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

• Published in: Plant molecular biology Vol. 38; no. 5; pp. 839 - 859
• Main Authors: Randolph-Anderson, B.L. (Duke Univ., Durham, NC (USA). Depts. of Botany and Zoology), Sato, R, Johnson, A.M, Harris, E.H, Hauser, C.R, Oeda, K, Ishige, F, Nishio, S, Gillham, N.W, Boynton, J.E
• Format: Journal Article
• Language: English
• Published: Netherlands Plant Mol Biol 01.11.1998; Springer Nature B.V
• Subjects: Amino Acid Sequence; amino acid sequences; Amino acids; analysis; Animals; Arabidopsis; Base Sequence; Blotting, Northern; Blotting, Southern; chemistry; CHLAMYDOMONAS REINHARDTII; Chlamydomonas reinhardtii - drug effects; Chlamydomonas reinhardtii - enzymology; Chlamydomonas reinhardtii - genetics; Clone Cells; Clone Cells - drug effects; Cloning, Molecular; complementary DNA; Cosmids; DNA; DNA - chemistry; DNA - genetics; DNA Mutational Analysis; DNA, Complementary; DNA, Complementary - chemistry; DNA, Complementary - genetics; DNA, Complementary - isolation & purification; drug effects; Drug Resistance; enzymology; Exons; GENE; GENES; Genes - drug effects; Genes - genetics; genetic complementation; genetic transformation; genetics; Genomic Library; HERBICIDAS; HERBICIDE; herbicide resistance; HERBICIDES; Herbicides - pharmacology; isolation & purification; Molecular Sequence Data; mutants; Mutation; Nucleic Acid Hybridization; NUCLEOTIDE SEQUENCE; OXIDOREDUCTASES; Oxidoreductases - genetics; Oxidoreductases Acting on CH-CH Group Donors; OXIDORREDUCTASAS; OXYDOREDUCTASE; pharmacology; Protoporphyrinogen Oxidase; RESISTANCE AUX PRODUITS CHIMIQUES; RESISTANCE TO CHEMICALS; RESISTENCIA A PRODUCTOS QUIMICOS; RNA; RNA - analysis; RNA - genetics; SECUENCIA NUCLEOTIDICA; Sequence Analysis, DNA; Sequence Homology, Amino Acid; SEQUENCE NUCLEOTIDIQUE; structural genes; Transcription, Genetic; Transformation, Genetic
• ISSN: 0167-4412; 1573-5028
• DOI: 10.1023/a:1006085026294

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 A rabidopsis 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.