ARS | Publication request: ARABIDOPSIS PLANTS WITH AN INDUCED RNAI HAIRPIN OR A CONSTITUTIVELY EXPRESSED DOMINANT-NEGATIVE ALLELE OF THE SERINE PALMITOYLTRANSFERASE LCB2 GENE HAVE ALTERED SPHINGOLIPID CONTENT AND DEVELOPMENT

Submitted to: International Symposium on Plant Lipids
Publication Type: Abstract Only
Publication Acceptance Date: October 26, 2005
Publication Date: October 26, 2005
Citation: Dietrich, C.R., Chen, M., Han, G., Dunn, T.M., Cahoon, E.B. 2005. Arabidopsis plants with an induced rnai hairpin or a constitutively expressed dominant-negative allele of the serine palmitoyltransferase lcb2 gene have altered sphingolipid content and development. International Symposium on Plant Lipids.

Technical Abstract: The first step in sphingolipid biosynthesis is the condensation of palmitoyl-CoA and serine to form 3-ketosphinganine. This reaction is catalyzed by serine palmitoyltransferase (SPT), a pyridoxal 5’ phosphate-dependent enzyme. In yeast, SPT is a heterodimer that consists of the polypeptides LCB1 and LCB2. Arabidopsis contains one LCB1 homolog and two LCB2 homologs (AtLCB2a and AtLCB2b). As a first step towards the characterization of sphingolipid function in plants, Arabidopsis T-DNA lines carrying genetic mutations in the AtLCB2 genes were examined. A single mutation in either AtLCB2 gene had no effect on plant development, but double mutants of the two AtLCB2 genes were not viable. Plants homozygous for one LCB2 T-DNA mutant and heterozygous for the other exhibit both male and female gametoyphtic lethality resulting in reduced transmission of the T-DNA alleles to progeny. As an alternative approach for obtaining suppression lines for both AtLCB2 genes, an RNAi hairpin cassette for the AtLCB2a gene was placed under the control of an inducible promoter in an AtLCB2b homozygous T-DNA mutant. Chemical induction of the hairpin resulted in silencing of the AtLCB2a gene, which in turn blocked all LCB2-mediated sphingolipid synthesis and resulted in gradual lethality. Arabidopsis lines with non-lethal reductions in sphingolipid content were obtained by over-expression of an AtLCB2a gene containing a Lys to Ala mutation within the pyridoxal 5’ phosphate binding domain. The resulting AtLCB2 dominant-negative plants displayed distinct growth phenotypes and altered responses to various stresses. It is anticipated that the detailed characterization of these plants will provide a further understanding of the role of sphingolipids in plant growth and development.