Publications by authors named "Jantana Keereetaweep"

22 Publications

  • Page 1 of 1

The Role of Sugar Signaling in Regulating Plant Fatty Acid Synthesis.

Front Plant Sci 2021 22;12:643843. Epub 2021 Mar 22.

Biology Department, Brookhaven National Laboratory, Upton, NY, United States.

Photosynthates such as glucose, sucrose, and some of their derivatives play dual roles as metabolic intermediates and signaling molecules that influence plant cell metabolism. Such sugars provide substrates for fatty acid (FA) biosynthesis. However, compared with the well-defined examples of sugar signaling in starch and anthocyanin synthesis, until recently relatively little was known about the role of signaling in regulating FA and lipid biosynthesis. Recent research progress shows that trehalose 6-phosphate and 2-oxoglutarate (2-OG) play direct signaling roles in the regulation of FA biosynthesis by modulating transcription factor stability and enzymatic activities involved in FA biosynthesis. Specifically, mechanistic links between sucrose non-fermenting-1-related protein kinase 1 (SnRK1)-mediated trehalose 6-phosphate (T6P) sensing and its regulation by phosphorylation of WRI1 stability, diacylglycerol acyltransferase 1 (DGAT1) enzyme activity, and of 2-OG-mediated relief of inhibition of acetyl-CoA carboxylase (ACCase) activity by protein PII are exemplified in detail in this review.
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http://dx.doi.org/10.3389/fpls.2021.643843DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8020596PMC
March 2021

Biotin attachment domain-containing proteins mediate hydroxy fatty acid-dependent inhibition of acetyl CoA carboxylase.

Plant Physiol 2021 Apr;185(3):892-901

Biology Department, Brookhaven National Laboratory, Upton, NY 11973, USA.

Hundreds of naturally occurring specialized fatty acids (FAs) have potential as desirable chemical feedstocks if they could be produced at large scale by crop plants; however, transgenic expression of their biosynthetic genes has generally been accompanied by dramatic reductions in oil yield. For example, expression of castor (Ricinus communis) FA hydroxylase (FAH) in the Arabidopsis thaliana FA elongation mutant fae1 resulted in a 50% reduction of FA synthesis rate that was attributed to inhibition of acetyl-CoA carboxylase (ACCase) by an undefined mechanism. Here, we tested the hypothesis that the ricinoleic acid-dependent decrease in ACCase activity is mediated by biotin attachment domain-containing (BADC) proteins. BADCs are inactive homologs of biotin carboxy carrier protein that lack a biotin cofactor and can inhibit ACCase. Arabidopsis contains three BADC genes. To reduce expression levels of BADC1 and BADC3 in fae1/FAH plants, a homozygous badc1,3/fae1/FAH line was created. The rate of FA synthesis in badc1,3/fae1/FAH seeds doubled relative to fae1/FAH, restoring it to fae1 levels, increasing both native FA and HFA accumulation. Total FA per seed, seed oil content, and seed yield per plant all increased in badc1,3/fae1/FAH, to 5.8 µg, 37%, and 162 mg, respectively, relative to 4.9 µg, 33%, and 126 mg, respectively, for fae1/FAH. Transcript levels of FA synthesis-related genes, including those encoding ACCase subunits, did not significantly differ between badc1,3/fae1/FAH and fae1/FAH. These results demonstrate that BADC1 and BADC3 mediate ricinoleic acid-dependent inhibition of FA synthesis. We propose that BADC-mediated FAS inhibition as a general mechanism that limits FA accumulation in specialized FA-accumulating seeds.
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http://dx.doi.org/10.1093/plphys/kiaa109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8133645PMC
April 2021

Expression of a Bacterial Trehalose-6-phosphate Synthase otsA Increases Oil Accumulation in Plant Seeds and Vegetative Tissues.

Front Plant Sci 2021 10;12:656962. Epub 2021 Mar 10.

Department of Biology, Brookhaven National Laboratory, Upton, NY, United States.

We previously demonstrated that exogenous trehalose 6-phosphate (T6P) treatment stabilized WRINKLED1 (WRI1), a master transcriptional regulator of fatty acid (FA) synthesis and increased total FA content in () embryo suspension cell culture. Here, we explore lines heterologously expressing the T6P synthase (otsA) or T6P phosphatase (otsB) to refine our understanding regarding the role of T6P in regulating fatty acid synthesis both in seeds and vegetative tissues. 35S: transgenic seeds showed an increase of 13% in fatty acid content compared to those of wild type (WT), while seeds of 35: transgenic seeds showed a reduction of 12% in fatty acid content compared to WT. Expression of otsB significantly reduced the level of WRI1 and expression of its target genes in developing seeds. Like seeds constitutively expressing otsA, transient expression of otsA in leaves resulted in strongly elevated levels of T6P. This was accompanied by an increase of 29% in fatty acid synthesis rate, a 2.3-fold increase in triacylglycerol (TAG) and a 20% increase in total fatty acid content relative to empty vector (EV) controls. Taken together, these data support the heterologous expression of otsA as an approach to increasing TAG accumulation in plant seeds and vegetative tissues.
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http://dx.doi.org/10.3389/fpls.2021.656962DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7988188PMC
March 2021

Seedling Chloroplast Responses Induced by -Linolenoylethanolamine Require Intact G-Protein Complexes.

