Specific Adduction of Plant Lipid Transfer Protein by an Allene Oxide Generated by 9-Lipoxygenase and Allene Oxide Synthase

Abstract

Lipid transfer proteins (LTPs) are ubiquitous plant lipid-binding proteins that have been associated with multiple developmental and stress responses. Although LTPs typically bind fatty acids and fatty acid derivatives in a non-covalent way, studies on the LTPs of barley seeds have identified an abundantly occurring covalently modified form, LTP1b, the lipid ligand of which has resisted clarification. In the present study, this adduct was identified as the α-ketol 9-hydroxy-10-oxo-12(Z)-octadecenoic acid. Further studies on the formation of LTP1b demonstrated that the ligand was introduced by nucleophilic attack of the free carboxylate group of the Asp-7 residue of the protein at carbon-9 of the allene oxide fatty acid 9(S),10-epoxy-10,12(Z)-octadecadienoic acid. This reactive oxylipin was produced in barley seeds by oxygenation of linoleic acid by 9-lipoxygenase followed by dehydration of the resulting hydroperoxide by allene oxide synthase. The generation of protein-oxylipin adducts represents a new function for plant allene oxide synthases, enzymes that have earlier been implicated mainly in the biosynthesis of the jasmonate family of plant hormones. Additionally, the LTP-allene oxide synthase interaction opens new perspectives regarding the roles of LTPs in the signaling of plant defense and development. Lipid transfer proteins (LTPs) are ubiquitous plant lipid-binding proteins that have been associated with multiple developmental and stress responses. Although LTPs typically bind fatty acids and fatty acid derivatives in a non-covalent way, studies on the LTPs of barley seeds have identified an abundantly occurring covalently modified form, LTP1b, the lipid ligand of which has resisted clarification. In the present study, this adduct was identified as the α-ketol 9-hydroxy-10-oxo-12(Z)-octadecenoic acid. Further studies on the formation of LTP1b demonstrated that the ligand was introduced by nucleophilic attack of the free carboxylate group of the Asp-7 residue of the protein at carbon-9 of the allene oxide fatty acid 9(S),10-epoxy-10,12(Z)-octadecadienoic acid. This reactive oxylipin was produced in barley seeds by oxygenation of linoleic acid by 9-lipoxygenase followed by dehydration of the resulting hydroperoxide by allene oxide synthase. The generation of protein-oxylipin adducts represents a new function for plant allene oxide synthases, enzymes that have earlier been implicated mainly in the biosynthesis of the jasmonate family of plant hormones. Additionally, the LTP-allene oxide synthase interaction opens new perspectives regarding the roles of LTPs in the signaling of plant defense and development. Lipid transfer proteins (LTPs) 2The abbreviations and trivial names used are: LTP, lipid transfer protein; AOS, allene oxide synthase; LOX, lipoxygenase; NDGA, nordihydroguaiaretic acid; HPOD, hydroperoxy-octadecadienoic acid; ELISA, enzyme-linked immunosorbent assay; ESI, electrospray mass ionization; MS, mass spectrometry; MS/MS, tandem mass spectrometry; LC-MS, liquid chromatography-MS; GC, gas chromatography; HPLC, high pressure liquid chromatography; MALDI-MS, matrix-assisted laser desorption/ionization-MS; HPOD, 9,10-allene oxide, 9(S),10-epoxy-10,12(Z)-octadecadienoic acid; 9,10-ketol, 9-hydroxy-10-oxo-12(Z)-octadecenoic acid. are ubiquitous plant lipid-binding proteins. They form a multigenic family composed by two subfamilies, LTP1 and LTP2, whose members have molecular masses around 9,000 and 7,000, respectively. LTP1 and LTP2 display low or no sequence identities except for a common motif of 8 cysteines forming four disulfide bonds, which is now considered as the LTP signature. This signature is related to an α-helical fold in which a large hydrophobic tunnel or cavity allows the binding of different types of lipids and hydrophobic molecules (1Douliez J.P. Michon T. Marion D. J. Cereal Sci. 2000; 32: 1-20Crossref Scopus (301) Google Scholar, 2Marion D. Douliez J.