Acyl lipids in Arabidopsis and all other plants have a myriad of diverse functions. These include providing the core diffusion barrier of the membranes that separates cells and subcellular organelles. This function alone involves more than 10 membrane lipid classes, including the phospholipids, galactolipids, and sphingolipids, and within each class the variations in acyl chain composition expand the number of structures to several hundred possible molecular species. Acyl lipids in the form of triacylglycerol account for 35% of the weight of Arabidopsis seeds and represent their major form of carbon and energy storage. A layer of cutin and cuticular waxes that restricts the loss of water and provides protection from invasions by pathogens and other stresses covers the entire aerial surface of Arabidopsis. Similar functions are provided by suberin and its associated waxes that are localized in roots, seed coats, and abscission zones and are produced in response to wounding. This chapter focuses on the metabolic pathways that are associated with the biosynthesis and degradation of the acyl lipids mentioned above. These pathways, enzymes, and genes are also presented in detail in an associated website (ARALIP: http://aralip.plantbiology.msu.edu/). Protocols and methods used for analysis of Arabidopsis lipids are provided. Finally, a detailed summary of the composition of Arabidopsis lipids is provided in three figures and 15 tables.

Acyl lipids in Arabidopsis and all other plants have a myriad of diverse functions. These include providing the core diffusion barrier of the membranes that separates cells and subcellular organelles. This function alone involves more than 10 membrane lipid classes, including the phospholipids, galactolipids, and sphingolipids, and within each class the variations in acyl chain composition expand the number of structures to several hundred possible molecular species. Acyl lipids in the form of triacylglycerol account for 35% of the weight of Arabidopsis seeds and represent their major form of carbon and energy storage. A layer of cutin and cuticular waxes that restricts the loss of water and provides protection from invasions by pathogens and other stresses covers the entire aerial surface of Arabidopsis. Similar functions are provided by suberin and its associated waxes that are localized in roots, seed coats, and abscission zones and are produced in response to wounding. This chapter focuses on the metabolic pathways that are associated with the biosynthesis and degradation of the acyl lipids mentioned above. These pathways, enzymes, and genes are also presented in detail in an associated website (ARALIP: http://aralip.plantbiology.msu.edu/). Protocols and methods used for analysis of Arabidopsis lipids are provided. Finally, a detailed summary of the composition of Arabidopsis lipids is provided in three figures and 15 tables.

Sphingolipids are major components of the plasma membrane, tonoplast, and other endomembranes of plant cells. Previous compositional analyses have focused only on individual sphingolipid classes because of the widely differing polarities of plant sphingolipids. Consequently, the total content of sphingolipid classes in plants has yet to be quantified. In addition, the major polar sphingolipid class in the model plant Arabidopsis thaliana has not been previously determined. In this report, we describe the separation and quantification of sphingolipid classes from A. thaliana leaves using hydrolysis of sphingolipids and high performance liquid chromatography (HPLC) analysis of o-phthaldialdehyde derivatives of the released long-chain bases to monitor the separation steps. An extraction solvent that contained substantial proportions of water was used to solubilized >95% of the sphingolipids from leaves. Neutral and charged sphingolipids were then partitioned by anion exchange solid phase extraction. HPLC analysis of the charged lipid fraction from A. thaliana revealed only one major anionic sphingolipid class, which was identified by mass spectrometry as hexose-hexuronic-inositolphosphoceramide. The neutral sphingolipids were predominantly composed of monohexosylceramide with lesser amounts of ceramides. Extraction and separation of sphingolipids from soybean and tomato showed that, like A. thaliana, the neutral sphingolipids consisted of ceramide and monohexosylceramides; however, the major polar sphingolipid was found to be N-acetyl-hexosamine-hexuronic-inositolphosphoceramide. In extracts from A. thaliana leaves, hexosehexuronic-inositolphosphoceramides, monohexosylceramides, and ceramides accounted for ϳ64, 34, and 2% of the total sphingolipids, respectively, suggesting an important role for the anionic sphingolipids in plant membranes.Sphingolipids are recognized as universal components of eukaryotic membranes with a diverse array of functions (1-3). Recent interest in sphingolipids from plants has been stimulated by the realization that they may form a significant proportion of the plasma membrane (4), potentially as lipid rafts (5, 6), are involved in signaling a plant's response to drought (7,8), and regulate the ultimate fate of plant cells through programmed cell death (9, 10). In order to understand the role of plant sphingolipids in this diverse array of already discovered roles and to determine what other biological functions sphingolipids may have in plants, it is necessary to be able to measure the sphingolipid content in a qualitative and quantitative way (11). Sphingolipid signaling is thought to be a complex multifactorial signal derived from the interaction of several different sphingolipids (12), making the examination of all sphingolipids a critical factor in the dissection of sphingolipid function. Thus, the emerging field of sphingolipidomics has received much attention in animal biology but remains neglected in plants (13).Previous studies on plant sphingolipids have exclusively concentrated...

Changes in sphingolipids have been associated with profound effects in cell fate and development in both plants and animals. Sphingolipids as a group consist of a large number of different compound classes of which numerous individual species may vary in response to environmental stimuli to affect cellular responses. The ability to measure all sphingolipids simultaneously is, therefore, essential to an understanding of the biochemical regulation of sphingolipid metabolism and signaling molecules derived from it. In the model plant Arabidopsis thaliana, the major sphingolipid classes are glycosylinositolphosphoceramides, glucosylceramides, hydroxyceramides and ceramides. Other minor but potentially important sphingolipids are free long-chain bases and their phosphorylated derivates. By using a single solvent system with reversed-phase high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry detection we have been able to separate and measure 168 sphingolipids from a crude sample. This greatly speeds up and simplifies the analysis of plant sphingolipids and should pave the way for a better understanding of their role in plant performance.