Plant Physiol 2020 09 14;184(1):459-477. Epub 2020 Jul 14.

Department of Biological Sciences and BioDiscovery Institute, University of North Texas, Denton, Texas 76203

In animals, several long-chain -acylethanolamines (NAEs) have been identified as endocannabinoids and are autocrine signals that operate through cell surface G-protein-coupled cannabinoid receptors. Despite the occurrence of NAEs in land plants, including nonvascular plants, their precise signaling properties and molecular targets are not well defined. Here we show that the activity of -linolenoylethanolamine (NAE 18:3) requires an intact G-protein complex. Specifically, genetic ablation of the Gβγ dimer or loss of the full set of atypical Gα subunits strongly attenuates an NAE-18:3-induced degreening of cotyledons in Arabidopsis () seedlings. This effect involves, at least in part, transcriptional regulation of chlorophyll biosynthesis and catabolism genes. In addition, there is feedforward transcriptional control of G-protein signaling components and G-protein interactors. These results are consistent with NAE 18:3 being a lipid signaling molecule in plants with a requirement for G-proteins to mediate signal transduction, a situation similar, but not identical, to the action of NAE endocannabinoids in animal systems.
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http://dx.doi.org/10.1104/pp.19.01552DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7479873PMC
September 2020

Castor Stearoyl-ACP Desaturase Can Synthesize a Vicinal Diol by Dioxygenase Chemistry.

Plant Physiol 2020 02 5;182(2):730-738. Epub 2019 Dec 5.

Biology Department, Brookhaven National Laboratory, Upton, New York 11973

In previous work, we identified a triple mutant of the castor () stearoyl-Acyl Carrier Protein desaturase (T117R/G188L/D280K) that, in addition to introducing a double bond into stearate to produce oleate, performed an additional round of oxidation to convert oleate to a trans allylic alcohol acid. To determine the contributions of each mutation, in this work we generated individual castor desaturase mutants carrying residue changes corresponding to those in the triple mutant and investigated their catalytic activities. We observed that T117R, and to a lesser extent D280K, accumulated a novel product, namely -9,10-dihydroxystearate, that we identified via its methyl ester through gas chromatography-mass spectrometry and comparison with authentic standards. The use of O labeling showed that the oxygens of both hydroxyl moieties originate from molecular oxygen rather than water. Incubation with an equimolar mixture of O and O demonstrated that both hydroxyl oxygens originate from a single molecule of O, proving the product is the result of dioxygenase catalysis. Using prolonged incubation, we discovered that wild-type castor desaturase is also capable of forming -9,10-dihydroxystearate, which presents a likely explanation for its accumulation to ∼0.7% in castor oil, the biosynthetic origin of which had remained enigmatic for decades. In summary, the findings presented here expand the documented constellation of di-iron enzyme catalysis to include a dioxygenase reactivity in which an unactivated alkene is converted to a vicinal diol.
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http://dx.doi.org/10.1104/pp.19.01111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6997704PMC
February 2020

WRINKLED1 Regulates BIOTIN ATTACHMENT DOMAIN-CONTAINING Proteins that Inhibit Fatty Acid Synthesis.

Plant Physiol 2019 09 17;181(1):55-62. Epub 2019 Jun 17.

Department of Biology, Brookhaven National Laboratory 463, Upton, New York 11973

WRINKLED1 (WRI1) is a transcriptional activator that binds to a conserved sequence (designated as AW box) boxes in the promoters of many genes from central metabolism and fatty acid (FA) synthesis, resulting in their transcription. BIOTIN ATTACHMENT DOMAIN-CONTAINING (BADC) proteins lack a biotin-attachment domain and are therefore inactive, but in the presence of excess FA, BADC1 and BADC3 are primarily responsible for the observed long-term irreversible inhibition of ACETYL-COA CARBOXYLASE, and consequently FA synthesis. Here, we tested the interaction of WRI1 with genes in Arabidopsis () and found purified WRI1 bound with high affinity to canonical AW boxes from the promoters of all three genes. Consistent with this observation, both expression of , , and genes and BADC1 protein levels were reduced in relative to the wild type, and elevated upon overexpression. The double mutant phenocopied with respect to both reduction in root length and elevation of indole-3-acetic acid-Asp levels relative to the wild type. Overexpression of in decreased indole-3-acetic acid-Asp content and partially rescued its short-root phenotype, demonstrating a role for BADCs in seedling establishment. That WRI1 positively regulates genes encoding both FA synthesis and BADC proteins (i.e. conditional inhibitors of FA synthesis), represents a coordinated mechanism to achieve lipid homeostasis in which plants couple the transcription of their FA synthetic capacity with their capacity to biochemically downregulate it.
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http://dx.doi.org/10.1104/pp.19.00587DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6716254PMC
September 2019

Trehalose 6-Phosphate Positively Regulates Fatty Acid Synthesis by Stabilizing WRINKLED1.