-P. Gautier M.-F. Elmorjani K. Mills E.N.C. Shewry P.R. Plant Food Allergens. Blackwell Science, Oxford, UK2004: 57-69Google Scholar). LTPs have been associated with a multitude of developmental and stress responses, and their precise function is still unknown. Concerning plant development, it has been suggested that LTPs are involved in the mobilization of seed storage lipids (3Edqvist J. Farbos I. Planta. 2002; 215: 41-50Crossref PubMed Scopus (33) Google Scholar), in the formation of cuticles (4Sterk P. Booij H. Schellekens G.A. Van Kammen A. De Vries S.C. Plant Cell. 1991; 3: 907-921Crossref PubMed Scopus (436) Google Scholar, 5Hollenbach B. Schreiber L. Hartung W. Dietz K.J. Planta. 1997; 203: 9-19Crossref PubMed Scopus (120) Google Scholar), in pollen tube adhesion (6Mollet J.C. Park S.Y. Nothnagel E.A. Lord E.M. Plant Cell. 2000; 12: 1737-1750Crossref PubMed Scopus (150) Google Scholar, 7Park S.Y. Jauh G.Y. Mollet J.C. Eckard K.J. Nothnagel E.A. Walling L.L. Lord E.M. Plant Cell. 2000; 12: 151-164Crossref PubMed Google Scholar), in endosperm programmed cell death (8Eklund D.M. Edqvist J. Plant Physiol. 2003; 132: 1249-1259Crossref PubMed Scopus (45) Google Scholar), and in cell wall loosening (9Nieuwland J. Feron R. Huisman B.A. Fasolino A. Hilbers C.W. Derksen J. Mariani C. Plant Cell. 2005; 17: 2009-2019Crossref PubMed Scopus (176) Google Scholar). Concerning plant defense against biotic and abiotic stress, it has been shown that LTPs are associated with cold and wounding stress (10Yubero-Serrano E.M. Moyano E. Medina-Escobar N. Munoz-Blanco J. Caballero J.L. J. Exp. Bot. 2003; 54: 1865-1877Crossref PubMed Scopus (69) Google Scholar, 11Pearce R.S. Houlston C.E. Atherton K.M. Rixon J.E. Harrison P. Hughes M.A. Alison Dunn M. Plant Physiol. 1998; 117: 787-795Crossref PubMed Scopus (76) Google Scholar), are induced by microbial pathogens (12Molina A. Segura A. Garcia-Olmedo F. FEBS Lett. 1993; 316: 119-122Crossref PubMed Scopus (321) Google Scholar), and can inhibit fungal growth in vitro (13Garcia-Olmedo F. Molina A. Alamillo J.M. Rodriguez-Palenzuela P. Biopolymers. 1998; 47: 479-491Crossref PubMed Scopus (460) Google Scholar, 14Broekaert W.F. Cammue B.P.A. DeBolle M.F.C. Thevissen K. DeSamblanx G.W. Osborn R.W. CRC Crit. Rev. Plant Sci. 1997; 16: 297-323Crossref Scopus (595) Google Scholar). Recently, it has been shown that LTPs and oomycetous elicitins compete for a plasma membrane receptor involved in the hypersensitive response and systemic acquired resistance (15Blein J.P. Coutos-Thevenot P. Marion D. Ponchet M. Trends Plant Sci. 2002; 7: 293-296Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar, 16Buhot N. Douliez J.P. Jacquemard A. Marion D. Tran V. Maume B.F. Milat M.L. Ponchet M. Mikes V. Kader J.C. Blein J.P. FEBS Lett. 2001; 509: 27-30Crossref PubMed Scopus (137) Google Scholar). Besides, the observation that a mutant of Arabidopsis thaliana affected in an LTP2 gene was unable to develop systemic acquired resistance (17Maldonado A.M. Doerner P. Dixon R.A. Lamb C.J. Cameron R.K. Nature. 