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• W2555348599 abstract "N-Acylethanolamines (NAEs) are bioactive fatty acid derivatives present in trace amounts in many eukaryotes. Although NAEs have signaling and physiological roles in animals, little is known about their metabolic fate in plants. Our previous microarray analyses showed that inhibition of Arabidopsis thaliana seedling growth by exogenous N-lauroylethanolamine (NAE 12:0) was accompanied by the differential expression of multiple genes encoding small molecule-modifying enzymes. We focused on the gene At5g39050, which encodes a phenolic glucoside malonyltransferase 1 (PMAT1), to better understand the biological significance of NAE 12:0-induced gene expression changes. PMAT1 expression was induced 3–5-fold by exogenous NAE 12:0. PMAT1 knockouts (pmat1) had reduced sensitivity to the growth-inhibitory effects of NAE 12:0 compared with wild type leading to the hypothesis that PMAT1 might be a previously uncharacterized regulator of NAE metabolism in plants. To test this hypothesis, metabolic profiling of wild-type and pmat1 seedlings treated with NAE 12:0 was conducted. Wild-type seedlings treated with NAE 12:0 accumulated glucosylated and malonylated forms of this NAE species, and structures were confirmed using nuclear magnetic resonance (NMR) spectroscopy. By contrast, only the peak corresponding to NAE 12:0-glucoside was detected in pmat1. Recombinant PMAT1 catalyzed the reaction converting NAE 12:0-glucoside to NAE 12:0-mono- or -dimalonylglucosides providing direct evidence that this enzyme is involved in NAE 12:0-glucose malonylation. Taken together, our results indicate that glucosylation of NAE 12:0 by a yet to be determined glucosyltransferase and its subsequent malonylation by PMAT1 could represent a mechanism for modulating the biological activities of NAEs in plants. N-Acylethanolamines (NAEs) are bioactive fatty acid derivatives present in trace amounts in many eukaryotes. Although NAEs have signaling and physiological roles in animals, little is known about their metabolic fate in plants. Our previous microarray analyses showed that inhibition of Arabidopsis thaliana seedling growth by exogenous N-lauroylethanolamine (NAE 12:0) was accompanied by the differential expression of multiple genes encoding small molecule-modifying enzymes. We focused on the gene At5g39050, which encodes a phenolic glucoside malonyltransferase 1 (PMAT1), to better understand the biological significance of NAE 12:0-induced gene expression changes. PMAT1 expression was induced 3–5-fold by exogenous NAE 12:0. PMAT1 knockouts (pmat1) had reduced sensitivity to the growth-inhibitory effects of NAE 12:0 compared with wild type leading to the hypothesis that PMAT1 might be a previously uncharacterized regulator of NAE metabolism in plants. To test this hypothesis, metabolic profiling of wild-type and pmat1 seedlings treated with NAE 12:0 was conducted. Wild-type seedlings treated with NAE 12:0 accumulated glucosylated and malonylated forms of this NAE species, and structures were confirmed using nuclear magnetic resonance (NMR) spectroscopy. By contrast, only the peak corresponding to NAE 12:0-glucoside was detected in pmat1. Recombinant PMAT1 catalyzed the reaction converting NAE 12:0-glucoside to NAE 12:0-mono- or -dimalonylglucosides providing direct evidence that this enzyme is involved in NAE 12:0-glucose malonylation. Taken together, our results indicate that glucosylation of NAE 12:0 by a yet to be determined glucosyltransferase and its subsequent malonylation by PMAT1 could represent a mechanism for modulating the biological activities of NAEs in plants. N-Acylethanolamines (NAEs) 2The abbreviations used are: NAE, N-acylethanolamine; CB, cannabinoid; UHPLC, ultra-high