Plant Cell 2018 10 24;30(10):2616-2627. Epub 2018 Sep 24.

Department of Biology, Brookhaven National Laboratory, Upton, New York 11973

WRINKLED1 (WRI1), the transcriptional activator of fatty acid synthesis, was recently identified as a target of KIN10, a catalytic α-subunit of the SUCROSE-NON-FERMENTING1-RELATED PROTEIN KINASE1 (SnRK1). We tested the hypothesis that trehalose 6-phosphate (T6P), a signal of cellular sucrose status, can regulate fatty acid synthesis by inhibiting SnRK1. Incubation of suspension cells in medium containing T6P, or overexpression of the T6P synthase, OtsA, in , significantly increased T6P levels, WRI1 levels, and fatty acid synthesis rates. T6P directly bound to purified recombinant KIN10 with an equilibrium dissociation constant () of 32 ± 6 μM based on microscale thermophoresis. GEMINIVIRUS REP-INTERACTING KINASE1 (GRIK1) bound to KIN10 ( 19 ± 3 μM) and activated it by phosphorylation. In the presence of T6P, the GRIK1-KIN10 association was weakened by more than 3-fold ( 68 ± 9.8 μM), which reduced both the phosphorylation of KIN10 and its activity. T6P-dependent inhibition of SnRK1 activity was reduced in extracts of individual and mutants relative to the wild type, while SnRK1 activity in extracts was enhanced by T6P. These results indicate that the T6P sensitivity of SnRK1 in vivo is GRIK1/GRIK2 dependent. Based on our findings, we propose a mechanistic model that links sugar signaling and fatty acid homeostasis.
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http://dx.doi.org/10.1105/tpc.18.00521DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6241258PMC
October 2018

Biotin Attachment Domain-Containing Proteins Irreversibly Inhibit Acetyl CoA Carboxylase.

Plant Physiol 2018 05 6;177(1):208-215. Epub 2018 Apr 6.

Biology Department, Brookhaven National Laboratory, Upton, New York 11973

The first committed step in fatty acid synthesis is mediated by acetyl-CoA carboxylase (ACCase), a biotin-dependent enzyme that carboxylates acetyl-CoA to produce malonyl-CoA. ACCase can be feedback regulated by short-term or long-term exposure to fatty acids in the form of Tween 80 (predominantly containing oleic acid), which results in reversible or irreversible ACCase inhibition, respectively. Biotin attachment domain-containing (BADC) proteins are inactive analogs of biotin carboxyl transfer proteins that lack biotin, and their incorporation into ACCase down-regulates its activity by displacing active (biotin-containing) biotin carboxyltransferase protein subunits. Arabidopsis () lines containing T-DNA insertions in , , and were used to generate and double mutants. The mutant exhibited normal growth and development; however, ACCase activity was 26% higher in and its seeds contained 30.1% more fatty acids and 32.6% more triacylgycerol relative to wild-type plants. To assess whether BADC contributes to the irreversible phase of ACCase inhibition, cell suspension cultures were generated from the leaves of and wild-type plants and treated with 10 mm Tween 80. Reversible ACCase inhibition was similar in and wild-type cultures after 2 d of Tween 80 treatment, but irreversible inhibition was reduced by 50% in relative to wild-type plants following 4 d of Tween 80 treatment. In this study, we present evidence for two important homeostatic roles for BADC proteins in down-regulating ACCase activity: by acting during normal growth and development and by contributing to its long-term irreversible feedback inhibition resulting from the oversupply of fatty acids.
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http://dx.doi.org/10.1104/pp.18.00216DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5933113PMC
May 2018

Thermal acclimation in American alligators: Effects of temperature regime on growth rate, mitochondrial function, and membrane composition.

J Therm Biol 2017 Aug 29;68(Pt A):45-54. Epub 2016 Jun 29.

Developmental Integrative Biology Research Group, Department of Biological Sciences, University of North Texas, Denton, TX 76203, USA.