2002; 419: 399-403Crossref PubMed Scopus (606) Google Scholar) suggests that, in planta, LTPs are probably involved in the signaling of plant defense mechanisms rather than in a direct inhibitory activity on microbial pathogens. Most of the putative functions of LTPs are related to their capabilities of binding lipids and other hydrophobic molecules. For example, it has been recently shown that LTPs can induce plant protection when they are loaded with jasmonic acid (but not with linolenic acid) (18Buhot N. Gomes E. Milat M.L. Ponchet M. Marion D. Lequeu J. Delrot S. Coutos-Thevenot P. Blein J.P. Mol. Biol. Cell. 2004; 15: 5047-5052Crossref PubMed Scopus (101) Google Scholar) and that the cell wall-loosening effect of LTP is modulated by lipid binding (9Nieuwland J. Feron R. Huisman B.A. Fasolino A. Hilbers C.W. Derksen J. Mariani C. Plant Cell. 2005; 17: 2009-2019Crossref PubMed Scopus (176) Google Scholar). Although LTPs typically bind lipids in a non-covalent way, we have previously found that an adduct is covalently bound to wheat LTP1 (19Douliez J.P. Jegou S. Pato C. Larre C. Molle D. Marion D. J. Agric. Food Chem. 2001; 49: 1805-1808Crossref PubMed Scopus (27) Google Scholar). The adduct with a molecular mass of about 294 Da has been also found in barley LTP1, where a structure has been proposed from NMR and mass spectrometry analysis. It was suggested that the adduct corresponds to a new post-translational modification (20Lindorff-Larsen K. Lerche M.H. Poulsen F.M. Roepstorff P. Winther J.R. J. Biol. Chem. 2001; 276: 33547-33553Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). The proposed structure was an oxygenated fatty acid derivative with an odd number of carbon atoms (C17) that does not fit with current fatty acid pathways described in plants. The history of allene oxide synthase (AOS) goes back to 1966, in which year Zimmerman reported the formation of the α-ketol fatty acid 13-hydroxy-12-oxo-9-octadecenoic acid from linoleic acid in a preparation of flaxseed (21Zimmerman D.C. Biochem. Biophys. Res. Commun. 1966; 23: 398-402Crossref PubMed Scopus (93) Google Scholar). Sequential actions of lipoxygenase (LOX) and a new enzyme termed “hydroperoxide isomerase” were implicated in this conversion. The reaction mechanism of hydroperoxide isomerase was studied by Hamberg (22Hamberg M. Biochim. Biophys. Acta. 1987; 920: 76-84Crossref Scopus (104) Google Scholar), who demonstrated that the true products are unstable allene oxides, which undergo spontaneous hydrolysis to provide the α-ketols isolated in the earlier studies. In subsequent studies, Brash et al. (23Brash A.R. Baertschi S.W. Ingram C.D. Harris T.M. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 3382-3386Crossref PubMed Scopus (110) Google Scholar) isolated the methyl esters of two allene oxides and additionally characterized and cloned the hydroperoxide isomerase (24Song W.C. Brash A.R. Science. 1991; 253: 781-784Crossref PubMed Scopus (230) Google Scholar, 25Song W.C. Funk C.D. Brash A.R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8519-8523Crossref PubMed Scopus (270) Google Scholar). This turned out to be a cytochrome P-450 protein related to mammalian thromboxane and prostacyclin synthases and was renamed allene oxide synthase. AOS is widely distributed in higher plants, and a catalytically distinct form related to catalases is present in coral (for reviews, see Refs. 26Tijet N. Brash A.R. Prostaglandins Other Lipid Mediat. 2002; 68–69: 423-431Crossref PubMed Scopus (63) Google Scholar and 27Feussner I. Wasternack C. Annu. Rev. Plant Biol. 2002; 53: 275-297Crossref PubMed Scopus (1156) Google Scholar). The primary function of AOS in higher plants is to provide the linolenic acid-derived allene oxide that, in the presence of allene oxide cyclase (28Hamberg M. Fahlstadius P. Arch Biochem. Biophys. 1990; 276: 518-526Crossref PubMed Scopus (109) Google Scholar), undergoes cyclization into 12-oxo-10,15-phytodienoic acid. This cyclopentenone serves as the precursor of the jasmonate subfamily of oxylipins (29Vick B.A. Zimmerman D.C. Biochem. Biophys. Res. Commun. 