Abstract
The green micro-alga Chlamydomonas reinhardtii is an elegant model organism to study all aspects of oxygenic photosynthesis. Chlorophyll (Chl) and heme are major tetrapyrroles that play an essential role in energy metabolism in photosynthetic organisms. These tetrapyrroles are synthesized via a common branched pathway that involves mainly nuclear encoded enzymes. One of the enzymes in the pathway is Mg chelatase (MgChel) which inserts 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.
Highlights
Chlamydomonas reinhardtii is a green micro-alga that can grow heterotrophically in the dark by metabolizing exogenous acetate
In Arabidopsis MgPPIX was hypothesized to be a retrograde signal from the chloroplast to the nucleus on the basis of data obtained with mutants that are defective in the norflurazon (NF) induced down-regulation of transcription of light harvesting complex protein B (LHCB)[gun phenotype]40,42
Cyanobacterial and higher plant GUN4 directly interacts with the CHLH subunit of Mg chelatase (MgChel) and binds PPIX and MgPPIX, the substrate and the reaction product of the MgChel[30,46,47,48,49,50]
Summary
Chlamydomonas reinhardtii is a green micro-alga that can grow heterotrophically in the dark by metabolizing exogenous acetate. It possesses a photosynthetic apparatus very similar to higher plants, has a short and simple haplontic life cycle, can synthesize Chl both light dependently and light independently (unlike most angiosperms) and its genome has been sequenced[1]. ALA is subsequently converted in six steps to PPIX, the last common precursor for both Chl and heme biosynthesis[4,5]. Insertion of Mg2+ in PPIX by the heterotrimeric MgChel (comprised of three subunits: CHLD, CHLH and CHLI6) leads to MgPPIX, the first biosynthetic intermediate in the Chl branch[6].
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