Manipulation of Arabidopsis fatty acid amide hydrolase expression modifies plant growth and sensitivity to N-acylethanolamines

Abstract

In vertebrates, the endocannabinoid signaling pathway is an important lipid regulatory pathway that modulates a variety of physiological and behavioral processes. N-Acylethanolamines (NAEs) comprise a group of fatty acid derivatives that function within this pathway, and their signaling activity is terminated by an enzyme called fatty acid amide hydrolase (FAAH), which hydrolyzes NAEs to ethanolamine and their corresponding free fatty acids. Bioinformatic approaches led to the identification of plant homologues of FAAH that are capable of hydrolyzing NAEs in vitro. To better understand the role of NAEs in plants, we identified T-DNA knockouts to Arabidopsis FAAH (AtFAAH; At5g64440) and generated plants overexpressing AtFAAH. Here we show that seeds of AtFAAH knockouts had elevated levels of endogenous NAEs, and seedling growth was hypersensitive to exogenously applied NAE. On the other hand, seeds and seedlings of AtFAAH overexpressors had lower endogenous NAE content, and seedlings were less sensitive to exogenous NAE. Moreover, AtFAAH overexpressors displayed enhanced seedling growth and increased cell size. AtFAAH expression and FAAH catalytic activity increased during seed germination and seedling growth, consistent with the timing of NAE depletion during seedling establishment. Collectively, our results show that AtFAAH is one, but not the only, modulator of endogenous NAE levels in plants, and that NAE depletion likely participates in the regulation of plant growth.

Fig. 1.

FAAH expression in Arabidopsis. (A) AtFAAH mRNA transcript levels quantified in seeds, seedlings, and different organs of 6-week-old Arabidopsis plants by quantitative real-time RT-PCR. ACT8 was used to normalize AtFAAH expression levels and plotted relative to transcript levels in inflorescence stems. AtFAAH was expressed in all plant organs with highest levels quantified in 4-d-old seedlings and siliques of 6-week-old plants. Data points represent mean ± SD of triplicates of an experiment. (B) AtFAAH::GUS is expressed strongly in embryos 24–48 h after imbibition and in 4-d-old seedlings. The depletion of total NAEs in germinating seeds (C) correlated strongly with increased FAAH enzyme activity toward NAE12:0 and NAE18:2 (D).

Fig. 2.

Characterization of NAE amidohydrolase activity and NAE sensitivity of AtFAAH knockouts. (A) Reduced NAE amidohydrolase activity in microsomes of knockouts is indicated by the absence of free fatty acid (FFA) peak. The amount of FFA formation was determined by incubating synthetic NAE18:2 and NAE12:0 with microsomes from seedlings. (B) NAE12:0 induced a dose-dependent reduction in seedling development in Arabidopsis wild type as indicated by reduced cotyledon area. The effects of NAE12:0 on Arabidopsis cotyledon area was more pronounced in AtFAAH knockouts. (C) Wild-type seedlings are able to recover from exogenous NAE12:0 2 weeks after germination, whereas AtFAAH knockouts remain severely stunted.

Fig. 3.

Characterization of NAE amidohydrolase activity and NAE sensitivity of AtFAAH overexpressors. (A) Increased NAE amidohydrolase activity in microsomes of three AtFAAH-overexpressing lines. The amount of free fatty acid formation was determined by incubating synthetic NAE18:2, NAE16:0, and NAE12:0 with microsomes from seedlings. Three independent AtFAAH overexpressors were able to sustain hypocotyl (B) and cotyledon (C) expansion despite elevated levels of exogenous NAE12:0. (D) Extended exposure to 500 μM NAE12:0 is strongly inhibitory to vector control and wild-type seedlings. AtFAAH overexpressors, on the other hand, display robust growth despite the high levels and extended exposure to exogenous NAE12:0.

Fig. 4.

Enhanced growth of AtFAAH overexpressors. (A) Representative images of vector controls and AtFAAH-overexpressing seedlings 11 days after germination. Primary root and hypocotyl length is longer (A, arrows) and cotyledon area is larger (B) in the AtFAAH overexpressors. One-month-old plants of the AtFAAH overexpressors are generally larger than vector controls when grown under short days (C) and generally bolt faster (D). The larger cotyledon area in AtFAAH overexpressors is due to larger average epidermal cell size (E) and increased number of epidermal cells with a larger cell area (F). (Scale bar in C, 1 mm.)

Fig. 5.

Comparison of NAE profiles in desiccated Arabidopsis seeds and seedlings of wild type, At5g64440 knockout (SALK_095108), and AtFAAH overexpressors (OE11). NAE types were quantified by isotope-dilution mass spectrometry and summed for total content (A) or plotted individually (B and C). Values represent mean + SD of three to six independent extractions from seeds and seedlings of plants that were grown and harvested at the same time (within a 3-month period). Seeds were stored under identical conditions, whereas seedlings were grown under liquid culture for 8 days before NAE quantification. P values were obtained by Student’s t test.