1983; 111: 470-477Crossref PubMed Scopus (279) Google Scholar). The present study is concerned with the structure and mode of formation of the covalently modified LTP1. We report that LTP1 traps an allene oxide formed from linoleic acid by sequential action of 9-LOX and AOS. Chemicals—Linoleic acid, [13C]linoleic acid, and nordihydroguaiaretic acid (NDGA) were obtained from Sigma. The oxylipins used including the furane fatty acid 10,13-epoxy-10,12-octadecadienoic acid and the α-ketol 9-hydroxy-10-oxo-12(Z)-octadecenoic acid were purchased from Larodan Fine Chemicals, Malmö, Sweden. Maize AOSs were prepared as described previously (28Hamberg M. Fahlstadius P. Arch Biochem. Biophys. 1990; 276: 518-526Crossref PubMed Scopus (109) Google Scholar). Plant Material—Barley seeds (Hordeum vulgare cv. Scarlett) were provided by Malteurop (Reims, France). Embryos were dissected from dry barley seeds and grounded with liquid nitrogen. For studies on germination, mature barley seeds were sterilized in a solution of sodium hypochloride as described previously (30Dubreil L. Gaborit T. Bouchet B. Gallant D.J. Broekaert W.F. Quillien L. Marion D. Plant Sci. 1998; 138: 121-135Crossref Scopus (140) Google Scholar) and were allowed to germinate in the dark at 25 °C on moist paper, and about 40 seedlings were collected daily, transferred in liquid nitrogen, and freeze-dried before LTP LTP the was in the of LTP1 and and LTP1b were as described previously D. Douliez J.P. Marion D. C. E. J. Biochem. PubMed Scopus Google Scholar). The of LTPs was by and electrospray mass spectrometry as in a K. V. A. H. Marion D. Biochem. Biophys. Res. Commun. 2004; 316: PubMed Scopus Google Scholar). the LTP1, the barley was with and the barley was for before and of the was by of LTP1b, the LTP1 with in W. H. M. H. Biochem. 1991; PubMed Scopus Google Scholar). The methyl was with The was with and with sodium was a The methyl esters were by of and the reaction for at LTP1b was also to as by the or to in on for For and and acid Chemicals, were used as were and identified on an gas with a and to an mass The was programmed at °C and °C to °C °C to °C and to °C to was at mass spectrometry was with an mass to an gas and a was used with as The °C and °C to °C °C to °C and to °C to was at The was at was used as an for mass and NMR were on a at a carbon of The of the carbon were from acid with methyl at The NMR were a of carbon with The carbon was to and a of was also the to by D.M. J. Chem. 90: Scopus Google Scholar). NMR was at with of of LTP1 was with of in a with for at °C and by of at for The protein was by as described and by mass of and LTP1b was to by in a for at The were by on a with a laser at The was with the preparation of acid and acid, at in and acid. The of LTP were by tandem mass spectrometry were with a mass with an The were in and with acid and were at a of of against four of LTP1 with or were produced as described by and C. Nature. PubMed Scopus Google Scholar) and were for the presence of against LTP1 or LTP1 by We an which a for the it not the LTP1. of were and at °C with of LTP1 or LTP1 in were with in were for at bound were by with and as of LTP1 by Sequential was by a of of barley for at with of of was to for the of LTP1. were with LTP1 and as described at was with LTP1 or with barley for at where to the and for at the were as described for of the was and with allows a of LTP1 and and no was with LTP1 as of from was isolated as described previously (19Douliez J.P. Jegou S. Pato C. Larre C. Molle D. Marion D. J. Agric. Food Chem. 2001; 49: 1805-1808Crossref PubMed Scopus (27) Google Scholar). In with the presence of a covalently bound lipid the with not the adduct not and (19Douliez J.P. Jegou S. Pato C. Larre C. Molle D. Marion D. J. Agric. Food Chem. 2001; 49: 1805-1808Crossref PubMed Scopus (27) Google Scholar) of and with or barley about the of and from of