batches.

Figure 2

Reduction of xylanase- or NAE-induced PAL mRNA transcript accumulation by mammalian CB receptor antagonists AM 281 and SR 144528. A, PAL2 transcript abundance was evaluated by northern blotting in experiments wherein tobacco leaves were infiltrated with water (control), xylanase (1.0 μg mL⁻¹) alone, or xylanase in combination with different concentrations of AM 281 or SR 144528 for 8 h. B, PAL2 transcript abundance was evaluated by northern blotting in experiments wherein tobacco leaves infiltrated with water (control, including 0.1% [v/v] DMSO), different concentrations of NAE 14:0 (as aqueous preparations diluted from DMSO stock, up to 0.1% [v/v] DMSO final) alone, or in combination with AM 281 (1.0 μm in 0.1% [v/v] DMSO) or SR 144528 (1.0 μm in 0.1% [v/v] DMSO). In both A and B, results are a quantitative representation of relative abundance of PAL mRNA transcripts (normalized to 28S rRNA by scanning densitometry and NIH imaging software). These results are presented as the percent of xylanase-induced levels of PAL, which was arbitrarily set to 100%. The data plotted correspond to the means ± sd of three independent experiments analyzed under identical conditions of electrophoresis, hybridization, and film exposure.

Figure 3

Analysis of PAL mRNA transcript accumulation after treatment of tobacco leaves with different NAE types and the mammalian CB analog WIN 55,212-2. All NAEs (NAEs 12:0, 14:0, 16:0, 18:0, 18:1, 18:2, 18:3α, and 20:4) were infiltrated into leaves at 10.0 μm, and WIN 55, 212-2 was infiltrated at 1.0 μm. PAL2 transcript abundance was evaluated by northern blotting. Results are a quantitative representation of relative abundance of PAL mRNA transcripts (means ± sd) in tobacco leaves subjected to the above treatments for 8 h (normalized to 28S rRNA and percent of xylanase-induced levels of PAL) in three independent experiments analyzed under identical conditions of electrophoresis, hybridization, and film exposure.

Figure 4

Specific binding of [³H]NAE 14:0 to tobacco suspension-cultured cells and microsomes isolated from tobacco leaves. A, Analysis of specific binding activity of [³H]NAE 14:0 to tobacco suspension cells (20 μg of protein) with concentrations ranging from 5 to 100 nm in the absence and presence of the mammalian CB receptor antagonist, AM 281 (100 nm). B, Analysis of specific binding activity of [³H]NAE 14:0 to tobacco leaf microsomes (50 μg of protein) with concentrations ranging from 5 to 100 nm in the presence and absence of AM 281. Specific binding was determined by subtracting nonspecific binding (binding in the presence of approximately 500× nonradioactive NAE 14:0) from total radioligand binding. Data shown are means and range of duplicate samples within a given experiment and are representative of results obtained in six replicate experiments.

Figure 5

Comparison of [³H]NAE 14:0-specific binding to microsomal membranes (A) or to DDM-solubilized microsomal proteins (B) of tobacco leaves in the presence of other NAE types and CB receptor antagonist. NAE 18:1, which induced PAL transcript accumulation; NAE 18:2, which did not induce PAL in tobacco leaves; and AM281, which blocked NAE and xylanase-induced PAL activation, were included at 100 nm in the [³H]NAE 14:0-binding assays for microsomes, and the radioligand concentration was varied from 10 to 100 nm. These same competitors as well as SR144528 were included at 10 nm in the [³H]NAE 14:0-binding assays for DDM-solubilized microsomal proteins, and the radioligand concentration was varied from 10 to 100 nm. A, Values represent the means (triplicate assays) of an individual experiment reproduced two times using different preparations of microsomes (50 μg of protein). One-way ANOVA was used (GraphPad InStat v3.0) to compare (for 100 nm of each treatment) the data for statistically significant differences (***, P < 0.001; **, P < 0.01; NS, non-significant) in specific NAE 14:0 binding (control) in the presence of different NAE types or the antagonist, AM 281. For NAE 14:0 versus NAE 14:0 + NAE 18:1, P < 0.001(***); for NAE 14:0 versus NAE 14:0 + NAE 18:2, P > 0.05 (NS); for NAE 14:0 versus + AM 281, P < 0.01 (**); for NAE 14:0 + NAE 18:1 versus NAE 14:0 + NAE 18:2, P < 0.01 (**); for NAE 14:0 + NAE 18:1 versus NAE 14:0 + AM 281, P > 0.10 (NS); and for NAE 14:0 + NAE 18:2 versus NAE 14:0 + AM 281, P < 0.01 (**). B, Values represent the means (triplicate assays) of an individual experiment reproduced four times using different preparations of DDM-solubilized microsomes (5–10 μg of solubilized protein). One-way ANOVA was used (GraphPad InStat v3.0) as above to compare (for 10 nm of each treatment) the data for statistically significant differences (***, P < 0.001; **, P < 0.01; NS, non-significant) for specific binding for NAE 14:0 alone (control, set at 100%) compared with NAE 14:0 in the presence of different NAE types or the antagonists, AM 281 and SR 144528. For NAE 14:0 versus NAE 14:0 + NAE 18:1, P < 0.05; NAE 14:0 versus NAE 14:0 + NAE 18:2, P < 0.001 (***); for NAE 14:0 versus NAE 14:0+ AM 281, P < 0.01 (**); for NAE 14:0 versus NAE 14:0+ SR 144528, P < 0.01 (**); for NAE 14:0 + NAE 18:1 versus NAE 14:0 + NAE 18:2, P < 0.01 (**); for NAE 14:0 + AM 281 versus NAE 14:0 + SR 144528, P > 0.05 (NS); NAE 14:0 + AM 281 versus NAE 14:0 + NAE 18:1, P > 0.05 (NS); NAE 14:0 + AM 281 versus NAE 14:0 + NAE 18:2, P > 0.05 (NS); NAE 14:0 + SR 144528 versus NAE 14:0 + NAE 18:1, P > 0.05 (NS); NAE 14:0 + SR 144528 versus NAE 14:0 + NAE 18:2, P > 0.05 (NS). In both A and B, error bars are omitted for clarity; sd was generally less than 15%.