performance liquid chromatography; ESI, electrospray ionization; QTOF, quadrupole time of flight; SPE, solid phase extraction; ANOVA, analysis of variance; qRT, quantitative RT; FAAH, fatty acid amide hydrolase; F, forward; R, reverse; ABA, abscisic acid; LOX, lipoxygenase. represent a group of conserved lipid amides that have diverse biological functions in many eukaryotes. Their basic structure consists of an ethanolamine head linked to a fatty acid tail via an amide bond (1.Schmid H.H. Schmid P.C. Natarajan V. The N-acylation-phosphodiesterase pathway and cell signalling.Chem. Phys. Lipids. 1996; 80: 133-142Crossref PubMed Scopus (157) Google Scholar, 2.Chapman K.D. Occurrence, metabolism, and prospective functions of N-acylethanolamines in plants.Prog. Lipid. Res. 2004; 43: 302-327Crossref PubMed Scopus (99) Google Scholar). In vertebrates, NAEs are best known for their role in the endocannabinoid signaling pathway where they function as endogenous ligands to G protein-coupled cannabinoid (CB) receptors. Binding of NAEs to CB receptors triggers a series of signaling cascades that modulate a plethora of behavioral and physiological processes such as appetite, mood, cardiovascular function, pain sensation, sleep, and reproduction (3.Pagotto U. Marsicano G. Cota D. Lutz B. Pasquali R. The emerging role of the endocannabinoid system in endocrine regulation and energy balance.Endocr. Rev. 2006; 27: 73-100Crossref PubMed Scopus (713) Google Scholar, 4.Butler H. Korbonits M. Cannabinoids for clinicians: the rise and fall of the cannabinoid antagonists.Eur. J. Endocrinol. 2009; 161: 655-662Crossref PubMed Scopus (41) Google Scholar5.De Petrocellis L. Di Marzo V. An introduction to the endocannabinoid system: from the early to the latest concepts.Best Pract. Res. Clin. Endocrinol. Metab. 2009; 23: 1-15Crossref PubMed Scopus (185) Google Scholar). NAEs in organisms where they have been detected consist of several molecular species, which are classified based on the number of carbons and double bonds in their fatty acid chain. For example, one of the most studied NAE species in animal systems with CB receptor-dependent functions is N-arachidonoylethanolamine or anandamide. Anandamide is also known as NAE 20:4 because it has 20 carbons and 4 double bonds in its fatty acyl chain (6.Kim S.C. Chapman K.D. Blancaflor E.B. Fatty acid amide lipid mediators in plants.Plant Sci. 2010; 178: 411-419Crossref Scopus (38) Google Scholar, 7.Blancaflor E.B. Kilaru A. Keereetaweep J. Khan B.R. Faure L. Chapman K.D. N-Acylethanolamines: lipid metabolites with functions in plant growth and development.Plant J. 2014; 79: 568-583Crossref PubMed Scopus (43) Google Scholar). Although the presence of NAEs in plants has been recognized for many years, their role in plant physiological processes is limited (2.Chapman K.D. Occurrence, metabolism, and prospective functions of N-acylethanolamines in plants.Prog. Lipid. Res. 2004; 43: 302-327Crossref PubMed Scopus (99) Google Scholar, 8.Kilaru A. Blancaflor E.B. Venables B.J. Tripathy S. Mysore K.S. Chapman K.D. The N-acylethanolamine-mediated regulatory pathway in plants.Chem. Biodivers. 2007; 4: 1933-1955Crossref PubMed Scopus (58) Google Scholar). The first evidence that NAEs might have biological functions in plants came from studies of endogenous NAE levels in desiccated cotton seeds. The endogenous levels of NAEs were shown to be highest in seeds, but they were rapidly depleted during seed imbibition. This observation led to the hypothesis that NAEs might be involved in germination and early seedling establishment possibly as negative growth regulators (6.Kim S.C. Chapman K.D. Blancaflor E.B. Fatty acid amide lipid mediators in plants.Plant Sci. 2010; 178: 411-419Crossref Scopus (38) Google Scholar, 8.Kilaru A. Blancaflor E.B. Venables B.J. Tripathy S. Mysore K.S. Chapman K.D. The N-acylethanolamine-mediated regulatory pathway in plants.Chem. Biodivers. 