N-Acylethanolamine metabolism interacts with abscisic acid signaling in Arabidopsis thaliana seedlings

Abstract

N-Acylethanolamines (NAEs) are bioactive acylamides that are present in a wide range of organisms. In plants, NAEs are generally elevated in desiccated seeds, suggesting that they may play a role in seed physiology. NAE and abscisic acid (ABA) levels were depleted during seed germination, and both metabolites inhibited the growth of Arabidopsis thaliana seedlings within a similar developmental window. Combined application of low levels of ABA and NAE produced a more dramatic reduction in germination and growth than either compound alone. Transcript profiling and gene expression studies in NAE-treated seedlings revealed elevated transcripts for a number of ABA-responsive genes and genes typically enriched in desiccated seeds. The levels of ABI3 transcripts were inversely associated with NAE-modulated growth. Overexpression of the Arabidopsis NAE degrading enzyme fatty acid amide hydrolase resulted in seedlings that were hypersensitive to ABA, whereas the ABA-insensitive mutants, abi1-1, abi2-1, and abi3-1, exhibited reduced sensitivity to NAE. Collectively, our data indicate that an intact ABA signaling pathway is required for NAE action and that NAE may intersect the ABA pathway downstream from ABA. We propose that NAE metabolism interacts with ABA in the negative regulation of seedling development and that normal seedling establishment depends on the reduction of the endogenous levels of both metabolites.

Figure 1.

ABA or NAE12:0 Negatively Regulates Seedling Growth, and This Inhibition Is Reversible and Occurs within a Narrow Developmental Window. (A) Treatment of seedlings with 0.5 μM ABA or 35 μM NAE arrests seedling growth. Data shown are representative of a single time course experiment. The trends of growth suppression by ABA and NAE compared with controls were similar in replicate experiments (six for NAE and five for ABA), although the absolute fresh weight gain at each time point varied somewhat from experiment to experiment. (B) Endogenous ABA and NAE levels drop precipitously with seedling emergence. Values for NAE are means and sd of six independent extractions. Values for ABA are means and sd of three independent extractions. Under these conditions, radicle emergence occurs at about day 3. FW, fresh weight. (C) The inhibitory effects of NAE (35 μM) on Arabidopsis seedling growth are reversible. Bars show the sd of three different liquid culture experiments to show the general range of variability. Inset compares 8-d-old seedlings grown in liquid cultures without (two on left) or with (two on right) NAE12:0. Bar = 10 mm. (D) Seedling growth responses to NAE12:0 fall within a narrow window of developmental sensitivity (<6 d), similar to the window of growth arrest previously characterized for ABA (Lopez-Molina et al., 2001, 2002). For example, treatment of 2-d-old seedlings with ABA (0.5 μM) or NAE (35 μM) severely stunts growth (measured after 14 d), whereas treatment of 10-d-old seedlings does not impact growth much at all (measured after 14 d). Seedling growth was normalized to original seed weight and plotted as a percentage of fresh weight of untreated seedlings after 14 d. Results for each time point are averages from three replicate experiments containing ∼2500 individuals (50 mg seed) each. Asterisks indicate a significant difference compared with untreated seedlings, which was determined by t test. The sensitivity of seedlings to NAE was statistically significant (P < 0.001) up to day 6 but not significant (NS; P > 0.1) thereafter. ABA sensitivity overlapped NAE sensitivity up to day 6, and it too was abolished by day 10.

Figure 2.

ABA and NAE Content in Seedlings at 4 and 8 d after Sowing Quantified by Isotope-Dilution Mass Spectrometry. (A) ABA content in seedlings treated without or with 35 μM NAE12:0. Deuterated ABA standard was added at the time of extraction. Values are means and sd of three independent measurements. (B) NAE content in seedlings treated without or with 0.5 μM ABA. Deuterated NAE (NAE12:0, NAE18:0, and NAE20:4) standards were added at the time of extraction. Values are averages and sd of six independent measurements. (C) NAE composition in ABA-treated and untreated seedlings. NAE contents in (B) were summed from individual NAE types, and NAE composition was calculated based on the relative percentage of each type. See (B) for total NAE content at 4 or 8 d without or with ABA.

Figure 3.

NAE-Modulated Changes in Transcript Abundance for Several ABA-Responsive (ABI3, HVA22B, ATEM1, and CRA1) and ABA-Nonresponsive (EXPR3 and FAAH) Genes. Transcript levels were quantified in total RNA extracts by real-time RT-PCR against 18S rRNA and plotted as fold difference (NAE treated versus untreated) using the ΔΔCT method (Livak and Schmittgen, 2001). Transcripts were quantified in total RNA extracts from seeds or seedlings at designated days after sowing in liquid media (same stages as in Figure 1). RNAs were targeted for amplification with gene-specific primers (see Methods). Values shown are averages of duplicate samples, and results from three independent experiments showed similar profiles. Oldest seedlings showed the greatest increase in ABA-responsive transcripts in NAE12:0-treated (35 μM) seedlings.

Figure 4.

