Ethanolamide oxylipins of linolenic acid can negatively regulate Arabidopsis seedling development

Abstract

N-Acylethanolamines (NAEs) are fatty-acid derivatives with potent biological activities in a wide range of eukaryotic organisms. Polyunsaturated NAEs are among the most abundant NAE types in seeds of Arabidopsis thaliana, and they can be metabolized by either fatty acid amide hydrolase (FAAH) or by lipoxygenase (LOX) to low levels during seedling establishment. Here, we identify and quantify endogenous oxylipin metabolites of N-linolenoylethanolamine (NAE 18:3) in Arabidopsis seedlings and show that their levels were higher in faah knockout seedlings. Quantification of oxylipin metabolites in lox mutants demonstrated altered partitioning of NAE 18:3 into 9- or 13-LOX pathways, and this was especially exaggerated when exogenous NAE was added to seedlings. When maintained at micromolar concentrations, NAE 18:3 specifically induced cotyledon bleaching of light-grown seedlings within a restricted stage of development. Comprehensive oxylipin profiling together with genetic and pharmacological interference with LOX activity suggested that both 9-hydroxy and 13-hydroxy linolenoylethanolamides, but not corresponding free fatty-acid metabolites, contributed to the reversible disruption of thylakoid membranes in chloroplasts of seedling cotyledons. We suggest that NAE oxylipins of linolenic acid represent a newly identified, endogenous set of bioactive compounds that may act in opposition to progression of normal seedling development and must be depleted for successful establishment.

Figure 1.

Structures of TMS Derivatives of Ethanolamide and Free Oxylipin Metabolites with Molecular Ions and Several Key Diagnostic Fragment Ions Indicated for Each Species. Representative spectra from GC-MS are shown in Supplemental Figure 1 online. (A) TMS derivatives of 9-hydroxy-10,12,15-octadecatrienoylethanolamide (9-NAE-HOT). (B) TMS derivatives of 13-hydroxy-9,11,15-octadecatrienoylethanolamide (13-NAE-HOT). (C) TMS derivatives of (13S,9Z,10E,15Z)-12-oxo-10,15-phytodienoylethanolamide. (D) TMS derivatives of 9-hydroxy-10,12, 15-octadecatrienoic acid (9-HOT). (E) TMS derivatives of 13-hydroxy-9,11,15-octadecatrienoic acid (13-HOT). (F) TMS derivatives of (13S,9Z,10E,15Z)-12-oxo-10,15-phytodienoic acid. [See online article for color version of this figure.]

Figure 2.

Endogenous Ethanolamide Oxylipin Metabolites Derived from NAE 18:3. Arabidopsis seedlings (the wild type, faah1, faah1 faah2, FAAH1 OE, lox1 lox5, and lox2) were grown for 11 d. Seedling tissues (∼5 g) were sampled at days 5, 6, 7, 9, and 11 after sowing and oxylipins were quantified. A single asterisk indicates a significant difference compared with wild-type seedlings, which was determined by Student’s t test (P < 0.05). A double asterisk indicates a significant difference compared with wild-type seedlings, which was determined by Student’s t test (P < 0.01). Values are presented as means ± sd of three biological replicates. FW, fresh weight. (A) 9-NAE-HOT. (B) 13-NAE-HOT. (C) NAE-OPDA.

Figure 3.

Ethanolamide Oxylipins in Seedlings after Addition of Exogenous NAE 18:3. Arabidopsis seedlings (the wild type, faah1, faah1 faah2, FAAH1 OE, lox1 lox5, and lox2) were grown for 4 d, transferred into media containing 100 μM NAE 18:3, and continued to grow for 72 h. Seedlings were subsequently transferred into fresh media as a recovery period without NAE for an additional 96 h. Seedlings (∼2 g) were collected at 24 h (day 5), 48 h (day 6), and 72 h (day 7) after addition of NAE 18:3 or 48 h (day 9) and 96 h (day 11) after removal to fresh medium without NAE 18:3. Values are presented as means ± sd of three biological replicates. A single asterisk indicates a significant difference compared with wild-type seedlings, which was determined by Student’s t test (P < 0.05). A double asterisk indicates a significant difference compared with wild-type seedlings, which was determined by Student’s t test (P < 0.01). FW, fresh weight. (A) 9-NAE-HOT. (B) 13-NAE-HOT. (C) NAE-OPDA.

Figure 4.

Endogenous Free Oxylipin Metabolites. Arabidopsis seedlings (the wild type, faah1, faah1 faah2 knockout, FAAH1 OE, lox1 lox5, and lox2) were grown for 11 d. Seedling tissues (∼5 g) were sampled at days 5, 6, 7, 9, and 11 after sowing and oxylipins were quantified. Values are presented as means ± sd of three biological replicates. FW, fresh weight. (A) 9-HOT. (B) 13-HOT. (C) OPDA.

