Functional characterization of a higher plant sphingolipid Delta4-desaturase: defining the role of sphingosine and sphingosine-1-phosphate in Arabidopsis.
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.
Michaelson
Louise V
LV
Biological Chemistry Department, Rothamsted Research, Harpenden, Hertshire AL5 2JQ, United Kingdom.
Zäuner
Simone
S
Markham
Jennifer E
JE
Haslam
Richard P
RP
Desikan
Radhika
R
Mugford
Sarah
S
Albrecht
Sandra
S
Warnecke
Dirk
D
Sperling
Petra
P
Heinz
E
E
Napier
Johnathan A
JA
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
D029681:Arabidopsis Proteins; D004269:DNA, Bacterial; D005963:Glucosylceramides; D008246:Lysophospholipids; C036483:T-DNA; C060506:sphingosine 1-phosphate; D010088:Oxidoreductases; C115532:sphingolipid desaturase; D013110:Sphingosine