The pc-1 phenotype of Chlamydomonas reinhardtii results from a deletion mutation in the nuclear gene for NADPH:protochlorophyllide oxidoreductase.
The pc-1 mutant of Chlamydomonas reinhardtii has been shown to be incapable of protochlorophyllide photoconversion in vivo and is thought to be defective in light-dependent NADPH:protochlorophyllide oxidoreductase activity. We have isolated and characterized the nuclear genes encoding this enzyme from wild-type and pc-1 mutant Chlamydomonas cells. The wild-type CRlpcr-1 gene encodes a 397 amino acid polypeptide of which the N-terminal 57 residues comprise the chloroplast transit sequence. The Chlamydomonas protochlorophyllide reductase has 66-70% identity (79-82% similarity) to the higher plant enzymes. Transcripts encoding protochlorophyllide reductase are abundant in dark-grown wild-type cells, but absent or at very low levels in cells grown in the light. Similarly, immunoreactive protochlorophyllide reductase protein is also present to a greater extent in dark- versus light-grown wild-type cells. Both pc-1 and pc-1 y-7 cells lack CRlpcr-1 mRNA and the major (36 kDa) immunodetectable form of protochlorophyllide reductase consistent with their inability to photoreduce protochlorophyllide. DNA sequence analysis revealed that the lpcr gene in pc-1 y-7 cells contains a two-nucleotide deletion within the fourth and fifth codons of the protochlorophyllide reductase precursor that causes a shift in the reading frame and results in premature termination of translation. The absence of protochlorophyllide reductase message in pc-1 and pc-1 y-7 cells is likely the consequence of this frameshift mutation in the lpcr gene. Introduction of the CRlpcr-1 gene into pc-1 y-7 cells by nuclear transformation was sufficient to restore the wild-type phenotype. Transformants contained both protochlorophyllide reductase mRNA and immunodetectable enzyme protein. These studies demonstrate that pc-1 was in fact a defect in protochlorophyllide reductase activity and provide the first in vivo molecular evidence that the lpcr gene product is essential for light-dependent protochlorophyllide reduction.
Li
J
J
Department of Biology, University of Virginia, Charlottesville, 22903, USA.
Timko
M P
MP
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002467:Cell Nucleus / Q000235:genetics*; D016825:Chlamydomonas reinhardtii / Q000201:enzymology / Q000235:genetics* / Q000528:radiation effects; D016368:Frameshift Mutation; D005816:Genetic Complementation Test; D015698:Genomic Library; D015151:Immunoblotting; D008027:Light; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000235:genetics*; D044925:Oxidoreductases Acting on CH-CH Group Donors; D010641:Phenotype; D012333:RNA, Messenger / Q000032:analysis; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D014170:Transformation, Genetic
D012333:RNA, Messenger; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; C023886:protochlorophyllide reductase