Plant Molecular Biology

, Volume 39, Issue 5, pp 891–906 | Cite as

Δ7-Sterol-C5-desaturase: molecular characterization and functional expression of wild-type and mutant alleles

  • Tania Husselstein
  • Hubert Schaller
  • Daniel Gachotte
  • Pierre Benveniste
Article

Abstract

An Arabidopsis thaliana recessive monogenic mutant (ste1-1) presenting a deficiency of the Δ7-sterol-C5(6)-desaturase step in the sterol pathway has been reported previously [12]. To further characterize ste1-1, Arabidopsis, Nicotiana tabacum and Homo sapiens cDNAs encoding Δ7-sterol-C5(6)-desaturases were isolated and identified on the basis of their ability to restore ergosterol synthesis in erg3, a yeast null mutant whose gene encoding the Δ7-sterol-C5(6)-desaturase was disrupted. Overexpression of the Arabidopsis cDNA driven by a 35S promoter in transgenic ste1-1 plants led to full complementation of the mutant. This result demonstrates that STE1 was the impaired component in the desaturation system. Four independent reverse transcriptions of ste1-1 RNA followed by polymerase chain reactions (RT-PCRs), yielded a single product. Alignment of the wild-type ORF with the RT-PCR derived ste1-1 ORF revealed a single amino acid substitution: Thr-114 in the wild-type is changed to Ile in ste1-1. Expression in erg3 resulted in a 6-fold lowered efficiency of the ste1-1 ORF in complementing the yeast biosynthetic pathway when compared to the wild-type ORF. The presence of this mutation in the mutant ste1-1 genomic sequence (and no additional modification between ste1-1 and wild-type genes) demonstrates that the change of the Thr-114 to Ile is necessary and sufficient to create the leaky allele ste1-1. The occurrence of a hydroxylated amino acid (Thr or Ser) at the position corresponding to Thr-114 in the five Δ7-sterol-C5(6)-desaturases identified so far suggests that this amino acid is important for normal enzymatic function.

sterol Δ7-sterol-C5(6)-desaturase deficient mutant complementation Arabidopsis 

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Copyright information

© Kluwer Academic Publishers 1999

Authors and Affiliations

  • Tania Husselstein
    • 1
  • Hubert Schaller
    • 1
  • Daniel Gachotte
    • 2
  • Pierre Benveniste
    • 1
  1. 1.Département d'Enzymologie Cellulaire et Moleculaire, UPR 406 du CNRSInstitut de Biologie Moléculaire des PlantesStrasbourg CédexFrance
  2. 2.Department of BiologyIndiana University-Purdue University at IndianapolisIndianapolisUSA

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