2007; 4: 1933-1955Crossref PubMed Scopus (58) Google Scholar). In support of this notion was the observation that Arabidopsis seeds germinated on exogenous N-lauroylethanolamine (NAE 12:0) led to stunted seedling development due in part to an altered cytoskeleton and endomembrane system (9.Blancaflor E.B. Hou G. Chapman K.D. Elevated levels of N-lauroylethanolamine, an endogenous constituent of desiccated seeds, disrupt normal root development in Arabidopsis thaliana seedlings.Planta. 2003; 217: 206-217Crossref PubMed Scopus (71) Google Scholar, 10.Motes C.M. Pechter P. Yoo C.M. Wang Y.S. Chapman K.D. Blancaflor E.B. Differential effects of two phospholipase D inhibitors, 1-butanol and N-acylethanolamine, on in vivo cytoskeletal organization and Arabidopsis seedling growth.Protoplasma. 2005; 226: 109-123Crossref PubMed Scopus (88) Google Scholar). Furthermore, young Arabidopsis seedlings exposed to N-linoleoylethanolamide (NAE 18:2) and N-linolenoylethanolamine (NAE 18:3) had disrupted root and chloroplast development, respectively, suggesting that NAEs collectively blocked crucial processes that are part of the transition from germinative to post-germinative seedling growth (11.Keereetaweep J. Blancaflor E.B. Hornung E. Feussner I. Chapman K.D. Ethanolamide oxylipins of linolenic acid negatively regulates Arabidopsis seedling development.Plant Cell. 2013; 25: 3824-3840Crossref PubMed Scopus (25) Google Scholar, 12.Keereetaweep J. Blancaflor E.B. Hornung E. Feussner I. Chapman K.D. Lipoxygenase derived 9-hydro(pero)xides of linoleoylethanolamide interact with ABA signaling to arrest root development during Arabidopsis seedling establishment.Plant J. 2015; 82: 315-327Crossref PubMed Scopus (17) Google Scholar). The potential role of NAEs in seed germination and early seedling development was further reinforced by transcriptomic studies of NAE 12:0-treated seedlings, which showed that genes related to abscisic acid (ABA) signaling were differentially regulated (13.Teaster N.D. Motes C.M. Tang Y. Wiant W.C. Cotter M.Q. Wang Y.S. Kilaru A. Venables B.J. Hasenstein K.H. Gonzalez G. Blancaflor E.B. Chapman K.D. N-Acylethanolamine metabolism interacts with abscisic acid signaling in Arabidopsis thaliana seedlings.Plant Cell. 2007; 19: 2454-2469Crossref PubMed Scopus (57) Google Scholar, 14.Cotter M.Q. Teaster N.D. Blancaflor E.B. Chapman K.D. N-acylethanolamine (NAE) inhibits growth in Arabidopsis thaliana seedlings via ABI3-dependent and independent pathways.Plant Signal. Behav. 2011; 6: 671-679Crossref PubMed Scopus (18) Google Scholar). It was also reported that NAE could act synergistically with ABA in inhibiting seed germination and seedling root development with the latter process regulated by the oxidative metabolism of NAE 18:2 (12.Keereetaweep J. Blancaflor E.B. Hornung E. Feussner I. Chapman K.D. Lipoxygenase derived 9-hydro(pero)xides of linoleoylethanolamide interact with ABA signaling to arrest root development during Arabidopsis seedling establishment.Plant J. 2015; 82: 315-327Crossref PubMed Scopus (17) Google Scholar, 13.Teaster N.D. Motes C.M. Tang Y. Wiant W.C. Cotter M.Q. Wang Y.S. Kilaru A. Venables B.J. Hasenstein K.H. Gonzalez G. Blancaflor E.B. Chapman K.D. N-Acylethanolamine metabolism interacts with abscisic acid signaling in Arabidopsis thaliana seedlings.Plant Cell. 2007; 19: 2454-2469Crossref PubMed Scopus (57) Google Scholar). One mechanism by which the signaling and/or biological activities of NAE are modulated is through the action of the fatty acid amide hydrolase (FAAH) enzyme. FAAH hydrolyzes NAE at its amide linkage to yield an ethanolamine group and its corresponding free fatty acid (15.Cravatt B.F. Giang D.K. Mayfield S.P. Boger D.L. Lerner R.A. Gilula N.B. Molecular characterization of an enzyme that degrades neuromodulatory fatty-acid amides.Nature. 