The pc-1 phenotype of Chlamydomonas reinhardtii results from a deletion mutation in the nuclear gene for NADPH:protochlorophyllide oxidoreductase

Plant Mol Biol. 1996 Jan;30(1):15-37. doi: 10.1007/BF00017800.

Abstract

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.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Cell Nucleus / genetics*
  • Chlamydomonas reinhardtii / enzymology
  • Chlamydomonas reinhardtii / genetics*
  • Chlamydomonas reinhardtii / radiation effects
  • Frameshift Mutation*
  • Genetic Complementation Test
  • Genomic Library
  • Immunoblotting
  • Light
  • Molecular Sequence Data
  • Oxidoreductases / genetics*
  • Oxidoreductases Acting on CH-CH Group Donors*
  • Phenotype
  • RNA, Messenger / analysis
  • Sequence Analysis, DNA
  • Sequence Deletion
  • Sequence Homology, Amino Acid
  • Transformation, Genetic

Substances

  • RNA, Messenger
  • Oxidoreductases
  • Oxidoreductases Acting on CH-CH Group Donors
  • protochlorophyllide reductase

Associated data

  • GENBANK/U36752