The NAE regulatory pathway is part of the endocannabinoid signaling system in vertebrates, and as such it has received considerable experimental attention. Targeted lipidomics provided new opportunities to gain a clearer understanding of the effects of perturbations of the intricate network of the NAE metabolic pathway in vertebrate systems (Astarita and Piomelli 2009
; Astarita et al. 2009
; Kilaru et al. 2010
; Kilaru et al. 2011b
). Results with mammalian systems suggested that the physiological effects of NAE metabolism might involve changes in other metabolites in the pathway, including NAPE. For example, selective lipidomic analysis of FAAH (−/−) mice revealed differences in content and composition of NAPE, NAE, and other major and minor phospholipids in brain and heart tissues, indicating differential regulation of the NAE metabolic pathway (Kilaru et al. 2010
). Despite the physiological implications that NAE metabolites have on plant growth and development, an in depth understanding of the pathway and its regulation in plants is essentially lacking. Here, we have extended methods developed for mammalian systems to plants and profiled the NAPE content and composition, along with NAE, PE, and other phospholipids, in Arabidopsis
seeds and seedlings (-).
, the metabolites NAPE, NAE, and PE were most abundant in seeds, and the levels declined by ~ 10-, 7-, and 2-fold, respectively, as seeds germinated and seedlings developed (, ). Modulation of NAE levels is considered important for proper seedling establishment (Wang et al. 2006
; Teaster et al. 2007
; Cotter et al. 2011
). However, metabolite analyses revealed that normal seedling establishment might require a coordinated decline in NAE metabolites, especially NAPE, which showed the greatest decline during normal seedling growth. Perhaps the lower NAPE content in FAAH
OE seeds, relative to WT seeds, in addition to reduced NAE content (; Wang et al. 2006
), together help to explain their enhanced growth phenotype, as observed in 8-day-old FAAH
OE seedlings grown in the presence or absence of NAE 12:0 treatment (; Wang et al. 2006
; Teaster et al. 2007
; Cotter et al. 2011
). Interestingly, 50 to 80 % of the total NAPE and NAE content in seeds was composed of PU-N
-acyl species (18:2 and 18:3; ), suggesting that it is specifically their decline in seedlings (,) that substantially contributes to seedling development. This concept is supported by the fact that faah
KO seeds, despite their slightly higher levels of NAE (but not NAPE), showed normal germination and growth as long as they retained the capacity to metabolize the large amount of PU-NAEs via
oxidation (Kilaru et al. 2011a
). However, if oxidation also was blocked by LOX inhibitors such as NAE 12:0 (-, (Keereetaweep et al. 2010
)) or nordihydroguaiaretic acid (Keereetaweep et al. 2010
), seedling development was substantially retarded, especially in faah
KO. Additionally, the NAPE and NAE metabolite profiles were markedly elevated and more similar to those in seeds than in seedlings. Taken together, this suggests that activation of PU-NAPE and -NAE metabolism is associated with normal seedling establishment.
Perhaps the most significant observation here was the dramatic elevation in NAPE levels in seedlings after application of exogenous NAE 12:0 (36-fold in faah
KO), which was much more than the increase in NAE content itself (8-fold; ). In addition to being a competitive inhibitor of LOXs (Keereetaweep et al. 2010
) and PLD-α (Austin-Brown and Chapman 2002
), NAE 12:0 is known to affect membrane trafficking and cytoskeletal organization (Blancaflor et al. 2003
; Motes et al. 2005
). It is possible that the physiological actions of NAE 12:0 may be, in part, attributed to increases in levels of NAPE, which has an unusual structural influence on membrane organization and function (Sandoval et al. 1995
). NAPE levels also were elevated during elicitor-induced signaling in tobacco cells (Chapman et al. 1995
) and chilling stress (Chapman and Sprinkle 1996
). Furthermore, a 13-fold increase in NAPE levels was observed in anoxia-stressed potato cells (Rawyler and Braendle 2001
). Together, these data suggest that, while more experimental evidence needs to be obtained, it may be important to take a closer look at the role of NAPE in cellular responses to stress, as this lipid class may have unrecognized roles in plant cell function.