1996; 384: 83-87Crossref PubMed Scopus (1789) Google Scholar, 16.McKinney M.K. Cravatt B.F. Structure and function of fatty acid amide hydrolase.Annu. Rev. Biochem. 2005; 74: 411-432Crossref PubMed Scopus (557) Google Scholar). The critical role of FAAH in regulating NAE signaling in mammals was demonstrated through a series of studies with mouse FAAH knock-out mutants. Mouse mutants with the FAAH gene inactivated resulted in the accumulation of endogenous NAEs in the central nervous system, which subsequently led to a decrease in sensitivity to pain and increased sensitivity to anandamide (17.Cravatt B.F. Demarest K. Patricelli M.P. Bracey M.H. Giang D.K. Martin B.R. Lichtman A.H. Supersensitivity to anandamide and enhanced endogenous cannabinoid signaling in mice lacking fatty acid amide hydrolase.Proc. Natl. Acad. Sci. U.S.A. 2001; 16: 9371-9376Crossref Scopus (1106) Google Scholar, 18.Lichtman A.H. Shelton C.C. Advani T. Cravatt B.F. Mice lacking fatty acid amide hydrolase exhibit a cannabinoid receptor-mediated phenotypic hypoalgesia.Pain. 2004; 109: 319-327Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar). The response of plants to NAE has also been shown to change when the expression level of a plant's FAAH orthologue is modified. For example, knockouts to Arabidopsis FAAH (faah) were more sensitive to the growth-inhibitory effects of NAE 12:0, whereas FAAH overexpressors were less sensitive to NAE 12:0-induced growth arrest (19.Wang Y.S. Shrestha R. Kilaru A. Wiant W. Venables B.J. Chapman K.D. Blancaflor E.B. Manipulation of Arabidopsis fatty acid amide hydrolase expression modifies plant growth and sensitivity to N-acylethanolamines.Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 12197-12202Crossref PubMed Scopus (63) Google Scholar). The observation that FAAH-altered plants had modified endogenous NAEs (i.e. faah had higher NAEs whereas FAAH overexpressors had lower NAEs) is consistent with FAAH's function as an important enzyme involved in plant NAE hydrolysis in vivo (19.Wang Y.S. Shrestha R. Kilaru A. Wiant W. Venables B.J. Chapman K.D. Blancaflor E.B. Manipulation of Arabidopsis fatty acid amide hydrolase expression modifies plant growth and sensitivity to N-acylethanolamines.Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 12197-12202Crossref PubMed Scopus (63) Google Scholar, 20.Teaster N.D. Keereetaweep J. Kilaru A. Wang Y.S. Tang Y. Tran C.N. Ayre B.G. Chapman K.D. Blancaflor E.B. Overexpression of fatty acid amide hydrolase induces early flowering in Arabidopsis thaliana.Front. Plant Sci. 2012; 3: 32Crossref PubMed Scopus (24) Google Scholar). Recently, the formation of bioactive NAE oxylipin metabolites from NAE 18:2 and NAE 18:3 in plants that disrupt root and chloroplast development was shown to be catalyzed by lipoxygenase (LOX) enzymes (11.Keereetaweep J. Blancaflor E.B. Hornung E. Feussner I. Chapman K.D. Ethanolamide oxylipins of linolenic acid negatively regulates Arabidopsis seedling development.Plant Cell. 2013; 25: 3824-3840Crossref PubMed Scopus (25) Google Scholar, 12.Keereetaweep J. Blancaflor E.B. Hornung E. Feussner I. Chapman K.D. Lipoxygenase derived 9-hydro(pero)xides of linoleoylethanolamide interact with ABA signaling to arrest root development during Arabidopsis seedling establishment.Plant J. 2015; 82: 315-327Crossref PubMed Scopus (17) Google Scholar). The LOX-mediated oxidation of polyunsaturated NAEs in plants is reminiscent of the eicosanoids, a class of mammalian hormones that are oxidation products of cyclooxygenase and LOX enzymes (21.Dennis E.A. Norris P.C. Eicosanoid storm in infection and inflammation.Nat. Rev. Immunol. 