Figure 5.

Free Oxylipins in Seedlings after Addition of Exogenous NAE 18:3. Arabidopsis seedlings (the wild type, faah1, faah1 faah2, FAAH1 OE, lox1 lox5, and lox2) were grown for 4 d, transferred into media containing 100 μM NAE 18:3, and continued to grow for 72 h. Seedlings were subsequently transferred into fresh media without NAE for 96 h. Seedlings (∼2 g) were collected at 24 h (day 5), 48 h (day 6), and 72 h (day 7) after addition of NAE 18:3 or 48 h (day 9) and 96 h (day 11) after removal to fresh medium without NAE 18:3. Values are presented as means ± sd of three biological replicates. A single asterisk indicates a significant difference compared with wild-type seedlings, which was determined by t test (P < 0.05). A double asterisk indicates a significant difference compared with wild-type seedlings, which was determined by Student’s t test (P < 0.01). FW, fresh weight. (A) 9-HOT. (B) 13-HOT. (C) OPDA.

Figure 6.

NAE 18:3 Induces Cotyledon Bleaching in Arabidopsis Seedlings Except in FAAH1 OE. Arabidopsis seedlings (the wild type [WT], faah1, faah1 faah2, FAAH1 OE, lox1 lox5, and lox2) were grown for 4 d, transferred into media containing 100 μM NAE 18:3 (or 0.07% DMSO as a solvent-only control), and allowed to grow for 72 h. Total chlorophyll contents were quantified spectrophotometrically after extraction in 80% acetone. (A) Representative images of 7-d-old Arabidopsis seedlings treated with 100 μM NAE 18:3. (B) Chlorophyll contents of seedling exposed to 100 μM NAE 18:3 for 72 h. Chlorophyll contents of FAAH1 OE were not affected by exposure to elevated NAE 18:3, while other genotypes show substantial reduction in their total chlorophyll contents. FW, fresh weight. (C) Chlorophyll (chl) contents of seedlings exposed to 0.07% DMSO (control). Data are the means ± sd of three biological replicates.

Figure 7.

NAE 18:3 Inhibits Primary Root Elongation in a Dose-Dependent Manner. Four-day-old wild-type seedlings were transferred onto media containing NAE 18:3 at varying concentrations (with 0.07% DMSO as solvent control). Primary root length data are the means ± sd of 20 seedlings. Three replicate experiments showed similar results. [See online article for color version of this figure.]

Figure 8.

NAE 18:3 Specifically Disrupts Chloroplasts in Cotyledons of Arabidopsis Seedlings. Four-day-old wild-type seedlings were transferred into media containing 0.07% DMSO (solvent control) (A), 100 µM NAE 18:3 (B), 100 µM NAE 18:2 (C), or 100 µM FFA 18:3 (D) for 72 h. Chloroplast disruption was specific for NAE 18:3; neither free linolenic acid (FFA 18:3) nor NAE 18:2 showed the same effect. Images are z-stacks of mesophyll cells visualized by confocal fluorescence microscopy showing chlorophyll autofluorescence in chloroplasts.

Figure 9.

NAE 18:3 Bleaching/Chloroplast Disruption Was Reversible. Effects were more severe in faah1 knockouts (right) than in the wild type (WT; left). NAE 18:3 (100 µM) was applied to 4-d-old wild-type and faah1 seedlings for 72 h. Seedlings were subsequently removed from treatment to fresh media for 4 d. Not only was there a marked reduction in seedling growth noted after 3 d of treatment (insets), but a profound loss of chlorophyll in the seedlings also was observed. (A) Four-day-old seedling before addition of NAE 18:3 (control). (B) to (D) Chloroplast autofluorescence 1, 2, and 3 d after transfer to media containing NAE 18:3. (E) and (F) Recovery of chloroplasts after seedlings had been removed to fresh media. Microscopy images are z-stacks of mesophyll cells visualized by confocal fluorescence microscopy showing chlorophyll autofluorescence in chloroplasts. (G) Bottom panels are chlorophyll quantification at different stages, consistent with bleaching of seedlings and the disappearance and reappearance of chloroplasts (left, the wild type; right; faah1). Data are means and sd of three replicate samples (of three to five seedlings each). FW, fresh weight.

Figure 10.

NAE12:0, a General LOX Inhibitor Can Prevent Seedling Bleaching. Four-day-old Arabidopsis wild-type seedlings grown in liquid media for 4 d were transferred to media for three additional days containing 100 μM NAE 18:3 alone or with NAE 12:0, NAE 16:0, or FFA 12:0 at 20 μM. The bottom panels show NAE 12:0, NAE 16:0, and FFA 12:0. At these concentrations, NAE 12:0 and partially NAE 16:0, but not FFA 12:0, prevented seedling bleaching.