We investigated the ability of juvenile American alligators (Alligator mississippiensis) to acclimate to temperature with respect to growth rate. We hypothesized that alligators would acclimate to cold temperature by increasing the metabolic capacity of skeletal muscles and the heart. Additionally, we hypothesized that lipid membranes in the thigh muscle and liver would respond to low temperature, either to maintain fluidity (via increased unsaturation) or to maintain enzyme reaction rates (via increased docosahexaenoic acid). Alligators were assigned to one of 3 temperature regimes beginning at 9 mo of age: constant warm (30°C), constant cold (20°C), and daily cycling for 12h at each temperature. Growth rate over the following 7 mo was highest in the cycling group, which we suggest occurred via high digestive function or feeding activity during warm periods and energy-saving during cold periods. The warm group also grew faster than the cold group. Heart and liver masses were proportional to body mass, while kidney was proportionately larger in the cold group compared to the warm animals. Whole-animal metabolic rate was higher in the warm and cycling groups compared to the cold group - even when controlling for body mass - when assayed at 30°C, but not at 20°C. Mitochondrial oxidative phosphorylation capacity in permeabilized fibers of thigh muscle and heart did not differ among treatments. Membrane fatty acid composition of the brain was largely unaffected by temperature treatment, but adjustments were made in the phospholipid headgroup composition that are consistent with homeoviscous adaptation. Thigh muscle cell membranes had elevated polyunsaturated fatty acids in the cold group relative to the cycling group, but this was not the case for thigh muscle mitochondrial membranes. Liver mitochondria from cold alligators had elevated docosahexaenoic acid, which might be important for maintenance of reaction rates of membrane-bound enzymes.
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http://dx.doi.org/10.1016/j.jtherbio.2016.06.016DOI Listing
August 2017

Changes in Retinal N-Acylethanolamines and their Oxylipin Derivatives During the Development of Visual Impairment in a Mouse Model for Glaucoma.

Lipids 2016 07 24;51(7):857-66. Epub 2016 May 24.

Department of Ophthalmology, Vision Research Center, School of Medicine, University of Missouri-Kansas City, Kansas City, MO, 64108, USA.

Neurons are especially susceptible to oxidative damage, which is increasingly implicated in neurodegenerative disease. Certain N-acylethanolamines (NAEs) have been shown to protect neurons from oxidative stress. Since glaucoma may be considered a neurodegenerative disorder and the survival of retinal neurons could also be influenced by N-acylethanolamines, our goal was to quantify changes in certain N-acylethanolamine species and their oxylipin derivatives in the retina of a mouse model for glaucoma. We also sought to identify relationships between these and parameters of glaucoma disease development, specifically intraocular pressure, visual acuity, and contrast sensitivity. Five N-acylethanolamine species and three NAE oxylipin derivatives were quantified in retina from young and aged DBA/2Crl mice. N-Acylethanolamines and NAE-oxylipins in retinal extracts were quantified against deuterated standards by isotope dilution gas chromatography-mass spectrometry. Levels (nmol/g dry weight) of N-arachidonoylethanolamine (anandamide; NAE 20:4) were significantly (p = 0.008) decreased in aged (2.875 ± 0.6702) compared to young animals (5.175 ± 0.971). Conversely, the anandamide oxylipin, 15(S)-HETE ethanolamide (15(S)-HETE EA), was significantly (p = 0.042) increased in aged (0.063 ± 0.009) compared to young animals (0.039 ± 0.011). Enzymatic depletion of the anandamide pool by 15-lipoxygenase and consequent accumulation of 15(S)-HETE ethanolamine may contribute to decreased visual function in glaucomatous mice. Since N-acylethanolamines effectively attenuate glaucoma pathogenesis and associated visual impairment, our data provides additional rationale and novel targets for glaucoma therapies.
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http://dx.doi.org/10.1007/s11745-016-4161-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4911801PMC
July 2016

Lipidomic Analysis of Endocannabinoid Signaling: Targeted Metabolite Identification and Quantification.

Neural Plast 2016 29;2016:2426398. Epub 2015 Dec 29.

Department of Biological Sciences, Center for Plant Lipid Research, University of North Texas, Denton, TX 76203, USA.

The endocannabinoids N-arachidonoylethanolamide (or anandamide, AEA) and 2-arachidonoylglycerol (2-AG) belong to the larger groups of N-acylethanolamines (NAEs) and monoacylglycerol (MAG) lipid classes, respectively. They are biologically active lipid molecules that activate G-protein-coupled cannabinoid receptors found in various organisms. After AEA and 2-AG were discovered in the 1990s, they have been extensively documented to have a broad range of physiological functions. Along with AEA, several NAEs, for example, N-palmitoylethanolamine (PEA), N-stearoylethanolamine (SEA), and N-oleoylethanolamine (OEA) are also present in tissues, usually at much larger concentrations than AEA. Any perturbation that involves the endocannabinoid pathway may subsequently alter basal level or metabolism of these lipid mediators. Further, the altered levels of these molecules often reflect pathological conditions associated with tissue damage. Robust and sensitive methodologies to analyze these lipid mediators are essential to understanding how they act as endocannabinoids. The recent advances in mass spectrometry allow researchers to develop lipidomics approaches and several methodologies have been proposed to quantify endocannabinoids in various biological systems.
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http://dx.doi.org/10.1155/2016/2426398DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4709765PMC
November 2016

Facilitation of Fusarium graminearum Infection by 9-Lipoxygenases in Arabidopsis and Wheat.