NAE-Induced ABI3 Expression Is Inversely Associated with NAE-Regulated Seedling Growth. (A) Growth of wild-type and FAAH-altered seedlings in response to exogenous NAE 12:0 (35 μM). Values for gain in fresh weight are from a representative experiment, and replicate experiments exhibited similar trends (i.e., FAAH overexpressors tolerate NAE12:0, FAAH knockouts do not, and the wild types are intermediate; Wang et al., 2006). Insets show seedlings of the wild type (top left), knockout (bottom left), or the FAAH overexpressor, OE2, grown in 35 μM NAE12:0 for 12 d. Bars = 0.5 cm. (B) Representative agarose gel analysis of ABI3 amplification products in 2- or 10-d-old seedlings grown in 35 μM NAE12:0 (analyzed by semiquantitative RT-PCR). (C) Quantification of transcripts by real-time RT-PCR. Values for transcripts were averaged from two independent measurements at each time point (normalized to 18S rRNA) and are plotted as fold difference between NAE-treated (35 μM) and untreated seedlings.

Figure 5.

ABI3 Expression Is Associated with Growth Arrest and Is Responsive to NAE 12:0 (35 μM) or ABA (0.5 μM) Treatment Only within the NAE/ABA-Sensitive Interval (up to 6 d). (A) and (B) Treatment of early-stage seedlings (e.g., 2 d old) arrests growth (measured after 14 d), whereas treatment of late-stage seedlings (e.g., 10 d old) does not inhibit seedling growth (measured after 14 d). ABA treatment of late-stage seedlings shows characteristic promotion of root growth, but this is not evident in NAE-treated, late-stage seedlings. (C) Expression of ABI3 is apparent only in NAE- or ABA-arrested seedlings (treated at early stage of seedling development) after 14 d of growth. A representative agarose gel of duplicate samples following semiquantitative RT-PCR with either ABI3- or 18S rRNA gene-specific primers.

Figure 6.

FAAH Overexpressors Are Insensitive to NAE but Appear to Be Hypersensitive to Exogenous ABA. Representative seedling images of 14-d-old seedlings germinated and grown in 50 μM NAE12:0 (A) or 0.5 μM ABA (B). Two FAAH-overexpressing lines (OE2 and OE7) are shown. Radicle emergence after 3 d (C) or cotyledon expansion after 7 d (D) also showed ABA hypersensitivity for FAAH overexpressors compared with the wild type, particularly at higher ABA concentrations. Values plotted are means and se of six replicate experiments, each experiment consisting of 25 individuals. Asterisks indicate P < 0.001 versus the wild type.

Figure 7.

ABI Mutants Exhibit Increased Tolerance toward Exogenous NAE12:0. (A) Representative images of 6-d-old seedlings at increasing concentrations of NAE12:0 show that abi mutants (e.g., abi1-1) are larger than the wild type (Landsberg erecta). (B) Cotyledon area, quantified for 45 to 65 seedlings and plotted as a percentage of untreated controls, revealed a shift in the dose–response curve for NAE12:0-induced growth inhibition for abi1-1 and abi2-1 relative to the wild type. Both abi1-1 and abi2-1 were significantly more tolerant to NAE than the wild type (e.g., P < 0.0001 at 20, 30, and 40 μM). (C) Quantification of root length of 3-d-old seedlings showed that abi3-1 was significantly more tolerant (**; P < 0.0000001) to NAE 12:0 than the wild type plotted as either absolute length (left) or as relative length to untreated controls (right). n = ∼300 to 400 (3-d-old seedlings).

Figure 8.

ABA and NAE12:0 Have Synergistic Negative Effects on Seedling Growth in Liquid Media, but This Synergism Is Prevented in abi Mutants. (A) Low levels of NAE12:0 (10 μM) potentiate the inhibitory effects of low levels of ABA (0.1 μM) (both Landsberg erecta and Columbia ecotypes, first and third series of images). abi1-1 is insensitive to the synergistic inhibition. (B) NAE-tolerant seedlings (FAAH overexpressor) are hypersensitive to ABA, and this hypersentivitiy is not rescued by exogenous NAE12:0. (C) Quantification of the synergism between NAE12:0 and ABA by measuring primary root lengths of 10-d-old seedlings grown on solid media (bottom histogram). ABA-insensitive mutants (abi1-1, abi2-1, abi3-1, and abi5-1) did not exhibit the synergistic inhibition of root growth, indicating that NAE action requires an intact ABA-signaling pathway.

Figure 9.

Diagram Showing the Hypothetical Action of Elevated Levels of NAE on Seedling Growth and Its Relationship to ABA Action. NAE arrests seedling growth via a pathway dependent upon ABI1, ABI2, ABI3, and ABI5, and this is associated with upregulation of ABA-responsive genes, such as At HVA22b, CRA1, RAB18, EXGT-A4, ATEM1, and LEAM17. This suppression of seedling growth is reminiscent of the ABA-induced secondary dormancy program discovered by Chua and coworkers (Lopez-Molina et al., 2002), which is operable in young seedlings. We propose that NAE12:0 intersects the ABA signaling pathway downstream from ABA and that ABI3 expression is key to the suppression of growth induced by NAE metabolism. Independent of ABA signaling, NAE12:0 can influence gene expression in seeds and seedlings and suppress seedling growth. Ectopic overexpression of FAAH can reverse the growth suppression by NAE, but the interaction with ABA signaling in FAAH overexpressors is complex since FAAH overexpressors are hypersensitive to ABA. By contrast, the situation with abi mutants is more straightforward because they exhibited partial tolerance to NAE12:0 in addition to their ABA-tolerant phenotypes.