barley LTP1 was when the protein was from barley the barley LTP1, as shown by and by mass spectrometry of for the formation of LTP1 was by the LTP1 in protein from and barley seed no LTP1 was by in the of a not be in an In the was to the barley a that allowed the of barley and lipids were for the formation of of modified LTP1 on different barley seed barley in a new a of the of isolated and barley was for with LTP1 in the presence or of [13C]linoleic acid and mass we that when linoleic acid was in the reaction and that was in the LTP1 The an described suggested that the lipid adduct was an oxylipin formed the In with (19Douliez J.P. Jegou S. Pato C. Larre C. Molle D. Marion D. J. Agric. Food Chem. 2001; 49: 1805-1808Crossref PubMed Scopus (27) Google Scholar), the adduct was from LTP1 by and multiple products that in number with of This that the adduct was unstable as previously (20Lindorff-Larsen K. Lerche M.H. Poulsen F.M. Roepstorff P. Winther J.R. J. Biol. Chem. 2001; 276: 33547-33553Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). or in of the adduct to hydrolysis were direct was with on the freeze-dried with the adduct was by and mass The as as the that was to methyl a fatty acid methyl The of is in with of the 294 Da adduct from the and masses of the when free acid was with barley LTP1, no be by analysis. the acid was not present as in LTP1b was formed the of LTP1b with followed by that was by products and with were This was to that for the and of acid. of LTP1b to and to the of the derivative of 9-hydroxy-10-oxo-12(Z)-octadecenoic acid. In of LTP1b with sodium followed by hydrolysis produced acid, which was with at that this was the of the 9-hydroxy-10-oxo-12(Z)-octadecenoic acid with methyl the of the formed on of that the lipid adduct from barley LTP1b is This oxylipin is formed from linoleic acid in the presence of AOS M. Hughes M.A. Res. Google Scholar). of the to LTP1 was produced from barley LTP1 and barley as described [13C]linoleic acid. the presence of a at bound were to the and were also to as the were provided by the The structure was from as as their structure induced by bound and carbon were in the and to the acid and The group be from the by a with at a higher The and and the carbon at The be in the with a for the methyl group at that a by with the The structure from the with the 9-hydroxy-10-oxo-12(Z)-octadecenoic acid The of NMR suggested that the adduct has a high covalently to the the acid residue involved in the LTP1b was and to or of the of and barley LTP1 were of the mass by the modification of in LTP this is with a of not the modification the of the of were by tandem mass spectrometry to the and in the from LTP1 and LTP1b, Asp-7 was identified as involved in the LTP1 the the was with barley LTP1, no as by not are formed by hydrolysis of unstable allene oxides from in the presence of AOS, and it was that the allene oxide precursor of with LTP1 to This allene oxide, 9(S),10-epoxy-10,12(Z)-octadecadienoic acid has been previously characterized and found to have a of at °C M. Hughes M.A. Res. Google Scholar). this 9,10-allene oxide was by of with AOS at of the hydroperoxide into allene oxide is of and the 9,10-allene oxide with a of M. Hughes M.A. Res. Google Scholar). LTP1 was to at different the protein to the of 9,10-allene at of the at at at and were by in LTP1 was by the 9,10-allene oxide from of the of the of 9,10-allene oxide to LTP1 that the was a for the 9,10-allene oxide was by the that LTP1 in a to oxide from was not LTP1 seeds are in lipoxygenase activity and in and the activity of enzymes R.S. and of Cereal Scholar). LTPs were from seeds in the presence of NDGA, a We previously that this of LTP not of LTP1 was by from the LTP1 is in the K. R. F. J. Plant Mol. Biol. PubMed Scopus Google