Sphingolipids are a class of structural membrane lipids involved in membrane trafficking and cell polarity. Functional analysis of the ceramide synthase family in Arabidopsis thaliana demonstrates the existence of two activities selective for the length of the acyl chains. Very-long-acyl-chain (C > 18 carbons) but not long-chain sphingolipids are essential for plant development. Reduction of very-long-chain fatty acid sphingolipid levels leads in particular to auxin-dependent inhibition of lateral root emergence that is associated with selective aggregation of the plasma membrane auxin carriers AUX1 and PIN1 in the cytosol. Defective targeting of polar auxin carriers is characterized by specific aggregation of Rab-A2 a -and Rab-A1 elabeled early endosomes along the secretory pathway. These aggregates correlate with the accumulation of membrane structures and vesicle fragmentation in the cytosol. In conclusion, sphingolipids with very long acyl chains define a trafficking pathway with specific endomembrane compartments and polar auxin transport protein cargoes.

The Arabidopsis thaliana resistance gene RPW8 triggers the hypersensitive response (HR) to restrict powdery mildew infection via the salicylic acid-dependent signaling pathway. To further understand how RPW8 signaling is regulated, we have conducted a genetic screen to identify mutations enhancing RPW8-mediated HR-like cell death (designated erh). Here, we report the isolation and characterization of the Arabidopsis erh1 mutant, in which the At2g37940 locus is knocked out by a T-DNA insertion. Loss of function of ERH1 results in salicylic acid accumulation, enhanced transcription of RPW8 and RPW8-dependent spontaneous HR-like cell death in leaf tissues, and reduction in plant stature. Sequence analysis suggests that ERH1 may encode the long-sought Arabidopsis functional homolog of yeast and protozoan inositolphosphorylceramide synthase (IPCS), which converts ceramide to inositolphosphorylceramide. Indeed, ERH1 is able to rescue the yeast aur1 mutant, which lacks the IPCS, and the erh1 mutant plants display reduced (;53% of wild type) levels of leaf IPCS activity, indicating that ERH1 encodes a plant IPCS. Consistent with its biochemical function, the erh1 mutation causes ceramide accumulation in plants expressing RPW8. These data reinforce the concept that sphingolipid metabolism (specifically, ceramide accumulation) plays an important role in modulating plant programmed cell death associated with defense.

Very-long-chain fatty acids (VLCFAs) are synthesized as acyl-CoAs by the endoplasmic reticulum-localized elongase multiprotein complex. Two Arabidopsis genes are putative homologues of the recently identified yeast 3-hydroxy-acyl-CoA dehydratase (PHS1), the third enzyme of the elongase complex. We showed that Arabidopsis PASTICCINO2 (PAS2) was able to restore phs1 cytokinesis defects and sphingolipid long chain base overaccumulation. Conversely, the expression of PHS1 was able to complement the developmental defects and the accumulation of long chain bases of the pas2-1 mutant. The pas2-1 mutant was characterized by a general reduction of VLCFA pools in seed storage triacylglycerols, cuticular waxes, and complex sphingolipids. Most strikingly, the defective elongation cycle resulted in the accumulation of 3-hydroxy-acyl-CoA intermediates, indicating premature termination of fatty acid elongation and confirming the role of PAS2 in this process. We demonstrated by in vivo bimolecular fluorescence complementation that PAS2 was specifically associated in the endoplasmic reticulum with the enoyl-CoA reductase CER10, the fourth enzyme of the elongase complex. Finally, complete loss of PAS2 function is embryo lethal, and the ectopic expression of PHS1 led to enhanced levels of VLCFAs associated with severe developmental defects. Altogether these results demonstrate that the plant 3-hydroxy-acyl-CoA dehydratase PASTICCINO2 is an essential and limiting enzyme in VLCFA synthesis but also that PAS2-derived VLCFA homeostasis is required for specific developmental processes. cuticular wax ͉ elongase ͉ sphingolipid ͉ triacylglycerol ͉ leaf development

Sphingolipids are structural components of endomembranes and function through their metabolites as bioactive regulators of cellular processes such as programmed cell death. A characteristic feature of plant sphingolipids is their high content of trihydroxy long-chain bases (LCBs) that are produced by the LCB C-4 hydroxylase. To determine the functional significance of trihydroxy LCBs in plants, T-DNA double mutants and RNA interference suppression lines were generated for the two Arabidopsis thaliana LCB C-4 hydroxylase genes Sphingoid Base Hydroxylase1 (SBH1) and SBH2. These plants displayed reductions in growth that were dependent on the content of trihydroxy LCBs in sphingolipids. Double sbh1 sbh2 mutants, which completely lacked trihydroxy LCBs, were severely dwarfed, did not progress from vegetative to reproductive growth, and had enhanced expression of programmed cell death associated-genes. Furthermore, the total content of sphingolipids on a dry weight basis increased as the relative amounts of trihydroxy LCBs decreased. In trihydroxy LCB-null mutants, sphingolipid content was ;2.5-fold higher than that in wild-type plants. Increases in sphingolipid content resulted from the accumulation of molecular species with C16 fatty acids rather than with very-long-chain fatty acids, which are more commonly enriched in plant sphingolipids, and were accompanied by decreases in amounts of C16-containing species of chloroplast lipids. Overall, these results indicate that trihydroxy LCB synthesis plays a central role in maintaining growth and mediating the total content and fatty acid composition of sphingolipids in plants.