The contribution of polar sphingolipids to total sphingolipid content in food sources determined using a facile method for quantitation of long-chain bases.

Evidence indicates that dietary sphingolipids may influence health and disease, and increasingly are considered a functional food component. A facile method for quantifying total sphingolipid content in a wide variety of food samples would be valuable in nutrition research involving these lipid components. Such a method using basic HPLC instrumentation to quantify fluorescent derivatives of long-chain bases liberated from sphingolipids following direct hydrolysis of food samples is described. The results demonstrate that the sphingolipid content of plant-based foods obtained using direct hydrolysis is greater than that obtained using conventional extraction methods. Direct hydrolysis yields sphingolipid content for animal-based samples similar to more complicated conventional methods. With these advantages, direct hydrolysis is a valuable and broadly applicable method for quantifying the total sphingolipid content of both plant- and animal-based food samples.

Disruption of long-chain base hydroxylation alters growth and impacts sphingolipid synthesis in Physcomitrella patens.

Sphingolipids have roles as membrane structural components and as bioactive molecules in plants. In Physcomitrella patens, 4-hydroxysphinganine (phytosphingosine, t18:0) is the predominant sphingolipid long-chain base (LCB). To assess the functional significance of t18:0, CRISPR-Cas9 mutagenesis was used to generate mutant lines lacking the sole SPHINGOID BASE HYDROXYLASE (SBH) gene encoding the hydroxylase responsible for converting sphinganine (d18:0) to t18:0. Total sphingolipid content in sbh protonemata was 2.4-fold higher than in wild-type. Modest changes in glycosyl inositolphosphorylceramide (GIPC) glycosylation patterns occurred. Sphingolipidomic analyses of mutants lacking t18:0 indicated modest alterations in acyl-chain pairing with d18:0 in GIPCs and ceramides, but dramatic alterations in acyl-chain pairing in glucosylceramides, in which 4,8-sphingadienine (d18:2) was the principal LCB. A striking accumulation of free and phosphorylated LCBs accompanied loss of the hydroxylase. The sbh lines exhibited altered morphology, including smaller chloronemal cell size, irregular cell shape, reduced gametophore size, and increased pigmentation. In the presence of the synthetic trihydroxy LCB t17:0, the endogenous sphingolipid content of sbh lines decreased to wild-type levels, and the mutants exhibited phenotypes more similar to wild-type plants. These results demonstrate the importance of sphingolipid content and composition to Physcomitrella growth. They also illuminate similarities in regulating sphingolipid content but differences in regulating sphingolipid species composition between the bryophyte P. patens and angiosperm A. thaliana.

Group housing and nest building only slightly ameliorate the cold stress of typical housing in female C57BL/6J mice.

Huddling and nest building are two methods of behavioral thermoregulation used by mice under cold stress. In the laboratory, mice are typically housed at an ambient temperature (Ta) of 20°C, well below the lower end of their thermoneutral zone. We tested the hypothesis that the thermoregulatory benefits of huddling and nest building at a Ta of 20°C would ameliorate this cold stress compared with being singly housed at 20°C as assessed by heart rate (HR), blood pressure (BP), triiodothyronine (T3), brown adipose (BAT) expression of Elovl3 mRNA, and BAT lipid content. A series of experiments using C57BL/6J female mice exposed to 20°C in the presence or absence of nesting material and/or cage mates was used to test this hypothesis. Mice showed large differences in HR, BP, shivering, and core body temperature (Tb) when comparing singly housed mice at 20°C and 30°C, but only a modest reduction in HR with the inclusion of cage mates or bedding. However, group housing and/or nesting at 20°C decreased T3 levels compared with singly housed mice at 20°C. Singly housed mice at 20°C had a 22-fold higher level of BAT Elovl3 mRNA expression and a significantly lower triacylglycerol (TAG) content of BAT compared with singly housed mice at 30°C. Group housing at 20°C led to blunted changes in both Elovl3 mRNA and TAG levels. These findings suggest that huddling and nest building have a limited effect to ameliorate the cold stress associated with housing at 20°C.

Plant sphingolipids: function follows form.

Plant sphingolipids are structurally diverse molecules that are important as membrane components and bioactive molecules. An appreciation of the relationship between structural diversity and functional significance of plant sphingolipids is emerging through characterization of Arabidopsis mutants coupled with advanced analytical methods. It is increasingly apparent that modifications such as hydroxylation and desaturation of the sphingolipid nonpolar long-chain bases and fatty acids influence their metabolic routing to particular complex sphingolipid classes and their functions in signaling pathways and other cellular processes, such as membrane protein targeting. Here, we review recent reports investigating some of the more prevalent sphingolipid structural modifications and their functional importance in plants.