Mol Plant Microbe Interact 2015 Oct 6;28(10):1142-52. Epub 2015 Oct 6.

1 Department of Biological Sciences, University of North Texas, Denton, TX 76203, U.S.A.;

Fusarium graminearum causes Fusarium head blight, an important disease of wheat. F. graminearum can also cause disease in Arabidopsis thaliana. Here, we show that the Arabidopsis LOX1 and LOX5 genes, which encode 9-lipoxygenases (9-LOXs), are targeted during this interaction to facilitate infection. LOX1 and LOX5 expression were upregulated in F. graminearum-inoculated plants and loss of LOX1 or LOX5 function resulted in enhanced disease resistance in the corresponding mutant plants. The enhanced resistance to F. graminearum infection in the lox1 and lox5 mutants was accompanied by more robust induction of salicylic acid (SA) accumulation and signaling and attenuation of jasmonic acid (JA) signaling in response to infection. The lox1- and lox5-conferred resistance was diminished in plants expressing the SA-degrading salicylate hydroxylase or by the application of methyl-JA. Results presented here suggest that plant 9-LOXs are engaged during infection to control the balance between SA and JA signaling to facilitate infection. Furthermore, since silencing of TaLpx-1 encoding a 9-LOX with homology to LOX1 and LOX5, resulted in enhanced resistance against F. graminearum in wheat, we suggest that 9-LOXs have a conserved role as susceptibility factors in disease caused by this important fungus in Arabidopsis and wheat.
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http://dx.doi.org/10.1094/MPMI-04-15-0096-RDOI Listing
October 2015

Lipoxygenase-derived 9-hydro(pero)xides of linoleoylethanolamide interact with ABA signaling to arrest root development during Arabidopsis seedling establishment.

Plant J 2015 Apr;82(2):315-27

Department of Biological Sciences, Center for Plant Lipid Research, University of North Texas, Denton, TX, 76203, USA.

Ethanolamide-conjugated fatty acid derivatives, also known as N-acylethanolamines (NAEs), occur at low levels (μg per g) in desiccated seeds, and endogenous amounts decline rapidly with seedling growth. Linoleoylethanolamide (NAE18:2) is the most abundant of these NAEs in seeds of almost all plants, including Arabidopsis thaliana. In Arabidopsis, NAE18:2 may be oxidized by lipoxygenase (LOX) or hydrolyzed by fatty acid amide hydrolase (FAAH) during normal seedling establishment, and this contributes to the normal progression of NAE depletion that is coincident with the depletion of abscisic acid (ABA). Here we provide biochemical, genetic and pharmacological evidence that a specific 9-LOX metabolite of NAE18:2 [9-hydro(pero)xy linoleoylethanolamide (9-NAE-H(P)OD)] has a potent negative influence on seedling root elongation, and acts synergistically with ABA to modulate the transition from embryo to seedling growth. Genetic analyses using mutants in ABA synthesis (aba1 and aba2), perception (pyr1, pyl1, pyl2, pyl4, pyl5 and pyl8) or transcriptional activation (abi3-1) indicated that arrest of root growth by 9-NAE-H(P)OD requires an intact ABA signaling pathway, and probably operates to increase ABA synthesis as part of a positive feedback loop to modulate seedling establishment in response to adverse environmental conditions. These results identify a specific, bioactive ethanolamide oxylipin metabolite of NAE18:2, different from those of ethanolamide-conjugated linolenic acid (NAE18:3), as well as a molecular explanation for its inhibitory action, emphasizing the oxidative metabolism of NAEs as an important feature of seedling development.
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http://dx.doi.org/10.1111/tpj.12821DOI Listing
April 2015

CGI-58, a key regulator of lipid homeostasis and signaling in plants, also regulates polyamine metabolism.

Plant Signal Behav 2014 3;9(2):e27723. Epub 2014 Feb 3.

USDA-ARS; US Arid-Land Agricultural Research Center; Maricopa, AZ USA.

