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NAE Function

 

NAE 12:0 and Arabidopsis seedling growth and development

N-Acylethanolamines (NAE’s) are fatty acid amides that are derived from the hydrolysis of N-acylphosphatidylethanolamine (NAPE), by phospholipase D (PLD). This reaction in animal systems is part of the endocannabinoid signaling pathway, which regulates a variety of physiological processes. Desiccated seeds of plants contain NAE’s with chain lengths 12C to 18C. Total NAE content declines sharply after 4 to 8 h imbibition. During the same period of seed imbibition and seed germination the biosynthesis of NAPE is increased substantially. It may be that the rapid depletion of NAE’s during seed imbibition and germination is an important requisite for normal seedling growth. To test this hypothesis Arabidopsis thaliana ecotype Columbia seeds were germinated on levels of 12:0 NAE, N-lauroylethanolamine, above the physiological concentrations. Several morphological abnormalities were evident in seedling roots, although no substantial delay in seed germination was observed. Abnormalities in roots include a dose dependent reduction in the root elongation rate, a swelling of the root tip, and a reduction of the number of root hairs. All effects were specific for NAE 12:0 since seedlings grown in lauric acid (free fatty acid analogue of NAE 12:0) or N-palmitoylethanolamine (NAE 16:0) did not exhibit any significant effects that were observed with NAE 12:0. Characterization of the physiological responses of seedlings to exogenous NAE12:0 are described in Blancaflor et al., 2003).  Ongoing work on the role of NAEs in seedlings continues in collaboration with Dr. Elison Blancaflor at the S.R.Noble Foundation.  There are binding assay studies being conducted to identify binding protein/receptors that mediate NAE action (Tripathy et al., 2003), and other work indicates that NAEs are inhibitors of the alpha isoform of PLD (Austin-Brown and Chapman, 2002).

 

 

NAE and Seedling Growth and Development

         
 
NAEs Interefere with Normal Root Develeopment in Arabidopsis Seedlings
  • (NAE12:0, but not 12:0 FFA, affects root growth
nae and root development
    
 

NAEs interfere with primary root growth in Arabidopsis Seedlings

9 day old plants have drastically reduced primary root growth in 50mµM NAE with mitigated effects seen with 20µM NAE

nae and primary root growth in A. thaliana
    
         

 

NAE and Cellular Effects

       
 

Microtubule Organization is Altered by Elevated NAE

  • NAE 12:0 affects cortical microtubule organization in living Arabidopsis roots
  • GFP::MBD
  • ("Swollen" zone)
 microtubule organization  
         
 

F-Actin Organization is Altered by Elevated NAE

  • F-actin in visualized in living roots and root hairs by GFP::Fimbrin
f-acting organization  
       
 
NAEs Interfere with Endomembrane Organization and Membrane Trafficking

(Arabidopsis roots (ER-GFP) of 9d seedlings)
NAE Treated


Normal
       
 

Cell Wall Formation is Abnormal in NAE-Treated Seedling Roots

 

  • A,B Untreated
  • C,D,E NAE 12:0 Treated
 EM cell wall formation TEM
       

 

 

NAE and Molecular Changes

     
 
nae regulated genes in A. thaliana
  
 
 
nae up and down regulated genes
  
 

The above plot and graph show analysis of replicate microarray data from 4 day seedlings treated with NAE. The volcano plot shows genes with 2 fold or greater change in signal. Color coded data points are the genes selected for further investigation. The pie chart shows up and down regulated genes in a breakdown by function and cellular component.

 
 
 
  

 

N-Acylethanolamine Signaling in Elicitor Perception

N-acylethanolamines (NAEs) belong to a class of "endocannabinoid" lipid mediators that occur both in animal and plant membranes as acylamides of membrane bound N-acylphosphatidylethanolamine (NAPE).  NAEs are known to accumulate during membrane stress and mediate an array of biological activities in animal systems, including neurotransmission, embryo implantation, and immunomodulation.  In plants, within minutes of pathogen elicitor perception, NAPE is hydrolyzed by phospholipase-D (PLD), and NAEs accumulate extracellularly.  The released NAEs are NAE 14:0 and NAE 12:0 (identified by GC-MS), and they accumulate to about 100 nM in elicitor-treated leaves of tobacco.  Submicromolar concentrations of exogenous NAE 14:0 (but not 14:0 free fatty acid) are sufficient to activate phenylalanine-ammonia lyase (PAL 2) gene expression in a manner similar to, but independent of, pathogen elicitor perception.  Mammalian cannabinoid receptor antagonists block activation of PAL2 expression induced by NAE 14:0 (or by elicitors), suggesting the occurrence of an "endocannabinoid" signaling system in plant defense (summary hypothetical model, below). In addition to pathogen elicitor perception, NAEs appear to have lipid mediator roles in ABA-regulated processes (such as stomatal closure) and in seed/seedling development. Please see references below.