2015; 15: 511-523Crossref PubMed Scopus (845) Google Scholar). Information on other enzymes that regulate NAE levels in plants has remained elusive. To discover new players involved in regulating the metabolic fate of NAE in plants, we revisited our previous transcriptomic data of seedlings treated with NAE 12:0. In addition to ABA-related transcripts, genes encoding enzymes that modify low molecular mass compounds were up-regulated in seedlings exposed to NAE 12:0 (13.Teaster N.D. Motes C.M. Tang Y. Wiant W.C. Cotter M.Q. Wang Y.S. Kilaru A. Venables B.J. Hasenstein K.H. Gonzalez G. Blancaflor E.B. Chapman K.D. N-Acylethanolamine metabolism interacts with abscisic acid signaling in Arabidopsis thaliana seedlings.Plant Cell. 2007; 19: 2454-2469Crossref PubMed Scopus (57) Google Scholar). Modification of bioactive small molecules is a typical way of removing or inactivating potentially damaging compounds so the plant can protect itself (22.Sandermann Jr., H. Schmitt R. Eckey H. Bauknecht T. Plant biochemistry of xenobiotics: isolation and properties of soybean O- and N-glucosyl and O- and N-malonyltransferases for chlorinated phenols and anilines.Arch. Biochem. Biophys. 1991; 287: 341-350Crossref PubMed Scopus (62) Google Scholar23.Kreuz K. Tommasini R. Martinoia E. Old enzymes for a new job–herbicide detoxification in plants.Plant Physiol. 1996; 111: 349-353Crossref PubMed Scopus (235) Google Scholar, 24.Taguchi G. Shitchi Y. Shirasawa S. Yamamoto H. Hayashida N. Molecular cloning, characterization, and downregulation of an acyltransferase that catalyzes the malonylation of flavonoid and naphthol glucosides in tobacco cells.Plant J. 2005; 42: 481-491Crossref PubMed Scopus (29) Google Scholar25.Taguchi G. Ubukata T. Nozue H. Kobayashi Y. Takahi M. Yamamoto H. Hayashida N. Malonylation is a key reaction in the metabolism of xenobiotic phenolic glucosides in Arabidopsis and tobacco.Plant J. 2010; 63: 1031-1041Crossref PubMed Scopus (73) Google Scholar). Alternatively, small molecule modification could be a mechanism by which its signaling activity is regulated. Evidence for the latter comes from studies of the hormones, auxin and jasmonic acid. Both auxin and jasmonic acid are conjugated into biologically inactive forms by specific enzymes to dampen their activity within the cell (26.Ludwig-Müller J. Auxin conjugates: their role for plant development and in the evolution of land plants.J. Exp. Bot. 2011; 62: 1757-1773Crossref PubMed Scopus (407) Google Scholar, 27.Santino A. Taurino M. De Domenico S. Bonsegna S. Poltronieri P. Pastor V. Flors V. Jasmonates signaling in plant development and defense response to multiple (a)biotic stresses.Plant Cell Rep. 2013; 32: 1085-1098Crossref PubMed Scopus (217) Google Scholar). Conjugation of bioactive small molecules not only leads to their inactivation but also to their preferential transport to cellular compartments or to the extracellular matrix where they are spatially separated from their biological targets (28.Staswick P. Plant hormone conjugation.Plant Signal. Behav. 