Comparative Gene Identification-58 (CGI-58) is an α/β hydrolase-type protein that regulates lipid homeostasis and signaling in eukaryotes by interacting with and stimulating the activity of several different types of proteins, including a lipase in mammalian cells and a peroxisomal ABC transporter (PXA1) in plant cells. Here we show that plant CGI-58 also interacts with spermidine synthase 1 (SPDS1), an enzyme that plays a central role in polyamine metabolism by converting putrescine into spermidine. Analysis of polyamine contents in Arabidopsis thaliana plants revealed that spermidine levels were significantly reduced, and putrescine increased, in both cgi-58 and cgi-58/pxa1 mutant plants, relative to pxa1 mutant or wild-type plants. Evaluation of polyamine-related gene expression levels, however, revealed similar increases in transcript abundance in all mutants, including cgi-58, pxa1, and cgi-58/pxa1, in comparison to wild type. Taken together, the data support a model whereby CGI-58 and PXA1 contribute to the regulation of polyamine metabolism at the transcriptional level, perhaps through a shared lipid-signaling pathway, and that CGI-58 also acts independently of PXA1 to increase spermidine content at a post-transcriptional level, possibly through protein-protein interaction with SPDS1.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4091556PMC
http://dx.doi.org/10.4161/psb.27723DOI Listing
June 2015

N-Acylethanolamines: lipid metabolites with functions in plant growth and development.

Plant J 2014 Aug 25;79(4):568-83. Epub 2014 Feb 25.

Plant Biology Division, The Samuel Roberts Noble Foundation Inc., 2510 Sam Noble Parkway, Ardmore, OK, 73401, USA.

Twenty years ago, N-acylethanolamines (NAEs) were considered by many lipid chemists to be biological 'artifacts' of tissue damage, and were, at best, thought to be minor lipohilic constituents of various organisms. However, that changed dramatically in 1993, when anandamide, an NAE of arachidonic acid (N-arachidonylethanolamine), was shown to bind to the human cannabinoid receptor (CB1) and activate intracellular signal cascades in mammalian neurons. Now NAEs of various types have been identified in diverse multicellular organisms, in which they display profound biological effects. Although targets of NAEs are still being uncovered, and probably vary among eukaryotic species, there appears to be remarkable conservation of the machinery that metabolizes these bioactive fatty acid conjugates of ethanolamine. This review focuses on the metabolism and functions of NAEs in higher plants, with specific reference to the formation, hydrolysis and oxidation of these potent lipid mediators. The discussion centers mostly on early seedling growth and development, for which NAE metabolism has received the most attention, but also considers other areas of plant development in which NAE metabolism has been implicated. Where appropriate, we indicate cross-kingdom conservation in NAE metabolic pathways and metabolites, and suggest areas where opportunities for further investigation appear most pressing.
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http://dx.doi.org/10.1111/tpj.12427DOI Listing
August 2014

Ethanolamide oxylipins of linolenic acid can negatively regulate Arabidopsis seedling development.

Plant Cell 2013 Oct 22;25(10):3824-40. Epub 2013 Oct 22.

Department of Biological Sciences, University of North Texas, Center for Plant Lipid Research, Denton, Texas 76203.

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.
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http://dx.doi.org/10.1105/tpc.113.119024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3877782PMC
October 2013

N-Palmitoylethanolamine depot injection increased its tissue levels and those of other acylethanolamide lipids.

Drug Des Devel Ther 2013 12;7:747-52. Epub 2013 Aug 12.

Vision Research Center, Department of Ophthalmology, School of Medicine, University of Missouri-Kansas City, MO 64108, USA.

N-Palmitoylethanolamine (NAE 16:0) is an endogenous lipid signaling molecule that has limited water solubility, and its action is short-lived due to its rapid metabolism. This poses a problem for use in vivo as oral administration requires a high concentration for significant levels to reach target tissues, and injection of the compound in a dimethyl sulfoxide- or ethanol-based vehicle is usually not desirable during long-term treatment. A depot injection of NAE 16:0 was successfully emulsified in sterile corn oil (10 mg/kg) and administered in young DBA/2 mice in order to elevate baseline levels of NAE 16:0 in target tissues. NAE 16:0 levels were increased in various tissues, particularly in the retina, 24 and 48 hours following injections. Increases ranged between 22% and 215% (above basal levels) in blood serum, heart, brain, and retina and induced an entourage effect by increasing levels of other 18 carbon N-Acylethanolamines (NAEs), which ranged between 31% and 117% above baseline. These results indicate that NAE 16:0 can be used as a depot preparation, avoiding the use of inadequate vehicles, and can provide the basis for designing tissue-specific dosing regimens for therapies involving NAEs and related compounds.
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http://dx.doi.org/10.2147/DDDT.S48324DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3746786PMC
May 2014

The α/β hydrolase CGI-58 and peroxisomal transport protein PXA1 coregulate lipid homeostasis and signaling in Arabidopsis.