Figure 11.

NAE 12:0 Reversed the Effects of NAE 18:3 in a Dose-Dependent Manner. Four-day-old wild-type seedlings were treated with 100 μM NAE 18:3 for an additional 72 h in the presence of NAE 12:0 at 20, 30, 40, or 50 μM. chl, chlorophyll; FW, fresh weight. (A) Total chlorophyll contents were quantified spectrophotometrically following extraction with 80% acetone. Data are the means ± sd of three biological replicates. (B) Primary root lengths of seedlings treated with 100 μM NAE 18:3 in presence of NAE 12:0 were measured. Data are the means ± sd of n = 40. (C) Primary root lengths of NAE 12:0-treated seedlings were measured. Data are the means ± sd of n = 40. [See online article for color version of this figure.]

Figure 12.

NAE12:0, a General LOX Inhibitor, Blocks the Endogenous Formation of Oxylipins. Quantification of oxylipins in 7-d-old wild-type seedlings treated with 100 μM NAE 18:3 for 72 h in the presence of NAE 12:0 at 20, 30, 40, and 50 μM (cf. Figure 11). Data are means and sd of three replicate samples. A single asterisk indicates a significant difference compared with seedlings treated with NAE 18:3 alone, which was determined by Student’s t test (P < 0.05). A double asterisk indicates a significant difference compared with seedlings treated with NAE 18:3 alone, which was determined by Student’s t test (P < 0.01). FW, fresh weight. (A) Ethanolamide oxylipins. (B) FFA oxylipins. [See online article for color version of this figure.]

Figure 13.

Synthetic Ethanolamide Oxylipins, but Not Free Oxylipins, Bleached Seedling Cotyledons. Chlorophyll content in 7-d-old wild-type seedlings treated for 72 h with 100 μM NAE 18:3, 9-NAE-HOT, 13-NAE-HOT, a combination of 9- and 13-hydro(pero)xy-octadecatrienoylethanolamide (H[P]OT) (not fully reduced or fully reduced), or corresponding FFA oxylipins. Either 9- or 13-NAE-H(P)OT, but not corresponding free oxylipins, elicited bleaching in cotyledons. Data are means and sd of three replicate samples. chl, chlorophyll; FW, fresh weight. [See online article for color version of this figure.]

Figure 14.

Exogenous ABA-Induced Accumulation of NAE 18:3-Derived Oxylipins. Quantification of oxylipins in 7-d-old seedlings (wild type) treated with DMSO (control) or 0.5, 5, or 50 μM ABA on day 3. Concentrations of ABA were calculated based on the cis-isomer. Data are means and sd of three replicate samples. A single asterisk indicates a significant difference compared with untreated seedlings (DMSO), which was determined by Student’s t test (P < 0.05). A double asterisk indicates a significant difference compared with untreated seedlings, which was determined by Student’s t test (P < 0.01). FW, fresh weight.

Figure 15.

Summary of NAE 18:3 Metabolism via Hydrolysis, the 9-LOX or the 13-LOX Pathways, and the Relative Partitioning through These Pathways in the Various Mutants Used in These Studies. NAE 18:3 undergoes 13- and/or 9-LOX–mediated oxidation to generate corresponding hydroperoxides that are further reduced to hydroxides. Hydroperoxides and hydroxides of NAE 18:3 may also undergo hydrolysis and enter the larger pool of corresponding free hydroperoxides and hydroxides (as shown in blue). 13-NAE-HPOT is also metabolized by AOS and/or AOC to generate the unstable epoxide and NAE-OPDA, respectively. Corresponding FFA oxylipins are generated in the same LOX pathways (dashed lines). 9NAE-HPOT, (9S,12Z,10E,15Z)-9-hydroperoxy-10,12,15-octadecatrienoylethanolamide; 13-NAE-HPOT, (13S,9Z,11E,15Z)-13-hydroperoxy-9,11,15-octadecatrienoylethanolamide; 9NAE-HOT, (9S,12Z,10E,15Z)-9-hydroxy-10,12,15-octadecatrienoylethanolamide; 13-NAE-HOT, (13S,9Z,11E,15Z)-13-hydroxy-9,11,15-octadecatrienoylethanolamide; 12,13S-epoxy-NAE 18:3, (13S, 9Z, 11E, 15Z)-12,13-epoxy-9,11,15-octadecatrienoylethanolamide; NAE-OPDA, (13S,9Z,10E,15Z)-12-oxo-10,15-phytodienoylethanolamide (summarized from data in this article and consistent with previous data and speculation in Van Der Stelt et al., 2000; Shrestha et al., 2002; Kilaru et al., 2011). WT, the wild type.