Plant Molecular Biology

, Volume 38, Issue 5, pp 839–859 | Cite as

Isolation and characterization of a mutant protoporphyrinogen oxidase gene from Chlamydomonas reinhardtii conferring resistance to porphyric herbicides

  • Barbara L. Randolph-Anderson
  • Ryo Sato
  • Anita M. Johnson
  • Elizabeth H. Harris
  • Charles R. Hauser
  • Kenji Oeda
  • Fumiharu Ishige
  • Shoichi Nishio
  • Nicholas W. Gillham
  • John E. Boynton
Article

Abstract

In plant and algal cells, inhibition of the enzyme protoporphyrinogen oxidase (Protox) by the N-phenyl heterocyclic herbicide S-23142 causes massive protoporphyrin IX accumulation, resulting in membrane deterioration and cell lethality in the light. We have identified a 40.4 kb genomic fragment encoding S-23142 resistance by using transformation to screen an indexed cosmid library made from nuclear DNA of the dominant rs-3 mutant of Chlamydomonas reinhardtii. A 10.0 kb HindIII subclone (Hind10) of this insert yields a high frequency of herbicide-resistant transformants, consistent with frequent non-homologous integration of the complete RS-3 gene. A 3.4 kb XhoI subfragment (Xho3.4) yields rare herbicide-resistant transformants, suggestive of homologous integration of a portion of the coding sequence containing the mutation. Molecular and genetic analysis of the transformants localized the rs-3 mutation conferring S-23142 resistance to the Xho3.4 fragment, which was found to contain five putative exons encoding a protein with identity to the C-terminus of the Arabidopsis Protox enzyme. A cDNA clone containing a 1698 bp ORF that encodes a 563 amino acid peptide with 51% and 53% identity to Arabidopsis and tobacco Protox I, respectively, was isolated from a wild-type C. reinhardtii library. Comparison of the wild-type cDNA sequence with the putative exon sequences present in the mutant Xho3.4 fragment revealed a G→A change at 291 in the first putative exon, resulting in a Val→Met substitution at a conserved position equivalent to Val-389 of the wild-type C. reinhardtii cDNA. A sequence comparison of genomic Hind10 fragments from C. reinhardtii rs-3 and its wild-type progenitor CC-407 showed this G→A change at the equivalent position (5751) within exon 10.

Chlamydomonas complementation herbicide resistance indexed cosmid library nuclear transformation protoporphyrinogen oxidase 

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Copyright information

© Kluwer Academic Publishers 1998

Authors and Affiliations

  • Barbara L. Randolph-Anderson
    • 1
  • Ryo Sato
    • 1
  • Anita M. Johnson
    • 1
  • Elizabeth H. Harris
    • 1
  • Charles R. Hauser
    • 1
  • Kenji Oeda
    • 2
  • Fumiharu Ishige
    • 2
  • Shoichi Nishio
    • 2
  • Nicholas W. Gillham
    • 1
  • John E. Boynton
    • 1
  1. 1.Developmental, Cell and Molecular Biology Group, Departments of Botany and ZoologyDuke UniversityDurhamUSA
  2. 2.Sumitomo Chemical Company LimitedTakarazuka-Shi, Hygogo-KenJapan

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