Plant Cell 2013 May 10;25(5):1726-39. Epub 2013 May 10.

U.S. Department of Agriculture-Agricultural Research Service, U.S. Arid-Land Agricultural Research Center, Maricopa, Arizona 85138, USA.

COMPARATIVE GENE IDENTIFICATION-58 (CGI-58) is a key regulator of lipid metabolism and signaling in mammals, but its underlying mechanisms are unclear. Disruption of CGI-58 in either mammals or plants results in a significant increase in triacylglycerol (TAG), suggesting that CGI-58 activity is evolutionarily conserved. However, plants lack proteins that are important for CGI-58 activity in mammals. Here, we demonstrate that CGI-58 functions by interacting with the PEROXISOMAL ABC-TRANSPORTER1 (PXA1), a protein that transports a variety of substrates into peroxisomes for their subsequent metabolism by β-oxidation, including fatty acids and lipophilic hormone precursors of the jasmonate and auxin biosynthetic pathways. We also show that mutant cgi-58 plants display changes in jasmonate biosynthesis, auxin signaling, and lipid metabolism consistent with reduced PXA1 activity in planta and that, based on the double mutant cgi-58 pxa1, PXA1 is epistatic to CGI-58 in all of these processes. However, CGI-58 was not required for the PXA1-dependent breakdown of TAG in germinated seeds. Collectively, the results reveal that CGI-58 positively regulates many aspects of PXA1 activity in plants and that these two proteins function to coregulate lipid metabolism and signaling, particularly in nonseed vegetative tissues. Similarities and differences of CGI-58 activity in plants versus animals are discussed.
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http://dx.doi.org/10.1105/tpc.113.111898DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3694702PMC
May 2013

The green peach aphid, Myzus persicae, acquires a LIPOXYGENASE5-derived oxylipin from Arabidopsis thaliana, which promotes colonization of the host plant.

Plant Signal Behav 2013 Jan 6;8(1):e22735. Epub 2012 Dec 6.

Department of Biological Sciences and Center for Plant Lipid Research; University of North Texas; Denton, TX USA.

Oxylipins derived from lipoxygenase (LOX) activity play important roles in plant growth, development and stress response. In a recent study, we provided evidence that infestation of Arabidopsis thaliana foliage by the green peach aphid (GPA; Myzus persicae), a phloem sap-consuming insect, was promoted by plant LOX5-derived oxylipins. In comparison to the wild-type (WT) plant, GPA population was smaller on the Arabidopsis lox5 mutant. The insect spent less time feeding from the sieve element and xylem of the lox5 mutant compared with the WT plant. In addition, compared with insects feeding on the WT plant, when on the lox5 mutant, the GPA was unable to suppress an antibiotic activity that is present in Arabidopsis vascular sap. Roots are the critical source of a LOX5-derived oxylipin(s) that promotes colonization of the foliage by GPA. Here we show that the 9-hydoxy-10E, 12Z-octadecadienoic acid (9-HOD), a LOX5-derived oxylipin, accumulated in GPA that were reared on the WT, but not the lox5 mutant plant. However, 9-HOD accumulated in insects reared on lox5 mutant plants that were irrigated with 9-HOD, thus indicating that the insect ingests oxylipins from the host plant. We further demonstrate that the host plant requires LOX5 function to promote expression of the defense regulatory gene PHYTOALEXIN-DEFICIENT4 in the foliage. Taken together, our previous observations and results presented here indicate that while the host plant utilizes LOX5-dependent factors for promoting defense mechanisms, GPA has evolved to utilize plant 9-LOX-derived oxylipins as cues to facilitate infestation, thus suggesting a complex involvement of oxylipins in Arabidopsis interaction with GPA.
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http://dx.doi.org/10.4161/psb.22735DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3745579PMC
January 2013

Overexpression of Fatty Acid Amide Hydrolase Induces Early Flowering in Arabidopsis thaliana.

Front Plant Sci 2012 20;3:32. Epub 2012 Feb 20.

Plant Biology Division, The Samuel Roberts Noble Foundation Ardmore, OK, USA.

