SummaryIn Arabidopsis, the fatty acid moiety of sphingolipids is mainly a-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 a-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 aciddependent defence reactions.

Although the yeast Saccharomyces cerevisiae has only one sphingolipid class with a head group based on phosphoinositol, the yeast Pichia pastoris as well as many other fungi have a second class, glucosylceramide, which has a glucose head group. These two sphingolipid classes are in addition distinguished by a characteristic structure of their ceramide backbones. Here, we investigate the mechanisms controlling substrate entry into the glucosylceramide branch of the pathway. By a combination of enzymatic in vitro studies and lipid analysis of genetically engineered yeast strains, we show that the ceramide synthase Bar1p occupies a key branching point in sphingolipid biosynthesis in P. pastoris. By preferring dihydroxy sphingoid bases and C 16 /C 18 acyl-coenzyme A as substrates, Bar1p produces a structurally well defined group of ceramide species, which is the exclusive precursor for glucosylceramide biosynthesis. Correlating with the absence of glucosylceramide in this yeast, a gene encoding Bar1p is missing in S. cerevisiae. We could not successfully investigate the second ceramide synthase in P. pastoris that is orthologous to S. cerevisiae Lag1p/Lac1p. By analyzing the ceramide and glucosylceramide species in a collection of P. pastoris knock-out strains in which individual genes encoding enzymes involved in glucosylceramide biosynthesis were systematically deleted, we show that the ceramide species produced by Bar1p have to be modified by two additional enzymes, sphingolipid ⌬4-desaturase and fatty acid ␣-hydroxylase, before the final addition of the glucose head group by the glucosylceramide synthase. Together, this set of four enzymes specifically defines the pathway leading to glucosylceramide biosynthesis.