Received September 6, 2008
Accepted October 17, 2008
Functional characterisation of a higher plant sphingolipid 
4-desaturase: defining the role of sphingosine and sphingosine-1-phosphate in Arabidopsis thaliana
Louise V. Michaelson , Simone Zauner , Jonathan E. Markham , Richard Haslam , Radhika Desikan , Sarah Mugford , Sandra Albrecht , Dirk Warnecke , Petra Sperling , E. Heinz , and Johnathan A. Napier *
Biological Chemistry Department, Rothamsted Research, Harpenden, Herts AL5 2JQ, UK; Biocenter Klein Flottbek, University of Hamburg, Ohnhorststrasse 18, D- 22609 Hamburg, Germany; Donald Danforth Plant Science Center, St. Louis, Missouri 63132, USA; Division of Biology, Imperial College London, London SW7 2AZ, UK
* Corresponding author; email: johnathan.napier{at}bbsrc.ac.uk.
The role of 
4-unsaturated sphingolipid long chain bases such as sphingosine was investigated in Arabidopsis thaliana. Identification and functional characterisation of the sole Arabidopsis ortholog of the sphingolipid 4-desaturase was achieved by heterologous expression in Pichia pastoris. A P. pastoris mutant disrupted in the endogenous sphingolipid 4-desaturase gene was unable to synthesise glucosylceramides. Synthesis of glucosylceramides was restored by the expression of Arabidopsis gene At4g04930 and these sphingolipids were shown to contain 4-unsaturated long chain bases, confirming this open reading frame encodes the sphingolipid 4-desaturase. At4g04930 has a very restricted expression pattern, transcripts only being detected in pollen and floral tissues. Arabidopsis insertion mutants disrupted in the sphingolipid 4-desaturase At4g04930 were isolated and found to be phenotypically normal. Sphingolipidomic profiling of a T-DNA insertion mutant indicated the absence of 4-unsaturated sphingolipids in floral tissue, also resulting in the reduced accumulation of glucosylceramides. No difference in response to drought or water-loss was observed between wild-type plants and insertion mutants disrupted in the sphingolipid 4-desaturase At4g04930, nor was any difference observed in stomatal closure after treatment with ABA. No differences in pollen viability between wild-type and insertion mutants were detected. Based on these observations, it seems unlikely that 4-unsaturated sphingolipids and their metabolites such as sphingosine-1-phosphate play a significant role in Arabidopsis growth and development. However, 4-unsaturated ceramides may play a previously unrecognised role in the channelling of substrates for the synthesis of glucosylceramides.