2009; 4: 757-759Crossref PubMed Scopus (25) Google Scholar). In this regard, it is possible that some of the genes encoding small molecule-modifying enzymes up-regulated by NAE 12:0 could function as NAE-modifying enzymes. Here, we present genetic and biochemical evidence that phenolic glucoside malonyltransferase 1 (PMAT1), which was previously shown to have malonyltransferase activity to a range of phenolic glucosides (25.Taguchi G. Ubukata T. Nozue H. Kobayashi Y. Takahi M. Yamamoto H. Hayashida N. Malonylation is a key reaction in the metabolism of xenobiotic phenolic glucosides in Arabidopsis and tobacco.Plant J. 2010; 63: 1031-1041Crossref PubMed Scopus (73) Google Scholar), has a similar effect on NAE 12:0 glucosides. PMAT1 catalyzes the reaction converting glucosylated NAE 12:0 to NAE 12:0 malonylglucosides. We propose that the formation of an NAE glucoside and its subsequent malonylation by PMAT1 likely represents a pathway for the storage and/or metabolic inactivation of NAE in plants. A previously conducted microarray-based gene expression analysis on wild-type Arabidopsis seedlings treated with NAE 12:0 revealed the up-regulation of a number of genes encoding small molecule-modifying enzymes. Here, we focused on characterizing a gene (At5g39050) encoding PMAT1, which was shown by microarray data to be up-regulated 3-fold by NAE 12:0 treatment (13.Teaster N.D. Motes C.M. Tang Y. Wiant W.C. Cotter M.Q. Wang Y.S. Kilaru A. Venables B.J. Hasenstein K.H. Gonzalez G. Blancaflor E.B. Chapman K.D. N-Acylethanolamine metabolism interacts with abscisic acid signaling in Arabidopsis thaliana seedlings.Plant Cell. 2007; 19: 2454-2469Crossref PubMed Scopus (57) Google Scholar). Quantitative reverse transcriptase-PCR (qRT-PCR) revealed that PMAT1 gene expression was enhanced by about 5-fold in seedlings treated with NAE 12:0 validating the results obtained with microarrays (Fig. 1A). Exogenous NAE 12:0 inhibits the growth of Arabidopsis seedlings (9.Blancaflor E.B. Hou G. Chapman K.D. Elevated levels of N-lauroylethanolamine, an endogenous constituent of desiccated seeds, disrupt normal root development in Arabidopsis thaliana seedlings.Planta. 2003; 217: 206-217Crossref PubMed Scopus (71) Google Scholar, 10.Motes C.M. Pechter P. Yoo C.M. Wang Y.S. Chapman K.D. Blancaflor E.B. Differential effects of two phospholipase D inhibitors, 1-butanol and N-acylethanolamine, on in vivo cytoskeletal organization and Arabidopsis seedling growth.Protoplasma. 2005; 226: 109-123Crossref PubMed Scopus (88) Google Scholar). As such, NAE 12:0-induced seedling growth inhibition has served as a convenient biological readout for evaluating pathways involved in NAE metabolism in plants using gene knockouts and overexpressors (13.Teaster N.D. Motes C.M. Tang Y. Wiant W.C. Cotter M.Q. Wang Y.S. Kilaru A. Venables B.J. Hasenstein K.H. Gonzalez G. Blancaflor E.B. Chapman K.D. N-Acylethanolamine metabolism interacts with abscisic acid signaling in Arabidopsis thaliana seedlings.Plant Cell. 2007; 19: 2454-2469Crossref PubMed Scopus (57) Google Scholar, 19.Wang Y.S. Shrestha R. Kilaru A. Wiant W. Venables B.J. Chapman K.D. Blancaflor E.B. Manipulation of Arabidopsis fatty acid amide hydrolase expression modifies plant growth and sensitivity to N-acylethanolamines.Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 12197-12202Crossref PubMed Scopus (63) Google Scholar). Following similar genetic strategies, we tested whether altered PMAT1 expression had any effect on seedling growth when exposed to exogenous NAE 12:0. We obtained mutants to PMAT1 from the Arabidopsis Biological Research Center. The Arabidopsis PMAT1 gene consists of a single exon, and we identified two mutant alleles (SALK_110268 and SALK_007564) with transfer (T)-DNA insertions within the exon. We refer to these mutants as pmat1-1 and pmat1-2 following the nomenclature of Taguchi et al. (25.Taguchi G. Ubukata T. Nozue H. Kobayashi Y. Takahi M. Yamamoto H. Hayashida N. Malonylation is a key reaction in the metabolism of xenobiotic phenolic glucosides in Arabidopsis and tobacco.Plant J. 2010; 63: 1031-1041Crossref PubMed Scopus (73) Google Scholar) (Fig. 1B; supplemental Fig. S1). Semi-quantitative RT-PCR showed that pmat1-1 and pmat1-2 were null mutants as no transcripts were detected