Lipid signaling in Arabidopsis: no sphingosine? No problem!

The evidence for sphingosine-1-phosphate (S1P) as a signaling molecule in mammals has been extended to plants, where it is implicated in abscisic acid (ABA)-mediated stomatal closure. However, a recent description of sphingosine- and S1P-deficient mutants of Arabidopsis thaliana provides evidence against a unique role for S1P in ABA signaling in plants because guard cell behavior in response to ABA is unaffected in these mutants. These results and other recent publications indicate that other long-chain base-1-phosphates participate in ABA signaling in plants. As we discuss here, these findings also highlight the need for recognizing the large structural heterogeneity of plant long-chain bases (LCBs) when assigning functions, such as signaling, to sphingolipids and their metabolites in plants.

An inositolphosphorylceramide synthase is involved in regulation of plant programmed cell death associated with defense in Arabidopsis.

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 ( approximately 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.

Arabidopsis mutants lacking long chain base phosphate lyase are fumonisin-sensitive and accumulate trihydroxy-18:1 long chain base phosphate.

The sphingoid long chain bases (LCBs) and their phosphorylated derivatives (LCB-Ps) are important signaling molecules in eukaryotic organisms. The cellular levels of LCB-Ps are tightly controlled by the coordinated action of the LCB kinase activity responsible for their synthesis and the LCB-P phosphatase and lyase activities responsible for their catabolism. Although recent studies have implicated LCB-Ps as regulatory molecules in plants, in comparison with yeast and mammals, much less is known about their metabolism and function in plants. To investigate the functions of LCB-Ps in plants, we have undertaken the identification and characterization of Arabidopsis genes that encode the enzymes of LCB-P metabolism. In this study the Arabidopsis At1g27980 gene was shown to encode the only detectable LCB-P lyase activity in Arabidopsis. The LCB-P lyase activity was characterized, and mutant plant lines lacking the lyase were generated and analyzed. Whereas in other organisms loss of LCB-P lyase activity is associated with accumulation of high levels of LCB/LCB-Ps and developmental abnormalities, the sphingolipid profiles of the mutant plants were remarkably similar to those of wild-type plants, and no developmental abnormalities were observed. Thus, these studies indicate that the lyase plays a minor role in maintenance of sphingolipid metabolism during normal plant development and growth. However, a clear role for the lyase was revealed upon perturbation of sphingolipid synthesis by treatment with the inhibitor of ceramide synthase, fumonisin B(1).

Arabidopsis sphingosine kinase and the effects of phytosphingosine-1-phosphate on stomatal aperture.

Sphingolipids are a major component of membrane lipids and their metabolite sphingosine-1-phosphate (S1P) is a potent lipid mediator in animal cells. Recently, we have shown that the enzyme responsible for S1P production, sphingosine kinase (SphK), is stimulated by the phytohormone abscisic acid in guard cells of Arabidopsis (Arabidopsis thaliana) and that S1P is effective in regulating guard cell turgor. We have now characterized SphK from Arabidopsis leaves. SphK activity was mainly associated with the membrane fraction and phosphorylated predominantly the Delta4-unsaturated long-chain sphingoid bases sphingosine (Sph) and 4,8-sphingadienine, and to a lesser extent, the saturated long-chain sphingoid bases dihydrosphingosine and phytosphingosine (Phyto-Sph). 4-Hydroxy-8-sphingenine, which is a major sphingoid base in complex glycosphingolipids from Arabidopsis leaves, was a relatively poor substrate compared with the corresponding saturated Phyto-Sph. In contrast, mammalian SphK1 efficiently phosphorylated Sph, dihydrosphingosine, and 4,8-sphingadienine, but not the 4-hydroxylated long-chain bases Phyto-Sph and 4-hydroxy-8-sphingenine. Surface dilution kinetic analysis of Arabidopsis SphK with Sph presented in mixed Triton X-100 micelles indicated that SphK associates with the micellar surface and then with the substrate presented on the surface. In addition, measurements of SphK activity under different assay conditions combined with phylogenetic analysis suggest that multiple isoforms of SphK may be expressed in Arabidopsis. Importantly, we found that phytosphingosine-1-phosphate, similar to S1P, regulates stomatal apertures and that its action is impaired in guard cells of Arabidopsis plants harboring T-DNA null mutations in the sole prototypical G-protein alpha-subunit gene, GPA1.

A post-genomic approach to understanding sphingolipid metabolism in Arabidopsis thaliana.

