Plant Molecular Biology 38: 839–859, 1998.
© 1998 Kluwer Academic Publishers. Printed in the Netherlands.
Isolation and characterization of a mutant protoporphyrinogen oxidase
gene from Chlamydomonas reinhardtii conferring resistance to porphyric
Barbara L. Randolph-Anderson
, AnitaM. Johnson
, Elizabeth H. Harris
, Fumiharu Ishige
, Shoichi Nishio
, Nicholas W. Gillham
Developmental, Cell and Molecular Biology Group, Departments of Botany and Zoology, Box 91000, Duke
University, Durham, NC 27708-1000,USA(
Sumitomo Chemical Company Limited,
2-1,4-Chome, Takatsukasa, Takarazuka-Shi, Hygogo-Ken, 665-8555 Japan
Received 21 October 1997; accepted in revised form 22 June 1998
Key words: Chlamydomonas, complementation, herbicide resistance, indexed cosmid library, nuclear transforma-
tion, protoporphyrinogen oxidase
In plant and algal cells, inhibition of the enzyme protoporphyrinogen oxidase (Protox) by the N-phenyl hetero-
cyclic 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-resistanttransformants, consistent with frequentnon-homologousintegration of the complete RS-3 gene.
A3.4kbXhoI 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
aG→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 sequencecomparison of genomicHind10 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.
The enzyme protoporphyrinogen oxidase (Protox) is
the last enzyme in the common pathway of heme
The nucleotide sequence data reported will appear in the
EMBL, GenBank and DDBJ Nucleotide Sequence databases under
the accession numbers AF030179 (rs-3 Hind10 genomic sequence
from strain GB-2674), and AF068635 (wild-type cDNA sequence
from strain CC-621).
and chlorophyll synthesis and oxidizes protopor-
phyrinogen IX (Protogen IX) to protoporphyrin IX
(Proto IX) . Protox is accepted to be the site
of action for three main chemical classes of por-
phyric herbicides: (1) the diaryl ethers including the
diphenyl ethers such as aciﬂuorfen-methyl (AFM),
aciﬂuorfen-ethyl (AFE) and oxyﬂuorfen, (2) the
phenyl heterocycles such as the cyclic imide S-