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

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.

Molecular Genetic Analysis of Plastocyanin Biosynthesis in Chlamydomonas reinhardtii

“…The phenotypes of pcy1-1 and pcy1-2 can be accommodated within this model. A similar phenomenon was noted in strain pc-1 for the Lpcr1 transcript, which has a frameshift at the fourth codon resulting from a 2-nucleotide deletion (54). On the other hand, the dissimilar phenotype of pcy1-3 is surprising.…”

Bilin-Dependent Photoacclimation in Chlamydomonas reinhardtii

“…These include mutants of both the light-and the dark-dependent protochlorophyllide reductases (POR1 and DPOR) of the Mg-PPIX methyltransferase (CHLM) and of the three subunits of Mg chelatase (CHLH1, CHLI1/2, and CHLD). These mutants are photosensitive, severely chlorotic, and/or yellow in the dark (Li and Timko, 1996;Chekounova et al, 2001;Falciatore et al, 2005;von Gromoff et al, 2008;Meinecke et al, 2010;Grovenstein et al, 2013). It was recently demonstrated that the specific loss of chlorophyll b in Chlamydomonas does not disrupt accumulation of LHCI and LHCII polypeptides as long as chlorophyll a biosynthesis remains intact (Bujaldon et al, 2017).…”

Acclimation to Singlet Oxygen Stress in Chlamydomonas reinhardtii

“…In C. reinhardtii, there is both a light-dependent and a light-independent pathway for the conversion of protochlorophyllide to chlorophyllide (28). The pc1 y7 double mutant is blocked in both pathways, leading to the accumulation of protochlorophyllide and inability to grow under even LL conditions (57). In the presence of light, accumulated protochlorophyllide can act as an endogenous photosensitizer and generate 1 O 2 ‫ء‬ within the chloroplast (63).…”