N-acylethanolamines (NAEs) are bioactive lipids derived from the hydrolysis of the membrane phospholipid N-acylphosphatidylethanolamine (NAPE). In animal systems this reaction is part of the "endocannabinoid" signaling pathway, which regulates a variety of physiological processes. The signaling function of NAE is terminated by fatty acid amide hydrolase (FAAH), which hydrolyzes NAE to ethanolamine and free fatty acid. Our previous work in Arabidopsis thaliana showed that overexpression of AtFAAH (At5g64440) lowered endogenous levels of NAEs in seeds, consistent with its role in NAE signal termination. Reduced NAE levels were accompanied by an accelerated growth phenotype, increased sensitivity to abscisic acid (ABA), enhanced susceptibility to bacterial pathogens, and early flowering. Here we investigated the nature of the early flowering phenotype of AtFAAH overexpression. AtFAAH overexpressors flowered several days earlier than wild type and AtFAAH knockouts under both non-inductive short day (SD) and inductive long day (LD) conditions. Microarray analysis revealed that the FLOWERING LOCUS T (FT) gene, which plays a major role in regulating flowering time, and one target MADS box transcription factor, SEPATALLA3 (SEP3), were elevated in AtFAAH overexpressors. Furthermore, AtFAAH overexpressors, with the early flowering phenotype had lower endogenous NAE levels in leaves compared to wild type prior to flowering. Exogenous application of NAE 12:0, which was reduced by up to 30% in AtFAAH overexpressors, delayed the onset of flowering in wild type plants. We conclude that the early flowering phenotype of AtFAAH overexpressors is, in part, explained by elevated FT gene expression resulting from the enhanced NAE hydrolase activity of AtFAAH, suggesting that NAE metabolism may participate in floral signaling pathways.
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http://dx.doi.org/10.3389/fpls.2012.00032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3355813PMC
August 2012

Lipoxygenase-mediated oxidation of polyunsaturated N-acylethanolamines in Arabidopsis.

J Biol Chem 2011 Apr 3;286(17):15205-14. Epub 2011 Mar 3.

Center for Plant Lipid Research, Department of Biological Sciences, University of North Texas, Denton, Texas 76203, USA.

N-acylethanolamines (NAEs) are bioactive fatty acid derivatives that occur in all eukaryotes. In plants, NAEs have potent negative growth-regulating properties, and fatty acid amide hydrolase (FAAH)-mediated hydrolysis is a primary catabolic pathway that operates during seedling establishment to deplete these compounds. Alternatively, polyunsaturated (PU)-NAEs may serve as substrates for lipid oxidation. In Arabidopsis, PU-NAEs (NAE 18:2 and NAE 18:3) were the most abundant NAE species in seeds, and their levels were depleted during seedling growth even in FAAH tDNA knock-out plants. Therefore, we hypothesized that lipoxygenase (LOX) participated in the metabolism of PU-NAEs through the formation of NAE-oxylipins. Comprehensive chromatographic and mass spectrometric methods were developed to identify NAE hydroperoxides and -hydroxides. Recombinant Arabidopsis LOX enzymes expressed in Escherichia coli utilized NAE 18:2 and NAE 18:3 as substrates with AtLOX1 and AtLOX5 exhibiting 9-LOX activity and AtLOX2, AtLOX3, AtLOX4, and AtLOX6 showing predominantly 13-LOX activity. Feeding experiments with exogenous PU-NAEs showed they were converted to hydroxide metabolites indicating that indeed Arabidopsis seedlings had the capacity for LOX-mediated metabolism of PU-NAEs in planta. Detectable levels of endogenous NAE-oxylipin metabolites were identified in FAAH fatty acid amide hydrolase seedlings but not in wild-type or FAAH overexpressors, suggesting that NAE hydroxide pools normally do not accumulate unless flux through hydrolysis is substantially reduced. These data suggest that Arabidopsis LOXs indeed compete with FAAH to metabolize PU-NAEs during seedling establishment. Identification of endogenous amide-conjugated oxylipins suggests potential significance of these metabolites in vivo, and FAAH mutants may offer opportunities to address this in the future.
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http://dx.doi.org/10.1074/jbc.M110.217588DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3083184PMC
April 2011

Lauroylethanolamide is a potent competitive inhibitor of lipoxygenase activity.

FEBS Lett 2010 Jul 10;584(14):3215-22. Epub 2010 Jun 10.

University of North Texas, Center for Plant Lipid Research, Department of Biological Sciences, Denton, TX 76203, USA.

The lipoxygenase (LOX) pathway was proposed to compete with hydrolysis and be partly responsible for the metabolism of polyunsaturated N-acylethanolamines (PU-NAEs). Treatment of Arabidopsis seedlings with lauroylethanolamide (NAE 12:0) resulted in elevated levels of PU-NAE species, and this was most pronounced in plants with reduced NAE hydrolase activity. Enzyme activity assays revealed that NAE 12:0 inhibited LOX-mediated oxidation of PU lipid substrates in a dose-dependent and competitive manner. NAE 12:0 was 10-20 times more potent an inhibitor of LOX activities than lauric acid (FFA 12:0). Furthermore, treatment of intact Arabidopsis seedlings with NAE 12:0 (but not FFA 12:0) substantially blocked the wound-induced formation of jasmonic acid (JA), suggesting that NAE 12:0 may be used in planta to manipulate oxylipin metabolism.
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http://dx.doi.org/10.1016/j.febslet.2010.06.008DOI Listing
July 2010