Identification and molecular characterization of the second Chlamydomonas gun4 mutant, gun4-II

Abstract

The green micro-alga Chlamydomonas reinhardtii is an elegant model organism to study oxygenic photosynthesis. Chlorophyll (Chl) and heme are major tetrapyrroles that play an essential role in photosynthesis and respiration. These tetrapyrroles are synthesized via a common branched pathway that involves mainly enzymes, encoded by nuclear genes. One of the enzymes in the pathway is Mg chelatase (MgChel). MgChel catalyzes insertion of Mg 2+ into protoporphyrin IX (PPIX, proto) to form Magnesium-protoporphyrin IX (MgPPIX, Mgproto), the first biosynthetic intermediate in the Chl branch. The GUN4 (genomes uncoupled 4) protein is not essential for the MgChel activity but has been shown to significantly stimulate its activity. We have isolated a light sensitive mutant, 6F14, by random DNA insertional mutagenesis. 6F14 cannot tolerate light intensities higher than 90-100 μmol photons m -2 s -1. It shows a light intensity dependent progressive photo-bleaching. 6F14 is incapable of photo-autotrophic growth under light intensity higher than 100 μmol photons m -2 s -1. PCR based analyses show that in 6F14 the insertion of the plasmid outside the GUN4 locus has resulted in a genetic rearrangement of the GUN4 gene and possible deletions in the genomic region flanking the GUN4 gene. Our gun4 mutant has a Chl content very similar to that in the wild type in the dark and is very sensitive to fluctuations in the light intensity in the environment unlike the earlier identified Chlamydomonas gun4 mutant. Complementation with a functional copy of the GUN4 gene restored light tolerance, Chl biosynthesis and photo-autotrophic growth under high light intensities in 6F14. 6F14 is the second gun4 mutant to be identified in C. reinhardtii. Additionally, we show that our two gun4 complements over-express the GUN4 protein and show a higher Chl content per cell compared to that in the wild type strain.

Porphyrin Binding to Gun4 Protein, Facilitated by a Flexible Loop, Controls Metabolite Flow through the Chlorophyll Biosynthetic Pathway

“…Overexpression of Gun4 in plants causes an aberrant accumulation of Chl, accompanied by elevated steady state levels of P IX and MgP porphyrins (19). In the green alga Chlamydomonas reinhardtii, overexpression of Gun4 also increases Chl content per cell (38), whereas a C. reinhardtii strain possessing a low Gun4 content has reduced both Chl and P IX levels (15,20). Only after the complete elimination of Gun4 do cyanobacteria, algae, and plants accumulate aberrant concentrations of P IX (14,20,39).…”

“…However, the Gun4 mutations are generally more pronounced with increasing light intensity (14,19,38). The sensitivity of Gun4-less mutants to light and changes in ALA synthesizing capacity led to a hypothesis that Gun4 binds excessive porphyrins, which serves as a signal to down-regulate ALA formation (19,20).…”