using PMAT1 gene-specific primers (Fig. 1C). Wild-type, pmat1-1, and pmat1-2 seedlings on liquid medium under a 14-h light, 10-h dark cycle exhibited robust growth after 10 days (Fig. 2A). When 40 μm NAE 12:0 was included in the medium, growth of all three genotypes was strongly inhibited. We observed, however, that the extent of NAE 12:0-induced growth inhibition was less in the two pmat1 mutant alleles when compared with wild type. Whereas pmat1 mutants had green cotyledons on 40 μm NAE 12:0, those of wild-type seedlings were smaller and produced no or very little chlorophyll (Fig. 2, B and C). Growth of wild-type and pmat1 seedlings was quantified by measuring primary root length and cotyledon area in semi-solid medium with or without 40 μm NAE 12:0. In solid medium without NAE 12:0, primary root length and cotyledon area of wild-type and pmat1 seedlings were not different from each other. By contrast, values for primary root length and cotyledon area of pmat1 seedlings on 40 μm NAE 12:0 were significantly higher compared with wild-type seedlings (Fig. 2, D and E). The ability of pmat1 to tolerate growth on exogenous NAE 12:0 could not be overcome by simply increasing the concentration of NAE 12:0 in the media. Cotyledons of light-grown pmat1 seedlings exposed to NAE 12:0 concentrations as high as 250 μm remained larger and greener compared with wild type (supplemental Fig. S2). The extent of growth inhibition triggered by NAE 12:0 was more pronounced when seedlings were kept in total darkness. Compared with seedlings subjected to a 14-h light/10-h dark cycle, hypocotyls of seedlings kept in the dark elongate rapidly and produce no chlorophyll in their cotyledons (Fig. 2F). In the presence of exogenous NAE 12:0 and at concentrations much lower than those needed to inhibit seedling growth in the light, hypocotyl elongation of dark-grown wild-type seedlings was severely stunted. By contrast, dark-grown pmat1 seedlings on NAE 12:0 had significantly longer hypocotyls and cotyledons that expanded more compared with wild-type seedlings (Fig. 2, F and E). Taken together, our results indicate that loss of PMAT1 function rendered seedlings less sensitive to the growth-inhibitory effects of NAE 12:0. To confirm that loss of PMAT1 function is the cause for the increased tolerance of pmat1 seedlings to the growth-inhibitory effects of NAE 12:0, we expressed a 35S:PMAT1 construct in the pmat1-1 and pmat1-2 background. Expression of 35S:PMAT1 was verified by qRT-PCR analysis. Consistent with our semi-quantitative RT-PCR results (Fig. 1C), both pmat1 mutant alleles had no detectable PMAT1 transcripts. By contrast, PMAT1 transcripts were detected in two independent lines for each pmat1 mutant allele transformed with the 35S:PMAT1 construct (Fig. 3A). When pmat1-complemented seedlings were grown on exogenous NAE 12:0 in light or in darkness, they exhibited similar growth responses as wild-type seedlings (Fig. 3, B and C) indicating that loss of PMAT1 function is indeed responsible for the reduced growth sensitivity of seedlings to exogenous NAE 12:0. We also transformed wild type with the 35S:PMAT1 construct to determine whether PMAT1 overexpression had any effect on seedling growth with or without NAE 12:0. We selected two independent overexpressing lines that were determined by qRT-PCR to have PMAT1 expression at levels 10–15-fold more than wild type (Fig. 3A). The response of the PMAT1 overexpressors on NAE 12:0-supplemented media and their growth on media without NAE 12:0 were similar to wild type (Fig. 3, B and D). To better understand the biochemical basis for the enhanced tolerance of pmat1 to exogenous NAE 12:0, global metabolite profiles were obtained from NAE 12:0-treated and non-treated (DMSO control) wild-type