Sphingolipids desaturated at the Δ4-position are important signaling molecules in many eukaryotic organisms, including mammals. In a bioinformatics approach, we now identified a new family of protein sequences from animals, plants, and fungi and characterized these sequences biochemically by expression inSaccharomyces cerevisiae. This resulted in the identification of the enzyme sphingolipid Δ4-desaturase (dihydroceramide desaturase) from Homo sapiens, Mus musculus, Drosophila melanogaster, andCandida albicans, in addition to a bifunctional sphingolipid Δ4-desaturase/C-4-hydroxylase from M. musculus. Among the sequences investigated are the Homo sapiens membrane lipid desaturase, the M. musculus degenerative spermatocyte, and the Drosophila melanogaster degenerative spermatocyte proteins. During spermatogenesis, but not oogenesis of des mutant flies, both cell cycle and spermatid differentiation are specifically blocked at the entry into the first meiotic division, leading to male sterility. This mutant phenotype can be restored to wild-type by complementation with a functional copy of the des gene (Endo, K., Akiyama, T., Kobayashi S., and Okada, M. (1996) Mol. Gen. Genet. 253, 157–165). These results suggest that Δ4-desaturated sphingolipids provide an early signal necessary to trigger the entry into both meiotic and spermatid differentiation pathways during Drosophila spermatogenesis.

Ceramides are central intermediates of sphingolipid metabolism with critical functions in cell organization and survival. They are synthesized on the cytosolic surface of the endoplasmic reticulum (ER) and transported by ceramide transfer protein to the Golgi for conversion to sphingomyelin (SM) by SM synthase SMS1. In this study, we report the identification of an SMS1-related (SMSr) enzyme, which catalyses the synthesis of the SM analogue ceramide phosphoethanolamine (CPE) in the ER lumen. Strikingly, SMSr produces only trace amounts of CPE, i.e., 300-fold less than SMS1-derived SM. Nevertheless, blocking its catalytic activity causes a substantial rise in ER ceramide levels and a structural collapse of the early secretory pathway. We find that the latter phenotype is not caused by depletion of CPE but rather a consequence of ceramide accumulation in the ER. Our results establish SMSr as a key regulator of ceramide homeostasis that seems to operate as a sensor rather than a converter of ceramides in the ER.

• The bioactive lipid ceramide is produced by the enzyme ceramide synthase, which exists in several isoforms in most eukaryotic organisms. Here, we investigated functional differences between the three ceramide synthase isoforms in Arabidopsis thaliana. • The biochemical properties of the three ceramide synthases were investigated by comparing lipid profiles of yeast strains expressing LOH1, LOH2 or LOH3 with those of wild‐type and loh1, loh2 and loh3 knockout plants. Expression profiles of the ceramide synthases and of the pathogenesis‐related gene PR‐1 were investigated by real‐time PCR. • Each ceramide synthase isoform showed a characteristic preference regarding acyl‐CoA chain length as well as sphingoid base hydroxylation, which matches the pattern of ceramide and glucosylceramide species found in leaves. After extended culture under short‐day conditions, loh1 plants showed spontaneous cell death accompanied by enhanced expression of PR‐1. The levels of free trihydroxy sphingoid bases as well as ceramide and glucosylceramide species with C₁₆ fatty acid were significantly elevated while species with C₂₀–C₂₈ fatty acids were reduced. • These data suggest that spontaneous cell death in the loh1 line is triggered either by the accumulation of free trihydroxy sphingoid bases or ceramide species with C₁₆ fatty acid.

Summary: In Arabidopsis, the fatty acid moiety of sphingolipids is mainly α‐hydroxylated. The consequences of a reduction in this modification were analysed. Mutants of both Fatty Acid Hydroxylase genes (AtFAH1 and AtFAH2) were analysed for sphingolipid profiles. To elucidate further consequences of the mutations, metabolic analyses were performed and the influence on pathogen defence was determined. Ceramide and glucosylceramide profiles of double‐mutant plants showed a reduction in sphingolipids with α‐hydroxylated fatty acid moieties, and an accumulation of sphingolipids without these moieties. In addition, the free trihydroxylated long‐chain bases and ceramides were increased by five‐ and ten‐fold, respectively, whereas the amount of glucosylceramides was decreased by 25%. Metabolite analysis of the double mutant revealed salicylates as enriched metabolites. Infection experiments supported the metabolic changes, as the double mutant showed an enhanced disease‐resistant phenotype for infection with the obligate biotrophic pathogen Golovinomyces cichoracearum. In summary, these results suggest that fatty acid hydroxylation of ceramides is important for the biosynthesis of complex sphingolipids. Its absence leads to the accumulation of long‐chain bases and ceramides as their precursors. This increases salicylate levels and resistance towards obligate biotrophic fungal pathogens, confirming a role of sphingolipids in salicylic acid‐dependent defence reactions.