Scholar), lipids and enzymes are in the R.S. and of Cereal Scholar). germination, a cell by programmed cell death A. P. J. R. R. Plant Mol. Biol. 2000; PubMed Scopus Google Scholar), that allows LTP1 of LTP1 with a is a and than from studies (19Douliez J.P. Jegou S. Pato C. Larre C. Molle D. Marion D. J. Agric. Food Chem. 2001; 49: 1805-1808Crossref PubMed Scopus (27) Google Scholar, K. Lerche M.H. Poulsen F.M. Roepstorff P. Winther J.R. J. Biol. Chem. 2001; 276: 33547-33553Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). LTP1, and The is involved in LTP1 This was by of the structure of the an and by by LTP1 to linoleic acid and an of isolated barley or and AOS. 9-LOX and AOS are in the J. J. V. Plant J. 1998; 15: PubMed Scopus Google Scholar, C.W. D.C. Plant Physiol. PubMed Google Scholar), in with the that LTP1 the presence of the It be that 9-LOX is the in the of barley seeds G.A. FEBS Lett. 1991; PubMed Scopus Google Scholar, Van J.R. Plant Sci. 1997; Scopus Google Scholar) and that AOS is the enzyme in of barley C.W. D.C. Plant Physiol. PubMed Google Scholar). The of 9,10-allene oxide by LTP1 in an suggests binding of allene oxide to LTP1. of of and LTP1 that Asp-7 is involved in the an is also involved in the of Vries 2003; PubMed Scopus Google Scholar). In a nucleophilic attack of the carboxylate to the of the and formation of the the and the protein is out by is the of the and of the allene oxide for LTP1. This is by the that is in a of barley where different lipid of LTP1 are This be related to a of the allene oxide in an a The structure of the barley LTP1 with molecules of a cavity with the Asp-7 at the and where the of the is in the hydrophobic of this cavity at the of the group and the to of the allene oxide the in the of this binding with the carboxylate group of the allene oxide to in the This of the allene oxide suggests that a large of the allene oxide is on the protein In with a the for the LTP1 and not for the protein that the α-ketol composed a large of the the α-ketol is it to NMR with This of the is for the oxide that to a of the This the of LTP1 in the presence of the The high of LTP as as the of seed a of the In the of plants, reactive are programmed cell death A. P. V. R. J. Biol. Chem. 2002; 53: Scholar) with of LTP (8Eklund D.M. Edqvist J. Plant Physiol. 2003; 132: 1249-1259Crossref PubMed Scopus (45) Google Scholar). The products of the display cell V. H. J. Physiol. Mol. Plant 2001; Scopus Google Scholar, C. J.L. J.P. C. B. A. P. L. Blein J.P. C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, I. S. J. N. F. S. C. Hamberg M. J. Plant Physiol. 2005; PubMed Scopus Google Scholar), and of molecules by proteins cell In this have for of For example, the cyclopentenone is to and of the cell T. T. T. S. H. H. J. K. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). In as in T. T. T. S. H. H. J. K. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, C.J. T. D. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, V. Chem. 2004; PubMed Scopus Google Scholar), oxylipin to protein also in oxylipin as as LTPs have been associated with cell occurring on or in response to biotic and abiotic stress (1Douliez J.P. Michon T. Marion D. J. Cereal Sci. 2000; 32: 1-20Crossref Scopus (301) Google Scholar, 27Feussner I. Wasternack C. Annu. Rev. Plant Biol. 2002; 53: 275-297Crossref PubMed Scopus (1156) Google Scholar, E. Trends Plant Sci. 2002; 7: Full Text Full Text PDF PubMed Scopus Google Scholar), we have a new to the function of the multigenic and family of plant LTPs as as the function of AOS In this the in of LTPs with of or isolated enzymes and lipid provide a for studies on the of We are to for the in the of plant proteins and to Hamberg for the in the preparation and of We and for on the with