AIMS: To highlight the importance of sphingolipids and their metabolites in plant biology. SCOPE: The completion of the arabidopsis genome provides a platform for the identification and functional characterization of genes involved in sphingolipid biosynthesis. Using the yeast Saccharomyces cerevisiae as an experimental model, this review annotates arabidopsis open reading frames likely to be involved in sphingolipid metabolism. A number of these open reading frames have already been subject to functional characterization, though the majority still awaits investigation. Plant-specific aspects of sphingolipid biology (such as enhanced long chain base heterogeneity) are considered in the context of the emerging roles for these lipids in plant form and function. CONCLUSIONS: Arabidopsis provides an excellent genetic and post-genomic model for the characterization of the roles of sphingolipids in higher plants.

An introduction to plant sphingolipids and a review of recent advances in understanding their metabolism and function.

Sphingolipids are ubiquitous constituents of eukaryotic cells, and have been intensively investigated in mammals and yeast for decades. Aspects of sphingolipid biochemistry in plants have been explored only recently. To date, progress has been made in determining the structure and occurrence of sphingolipids in plant tissues; in characterizing the enzymatic steps involved in production and turnover of sphingolipids (and, in some cases, the genes encoding the relevant enzymes); and in identifying a variety of biological functions for sphingolipids in plants. Given that these efforts are far from complete and much remains to be learned, this review represents a status report on the burgeoning field of plant sphingolipid biochemistry. Contents Summary 677 I. Introduction 678 II. Plant sphingolipid structure 678 III. Sphingolipid metabolism in plants 683 IV. Sphingolipid functions in plants 693 V. Conclusions 696 Acknowledgements 696 References 696.

Complex sphingolipid synthesis in plants: characterization of inositolphosphorylceramide synthase activity in bean microsomes.

Complex glycophosphosphingolipids present in plants are composed of ceramide, inositolphosphate, and diverse polar oligosaccharide substituents. The activity of inositolphosphorylceramide (IPC) synthase (phosphatidylinositol:ceramide inositolphosphate transferase), the enzyme proposed to catalyze the initial committed step in the formation of these complex sphingolipids, was characterized in wax bean hypocotyl microsomes. Enzyme activity was assayed by monitoring the incorporation of fluorescent NBD-C(6) ceramide or [3H]inositolphosphate from radiolabeled phosphatidylinositol (PI) into product identified by TLC. IPC synthase was found to utilize nonhydroxy fatty acid-containing ceramide, hydroxy fatty acid-containing ceramide, and NBD-C(6) ceramide as substrate. Maximum product formation was observed at PI concentrations in excess of 600 microM (with half-maximum activity at approximately 200 microM). Both endogenous PI and ceramide appeared to serve as substrates. Aureobasidin A and rustmicin, two potent inhibitors of fungal IPC synthase, inhibited enzyme activity in bean microsomes with values for IC(50) of 0.4-0.8 and 16-20 nM, respectively. IPC synthase activity appeared most closely associated with the Golgi based on results using selected marker enzymes. Enzyme activity was detected in a variety of plant tissues. This report, the first to characterize IPC synthase in plant tissues, demonstrates the similarities between the plant enzyme and its yeast counterpart, and provides insight into plant glycophosphosphingolipid biology.

Synthesis of 4-hydroxysphinganine and characterization of sphinganine hydroxylase activity in corn.

Sphinganine and 4-hydroxysphinganine (phytosphingosine) are the predominant free long-chain bases in lipid extracts of plant tissues. While the synthesis of sphinganine in plants has been investigated, the metabolic origin of 4-hydroxysphinganine is not known. Three different approaches utilizing fumonisin B(1), an inhibitor of sphinganine acylation, alone or in combination with beta-chloroalanine, an inhibitor of sphinganine synthesis, were used to establish that free 4-hydroxysphinganine is produced in excised corn shoots by the direct hydroxylation of sphinganine and not from the breakdown of complex sphingolipids. Sphinganine hydroxylase activity was characterized in microsomes isolated from corn. The enzyme was found to utilize D-erythro-sphinganine (with half-maximal activity observed at a substrate concentration of approximately 60 microM) and either NADPH (K(m)=33 microM) or NADH (K(m)=58 microM) as substrates. Ceramide hydroxylation was also demonstrated in corn microsomes, and the lack of competition between ceramide and sphinganine suggests the presence of distinct enzymes responsible for hydroxylating these two substrates. Using marker assays, sphinganine hydroxylase activity was localized to the endoplasmic reticulum. Sphinganine hydroxylase activity in microsomes isolated from corn shoots treated with fumonisin B(1) increased more than 3-fold compared to controls. The results of this study shed light on sphingolipid long-chain base synthesis and modification in plant tissues and suggest a possible contribution of sphinganine hydroxylase in manifesting the effects of fumonisin in plants.