title: Functional characterization of a higher plant sphingolipid Delta4-desaturase: defining the role of sphingosine and sphingosine-1-phosphate in Arabidopsis.
abstract: The role of Delta4-unsaturated sphingolipid long-chain bases such as sphingosine was investigated in Arabidopsis (Arabidopsis thaliana). Identification and functional characterization of the sole Arabidopsis ortholog of the sphingolipid Delta4-desaturase was achieved by heterologous expression in Pichia pastoris. A P. pastoris mutant disrupted in the endogenous sphingolipid Delta4-desaturase gene was unable to synthesize glucosylceramides. Synthesis of glucosylceramides was restored by the expression of Arabidopsis gene At4g04930, and these sphingolipids were shown to contain Delta4-unsaturated long-chain bases, confirming that this open reading frame encodes the sphingolipid Delta4-desaturase. At4g04930 has a very restricted expression pattern, transcripts only being detected in pollen and floral tissues. Arabidopsis insertion mutants disrupted in the sphingolipid Delta4-desaturase At4g04930 were isolated and found to be phenotypically normal. Sphingolipidomic profiling of a T-DNA insertion mutant indicated the absence of Delta4-unsaturated sphingolipids in floral tissue, also resulting in the reduced accumulation of glucosylceramides. No difference in the response to drought or water loss was observed between wild-type plants and insertion mutants disrupted in the sphingolipid Delta4-desaturase At4g04930, nor was any difference observed in stomatal closure after treatment with abscisic acid. No differences in pollen viability between wild-type plants and insertion mutants were detected. Based on these observations, it seems unlikely that Delta4-unsaturated sphingolipids and their metabolites such as sphingosine-1-phosphate play a significant role in Arabidopsis growth and development. However, Delta4-unsaturated ceramides may play a previously unrecognized role in the channeling of substrates for the synthesis of glucosylceramides.
authors.affiliation: Biological Chemistry Department, Rothamsted Research, Harpenden, Hertshire AL5 2JQ, United Kingdom.
authors.forename: Louise V
authors.initials: LV
authors.lastname: Michaelson
authors.forename: Simone
authors.initials: S
authors.lastname: Zäuner
authors.forename: Jennifer E
authors.initials: JE
authors.lastname: Markham
authors.forename: Richard P
authors.initials: RP
authors.lastname: Haslam
authors.forename: Radhika
authors.initials: R
authors.lastname: Desikan
authors.forename: Sarah
authors.initials: S
authors.lastname: Mugford
authors.forename: Sandra
authors.initials: S
authors.lastname: Albrecht
authors.forename: Dirk
authors.initials: D
authors.lastname: Warnecke
authors.forename: Petra
authors.initials: P
authors.lastname: Sperling
authors.forename: E
authors.initials: E
authors.lastname: Heinz
authors.forename: Johnathan A
authors.initials: JA
authors.lastname: Napier
mesh_terms: D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D018506:Gene Expression Regulation, Plant; D005963:Glucosylceramides / Q000096:biosynthesis; D008246:Lysophospholipids / Q000378:metabolism*; D016254:Mutagenesis, Insertional; D016366:Open Reading Frames; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D010802:Phylogeny; D010843:Pichia / Q000235:genetics; D017384:Sequence Deletion; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000378:metabolism
chemical_list: D029681:Arabidopsis Proteins; D004269:DNA, Bacterial; D005963:Glucosylceramides; D008246:Lysophospholipids; C036483:T-DNA; C060506:sphingosine 1-phosphate; D010088:Oxidoreductases; C115532:sphingolipid desaturase; D013110:Sphingosine