Interestingly, feeding of exogenous NAE 12:0 affected mostly the levels of PU-N-acyl groups of the NAPE species (), similar to profiles of the NAEs (). The N-acyl groups of NAPE (and NAE) were dominated by N-18:2, N-18:3, and N-18:1, regardless of the genotype, whereas the glycerol backbone O-acyl patterns of NAPE remained similar among samples (), indicating that existing PE pools were utilized for NAPE synthesis. On the other hand, the results suggest that perhaps there was a preference for unsaturated 18C acyl-CoA/FFA for NAPE formation under NAE 12:0-stimulated conditions. Further, there was only a moderately higher amount of PE in NAE 12:0-treated seedlings compared to untreated seedlings. In fact, the ratio of PE to NAPE was greater than 6 to 1 in seeds (), but in treated seedlings this ratio was closer to 2 to 1 (). Overall amounts of the normally minor NAPE were as high in treated seedling tissues as the common and abundant PE was in untreated seedling tissues. The marked elevation of NAPE (much more than NAE on a percentage basis) suggests that NAE 12:0 treatment results in negative feedback regulation of the PLD-mediated hydrolysis of NAPE. Whether this is due mostly to a pharmacological inhibition of the PLD enzyme in situ, or an exaggeration of a normal biochemical regulatory process will require further examination. NAPE formation, on the other hand, seems to be under less tight control, allowing NAPE to accumulate in NAE-treated tissues, especially when FAAH activity was compromised (faah KO). Increased capacity for NAE turnover (FAAH OE) appeared to reduce the severity of the accumulation of NAE pathway metabolites (including NAPE), the re-direction of lipid metabolism toward NAPE formation, and the severity of inhibition of seedling growth caused by exogenous NAE treatment (-). Overall, the regulation of NAPE levels and composition in plants appears to require a constitutive metabolism of pathway intermediates, but this is perturbed by the inhibition of NAE catabolism. These data also suggest that the PLD-mediated formation of NAE is subject to substantial regulation and may require further attention as a control point of the NAE regulatory pathway in plants.
Comprehensive lipidomic analyses revealed significant reductions in the levels of galactolipids and phospholipids in FAAH
OE seeds, relative to WT seeds (). The reasons are unclear for the reductions in major membrane lipid classes in the FAAH
OE seeds on a seed mass basis. These differences may be due to differences in water content or the mass of something else other than lipid. We measured seed oil percentage of these genotypes by time-domain, 1
H-NMR, and the oil content did not vary significantly between genotypes. This suggests that the bulk storage lipid metabolism and compartmentation is not substantially influenced by changes in FAAH expression. Other studies have demonstrated that ABA signaling pathways and NAE metabolism interact during seedling establishment (Teaster et al. 2007
; Cotter et al. 2011
). It is possible that altered FAAH expression also influences embryo/seed development pathways through its interaction with ABA signaling, but this stage of development remains to be explored.
During seedling development, an increase in NAE content was associated with a marked reduction in galactolipid biosynthesis via the prokaryotic pathway (). For example, in NAE-treated seedlings, the levels of MGDG 34:6 (synthesized by the prokaryotic pathway) were severely reduced while MGDG 36:6 (synthesized by the eukaryotic pathway) levels were increased. This was especially obvious for wild type and faah knockout seedlings, whereas this trend was somewhat reversed, though not completely, in FAAH OE seedlings, suggesting that indeed it was related to alterations in the capacity for NAE turnover. The change in flux between chloroplast and endoplasmic reticulum membrane lipid synthesis pathways may be a reflection of the significant demand for PE and NAPE formation and their extraplastidial synthesis, such that overall membrane glycerolipid synthesis by the eukaryotic pathway was upregulated, resulting in a general increased availability of ER-derived glycerolipid to support chloroplast MGDG synthesis. On the other hand, DGDG species derived from the eukaryotic pathway were not altered in a similar manner to MGDG, as would be expected. Further, PC and PG metabolites seemed relatively unchanged (glycerolipid classes formed by eukaryotic and prokaryotic pathways, respectively, almost entirely). It is possible that the formation of DGDG from MGDG is unable to keep up with additional MGDG from the eukaryotic pathway, but this seems unlikely. Alternatively, the selective alteration in MGDG species might be a result of reduced seedling growth and stunted development of chloroplasts in NAE-treated seedlings (a reduced requirement for chloroplast lipids), but it is difficult to reconcile how this would result in a selective change within MGDG species and be exhibited not more broadly in chloroplast lipids. Perhaps exogenous NAE acts to inhibit a later step in plastidial MGDG formation, resulting in markedly reduced MGDG formation via the prokaryotic pathway, and increased MGDG formation via the eukaryotic pathway. Nonetheless, the evaluation of this alteration in flux between the prokaryotic and eukaryotic pathways may provide new insight into the functional significance of NAE metabolism in seedlings. In addition to gross morphological changes in seedlings grown in NAE 12:0, there may be a regulation of cellular lipid metabolism in general by the NAE regulatory pathway that remains to be explored in the future. In conclusion, comprehensive profiling of the metabolites of the NAE pathway in Arabidopsis has provided new insights into potential regulatory points of this pathway and how NAE-NAPE metabolism might influence seedling growth.