Although oxylipins can be synthesized from free fatty acids, recent evidence suggests that oxylipins are components of plastidlocalized polar complex lipids in Arabidopsis (Arabidopsis thaliana). Using a combination of electrospray ionization (ESI) collisionally induced dissociation time-of-flight mass spectrometry (MS) to identify acyl chains, ESI triple-quadrupole (Q) MS in the precursor mode to identify the nominal masses of complex polar lipids containing each acyl chain, and ESI Q-time-offlight MS to confirm the identifications of the complex polar lipid species, 17 species of oxylipin-containing phosphatidylglycerols, monogalactosyldiacylglycerols (MGDG), and digalactosyldiacylglycerols (DGDG) were identified. The oxylipins of these polar complex lipid species include oxophytodienoic acid (OPDA), dinor-OPDA (dnOPDA), 18-carbon ketol acids, and 16-carbon ketol acids. Using ESI triple-Q MS in the precursor mode, the accumulation of five OPDA-and/or dnOPDAcontaining MGDG and two OPDA-containing DGDG species were monitored as a function of time in mechanically wounded leaves. In unwounded leaves, the levels of these oxylipin-containing complex lipid species were low, between 0.001 and 0.023 nmol/mg dry weight. However, within the first 15 min after wounding, the levels of OPDA-dnOPDA MGDG, OPDA-OPDA MGDG, and OPDA-OPDA DGDG, each containing two oxylipin chains, increased 200-to 1,000-fold. In contrast, levels of OPDA-hexadecatrienoic acid MGDG, linolenic acid (18:3)-dnOPDA MGDG, OPDA-18:3 MGDG, and OPDA-18:3 DGDG, each containing a single oxylipin chain, rose 2-to 9-fold. The rapid accumulation of high levels of galactolipid species containing OPDA-OPDA and OPDA-dnOPDA in wounded leaves is consistent with these lipids being the primary products of plastidic oxylipin biosynthesis.

SummarySystemic acquired resistance (SAR) is an inducible defense mechanism that is activated throughout the plant, subsequent to localized inoculation with a pathogen. The establishment of SAR requires translocation of an unknown signal from the pathogen-inoculated leaf to the distal organs, where salicylic acid-dependent defenses are activated. We demonstrate here that petiole exudates (PeXs) collected from Arabidopsis leaves inoculated with an avirulent (Avr) Pseudomonas syringae strain promote resistance when applied to Arabidopsis, tomato (Lycopersicum esculentum) and wheat (Triticum aestivum). Arabidopsis FATTY ACID DESATURASE7 (FAD7), SUPPRESSOR OF FATTY ACID DESATURASE DEFICIENCY1 (SFD1) and SFD2 genes are required for accumulation of the SAR-inducing activity. In contrast to Avr PeX from wild-type plants, Avr PeXs from fad7, sfd1 and sfd2 mutants were unable to activate SAR when applied to wild-type plants. However, the SAR-inducing activity was reconstituted by mixing Avr PeXs collected from fad7 and sfd1 with Avr PeX from the SAR-deficient dir1 mutant. Since FAD7, SFD1 and SFD2 are involved in plastid glycerolipid biosynthesis and SAR is also compromised in the Arabidopsis monogalactosyldiacylglycerol synthase1 mutant we suggest that a plastid glycerolipid-dependent factor is required in Avr PeX along with the DIR1-encoded lipid transfer protein for long-distance signaling in SAR. FAD7-synthesized lipids provide fatty acids for synthesis of jasmonic acid (JA). However, co-infiltration of JA and methylJA with Avr PeX from fad7 and sfd1 did not reconstitute the SAR-inducing activity. In addition, JA did not co-purify with the SAR-inducing activity confirming that JA is not the mobile signal in SAR.

Summary Fusarium graminearum is the principal causative agent of Fusarium head blight (FHB), a devastating disease of wheat and barley. This fungus can also colonize Arabidopsis thaliana. Disease resistance was enhanced in transgenic wheat and Arabidopsis plants that constitutively over-express the NONEXPRESSOR OF PR GENES 1 (NPR1) gene, which regulates salicylic acid (SA) signaling and modulates the activation of jasmonic acid (JA)-dependent defenses. Here, we provide several lines of evidence that reveal an important role for SA and JA signaling in Arabidopsis defense against F. graminearum. SA level was elevated in fungus-inoculated leaves and SA application and biologically activated systemic acquired resistance enhanced resistance. Furthermore, the disruption of SA accumulation and signaling in the sid2 mutant and NahG transgenic plant, and the npr1 and wrky18 mutants, respectively, resulted in heightened susceptibility to this fungus in leaves and inflorescence. JA signaling was activated in parallel with SA signaling in the fungus-challenged plants. But, the hyper-resistance of the JA pathway mutants, opr3, coi1 and jar1 indicates that this pathway contributes to susceptibility. Genetic and biochemical experiments indicate that the JA pathway promotes disease by attenuating the activation of SA signaling in fungus-inoculated plants. However, the hyper-susceptibility of the jar1 npr1 double mutant compared to the npr1 mutant suggests that JAR1 also contributes to defense, signifying a dichotomous role of JA and JAR1-dependent mechanism in this interaction.