seedlings using ultra-high performance liquid chromatography (UHPLC) coupled to electrospray ionization (ESI)-MS. We focused our analyses on three major metabolites that were strongly induced in NAE 12:0-treated seedlings but absent in non-treated seedlings (Fig. 4A). The first metabolite (P1) had a retention time of 17.25 min and three major ions, m/z 428.265 [M + Na]+, m/z 406.280 [M + H]+, and m/z 244.225 [M + H − 162]+, corresponding to the loss of a hexose. The second metabolite (P2) had a retention time of 17.87 min and three major ions, m/z 514.259 [M + Na]+, 492.277 [M + H]+, and m/z 244.223 [M + H − 248]+, which corresponds to the loss of a malonyl hexose moiety. The third metabolite (P3) had a retention time of 18.56 min and three major ions, m/z 600.260 [M + Na]+, m/z of 578.279 [M + H]+, and m/z 244.223 [M + H −334]+, which corresponds to a loss of a dimalonyl hexose moiety. UHPLC-PDA-MS analyses indicated that these three unknown metabolites could represent glucose and malonyl conjugation products of NAE 12:0 (Fig. 4B; supplemental Table S1). The three unknown metabolites that accumulated in NAE 12:0-treated samples were purified using a Waters UHPLC coupled to a Bruker QTOF-MS and automated solid phase extraction (SPE). The purified analytes were then used for structural determination by NMR. NMR spectra indicated that the P1 metabolite was glucose-conjugated to NAE 12:0 at the hydroxyl position of the ethanolamine head through a glycosidic linkage. For the P2 metabolite, malonic acid was determined to be conjugated at the carbon 6 (C6) position of the glucose-NAE 12:0 molecule. The P3 metabolite had malonic acid conjugated to C6 and C3 of the glucose-NAE 12:0 molecule (Fig. 4C). Taken together, NMR spectra confirm that the exogenous NAE 12:0 is converted by seedlings into an NAE 12:0 glucoside and then malonylated at two separate positions on the glucose molecule (supplemental Figs. S3–S9). As noted, PMAT1 was previously shown to exhibit malonyltransferase activity to a range of phenolic glucosides (25.Taguchi G. Ubukata T. Nozue H. Kobayashi Y. Takahi M. Yamamoto H. Hayashida N. Malonylation is a key reaction in the metabolism of xenobiotic phenolic glucosides in Arabidopsis and tobacco.Plant J. 2010; 63: 1031-1041Crossref PubMed Scopus (73) Google Scholar). Based on our UHPLC-MS-SPE-NMR analyses of Arabidopsis seedlings treated with NAE 12:0, we hypothesized that PMAT1 might also function as a malonyltransferase for NAE 12:0 glucosides. To test this hypothesis, we compared metabolite profiles of pmat1 and wild-type seedlings treated with NAE 12:0. We found that the NAE 12:0 malonylglucosides corresponding to the P2 and P3 metabolites in NAE 12:0-treated wild-type seedlings were not detected in pmat1. Furthermore, the peak area of P1, NAE 12:0 glucoside, increased in pmat1, whereas the NAE 12:0 malonyl glucoside peaks were restored in pmat1 transformed with 35S:PMAT1 (Fig. 5). The absence of NAE 12:0 malonylglucosides in pmat1 strongly suggested that PMAT1 is responsible for the malonylation of NAE 12:0 glucoside. To obtain direct evidence that PMAT1 has NAE 12:0 glucoside malonyltransferase activity, we expressed PMAT1 in Escherichia coli and purified the recombinant protein for in vitro enzymatic studies (Fig. 6A). NAE 12:0 glucoside P1 was purified from extracts of wild-type seedlings treated with NAE 12:0 by UHPLC-QTOF-MS-SPE and used as a substrate for our enzymatic assays. Reaction with PMAT1 recombinant enzyme was initiated by the addition of the NAE 12:0 glucoside substrate. The reaction was incubated at 30 °C for 60 min and terminated by the addition of HCl. UHPLC-MS analyses of products from the reaction" @default.
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