     
  nae signal transduction pathway  
 
Hypothetical model illustrating the metabolism of NAPE/NAE in signal transduction of pathogen elicitor perception in tobacco.  Elicitors, such as xylanase (and cryptogein), are perceived by receptors at the cell surface (Hanania and Avni, 1997; Furman-Maturasso et al., 1999).  Elicitor perception activates, among other things, the PLD-mediated hydrolysis of cellular NAPE and the corresponding, rapid extracellular accumulation of NAE (Chapman et al., 1998).  Microsomal NAE formation in vitro was stimulated 20-fold by the addition of mastoparan implicating the involvement of G-proteins in elicitor-regulated NAPE-PLD activation (Chapman et al., 1998).  NAPE was hydrolyzed by either the PLD beta or gamma isoforms in vitro, but not by the PLD alpha isoform (Pappan et al., 1998), suggesting the two recently-discovered, polyphosphoinositide-regulated PLDs may be involved in elicitor signaling.  NAE14:0 levels rose 10 to 50 fold in elicitor treated tobacco leaves, and these in vivo concentrations were sufficient to activate the expression of phenylalanine-ammonia lyase (PAL) gene expression independent of elicitor treatment (Tripathy et al., 1999).  Based on pharmacological experiments with AM281 (Tripathy and Chapman, unpublished results), we postulate that NAE activation (+) of PAL expression is mediated by a CB-like receptor at the cell surface.  Intracellular NO production may be a downstream target of NAE signaling, since NO was recently shown to activate PAL gene expression, but not other classes of “defense genes” (Delledonne et al., 1998; Durner et al., 1998).  It is not yet clear whether NAE release is necessary for PAL activation, and there may be additional pathways independent of NAPE metabolism to activate PAL expression in response to elicitor perception (For complete references see Literature Cited in Chapman, 2000, Chem. Phys. Lipids 108: 221-229.
 
 
 
  

 

 

NAE Function Publications

  1. Chapman, K.D. and Sprinkle, W.B. 1996. Developmental, tissue-specific, and environmental factors regulate the biosynthesis of N-acylphosphatidylethanolamine in cotton (Gossypiumhirsutum L.). Journal of Plant Physiology 149: 277-284.
  2. Chapman, K.D., and Sriparameswaran, A. 1997. Intracellular localization of N-acylphosphatidylethanolamine synthesis in cotyledons of cotton seedlings. Plant and Cell Physiology; 38(12): 1359-1367.
  3. Pappan, K., Austin-Brown, S., Chapman, K.D., Wang, X. 1998. Substrate selectivities and lipid modulation of plant phospholipase Dalpha, beta, and gamma. Arch. Biochem. Biophys. 353: 131-140.
  4. Chapman, K.D. 1998. Phospholipase activity during plant growth and development and in response to environmental stress. Trends in Plant Science, 3(11): 419-426. (Review)
  5. Tripathy S, Venables BJ, Chapman K.D. (1999) N-acylethanolamines in signal transduction of elicitor perception: attenuation of alkalinization response and activation of defense gene expression. Plant Physiol 121:1299–1308.
  6. Chapman K.D (2000) Emerging physiological roles for N-acylphosphatidylethanolamine metabolism in plants: signal transduction and membrane protection. Chem Phys Lipids 108:221–230.
  7. Austin-Brown S., Chapman K.D. (2002) Inhibition of phospholipase Dalpha by N-acylethanolamines. Plant Physiol 129:1892–1898.
  8. Tripathy, S., Kleppinger-Sparace, K., Dixon, R.A., and Chapman, K.D. (2003)N-Acylethanolamine signaling in tobacco is mediated by a membrane-associated, high affinity binding protein. Plant Physiology 131: 1781-1791.
  9. Blancaflor, E.B., Hou, G., and Chapman, K.D. (2003) Elevated levels of N-lauroylethanolamine, an endogenous constituent of desiccated seeds, disrupt normal root development in Arabidopsis thaliana seedlings. Planta 217: 206-217.  INCLUDES SUPPLEMENTAL TIME-LAPSE VIDEO DATA OF ER-GFP.
  10. Chapman, K.D. (2004) Occurrence, metabolism and prospective functions of N-acylethanolamines in higher plants. Progress in Lipid Research, 2004 Jul;43(4):302-27. Review
  11. Motes, C.M., Pechter , P., Yoo, C.M., Wang, Y.S., Chapman,K.D., Blancaflor, E.B (2005) Differential effects of two phospholipase D inhibitors, 1-butanol and N- acylethanolamine (NAE), on in vivo cytoskeletal organization and Arabidopsis seedling growth. Protoplasma, 226:109-123.
  12. Wang Y-S, Shrestha, R., Wiant, W., Venables, B.J., Chapman, KD., Blancaflor, E.B. (2006) Manipulation of Arabidopsis Fatty Acid (Ethanol) Amide Hydrolase Expression Leads to Plants with Modified Growth Characteristics and Altered Sensitivity to N-Acylethanolamines. Proceedings of the National Academy of Sciences, 103(132): 12197-12202.
  13. Teaster ND, Motes CM, Tang Y, Wiant WC, Cotter MQ, Wang YS, Kilaru A, Venables BJ, Hasenstein KH, Gonzalez G, Blancaflor EB, Chapman KD (2007) N-Acylethanolamine metabolism interacts with abscisic acid signaling in Arabidopsis thaliana seedlings. Plant Cell 19(8):2454-69.