Analysis of the polar lipids of Toxoplasma gondii by electrospray ionization tandem mass spectrometry provides a detailed picture of the lipid molecular species of this parasitic protozoan. Most notably, T. gondii contains a relatively high level, estimated to about 2% of the total polar lipid, of ceramide phosphoethanolamine. The ceramide phosphoethanolamine has a fatty amide profile with only 16-and 18-carbon species. Compared with the host fibroblasts in which it was grown, T. gondii also has higher levels of phosphatidylcholine, but lower levels of sphingomyelin and phosphatidylserine. Analysis at the molecular species level indicated that T. gondii has greater amounts of shorter-chain fatty acid in its polar lipid molecular species than the host fibroblasts. Shorter-chain fatty acids with a combined total of 30 or fewer acyl carbons make up 21% of Toxoplasma's, but only 3% of the host's, diacyl phosphatidylcholine. Furthermore, diacyl phosphatidylcholine with two saturated acyl chains with 12, 14, or 16 carbons make up over 11% of parasite phosphatidylcholine, but less than 3% of the host phosphatidylcholine molecular species. The distinctive T. gondii tachyzoite lipid profile may be particularly suited to the function of parasitic membranes and the interaction of the parasite with the host cell and the host's immune system. Combined with T. gondii genomic data, these lipidomic data will assist in elucidation of metabolic pathways for lipid biosynthesis in this important human pathogen. † This work was supported by NIH NIAID R01s AI27530, AI43228, AI071319, the Research to Prevent Blindness Foundation, and gifts from the Kieweit, Blackmon, Brennan, Koshland, Langel, Morel, Rosenstein, Cussen, Kapnick, and Rooney-Alden families. Work and instrument acquisition at the Kansas Lipidomics Research Center Analytical Laboratory were supported by grants from NSF (MCB 0455318 and DBI 0521587) and NSF's EPSCoR program (EPS-0236913), with matching support from the State of Kansas through Kansas Technology Enterprise Corporation and Kansas State University, as well from NIH grant P20 RR016475 from the INBRE program of the National Center for Research Resources. This is contribution 07-308-J from the Kansas Agricultural Experiment Station.*To whom correspondence should be addressed: Rima McLeod, M.D., The University of Chicago, AMBH S206; 5841 S Maryland Avenue, Chicago, Illinois 60637, Email: rmcleod@midway.uchicago.edu. 1 Abbreviations: ceramide, Cer; ceramide phosphoethanolamine, PE-cer; dihexosylceramide, Dihex-cer; electrospray ionization, ESI; mass spectrometry, MS; phosphatidic acid, PA; phosphatidylcholine, PC; (alkyl or alkenyl)/acyl glycerophosphocholine, ePC; phosphatidylethanolamine, PE; (alkyl or alkenyl)/acyl glycerophosphoethanolamine, ePE; phosphatidylinositol, PI; (alkyl or alkenyl)/ acyl glycerophosphoserine, ePS; Hexosyl ceramide, Hex-cer; phosphatidylserine, PS; sphingomyelin, SM; tandem mass spectrometry, MS/MS. Toxoplasma gondii infects 2-3 billion people throughout the world and is known ...

Profiling of leaf extracts from mutants of Arabidopsis with defects in lipid desaturation demonstrates the utility of collision-induced dissociation time-of-flight mass spectrometry (CID-TOF MS) for screening biological samples for fatty acid compositional alterations. CID-TOF MS uses the collision cell of a quadrupole time-of-flight mass spectrometer to simultaneously fragment all of the ions produced by an ionization source. Electrospray ionization CID-TOF MS in the negative mode can be used to analyze fatty acyl anions derived from complex lipids as well as free fatty acids. Although acyl anion yield is shown to be a function of the lipid class and the position on the glycerol backbone, acyl compositional profiles can be determined, and the TOF detector provides resolution of nominally isobaric acyl species in the profiles. Good precision is obtained when data are acquired for z1 min per sample.-Esch, S. W., P. Tamura Fatty acyl composition is most commonly analyzed by gas chromatography after derivatization to fatty acid methyl esters. When flame ionization or a mass spectrometer is used as the detector, quantitative compositional data are obtained. Liquid chromatography provides an alternative means of separation, in which fatty acids can be detected by ultraviolet light absorption after chemical conversion to an appropriate derivative form (1). Separation and analytical techniques include adsorption chromatography, silver nitrate chromatography, TLC, and HPLC (2). Most gas chromatography and liquid chromatography protocols for fatty acyl species are optimized for normal chain fatty acyl species. Other fatty acids, such as those containing heteroatoms (e.g., oxygen), often require specialized procedures for analysis. Plant oxylipins, for example, have been analyzed by GC-MS or liquid chromatography after various extraction and derivatization steps designed to enable detection and minimize the degradation of these often lowlevel compounds (3-7).Recently, we used a novel negative ion mode mass spectrometric scanning method for rapid profiling of free and ester-linked fatty acyl moieties in crude lipid extracts (8). The collision-induced dissociation time-of-flight (CID-TOF) experiment involved simultaneous collision-induced dissociation of all members of a complex mixture of lipids in the collision cell of a quadrupole time-of-flight (Q-TOF) instrument. The TOF analyzer provided accurate mass characterization of all intact acyl fragments in a single spectrum. Using this method as a "discovery scan," we identified fatty acyl groups, including several oxygenated species that increased in response to wounding of the plant Arabidopsis thaliana (8). Here, we describe this methodology and investigate the accuracy and precision of collision-induced dissociation time-of-flight mass spectrometry (CID-TOF MS).

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