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 and are synthesized via a common branched tetrapyrrole biosynthetic pathway. 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. MgChel is a multimeric enzyme that consists of three subunits designated CHLD, CHLI and CHLH. Plants have two isozymes of CHLI (CHLI1 and CHLI2) which are 70%-81% identical in protein sequences. Although the functional role of CHLI1 is well characterized, that of CHLI2 is not. We have isolated a non-photosynthetic light sensitive mutant 5A7 by random DNA insertional mutagenesis that is devoid of any detectable Chl. PCR based analyses show that 5A7 is missing the CHLI1 gene and at least eight additional functionally uncharacterized genes. 5A7 has an intact CHLI2 gene. Complementation with a functional copy of the CHLI1 gene restored Chl biosynthesis, photo-autotrophic growth and light tolerance in 5A7. We have identified the first chli1 (chli1-1) mutant of Chlamydomonas reinhardtii and in green algae. Our results show that in the wild type Chlamydomonas CHLI2 protein amount is lower than that of CHLI1 and the chli1-1 mutant has a drastic reduction in CHLI2 protein levels although it possesses the CHLI2 gene. Our chli1-1 mutant opens up new avenues to explore the functional roles of CHLI1 and CHLI2 in Chl biosynthesis in Chlamydomonas, which has never been studied before.
Highlights
The green micro-alga Chlamydomonas reinhardtii possesses a photosynthetic apparatus very similar to that of higher plants, can grow photo-autotrophically and heterotrophically and possesses a completely sequenced genome[1]
Most of the data on CHLI comes from studies on Arabidopsis thaliana chli mutants and the functional significance of CHLI1 and CHLI2 has not been studied in green algae[10,11,12,13,14,15,16]
Generation and identification of the mutant 5A7 Mutant 5A7 was generated by random insertional mutagenesis of the Chlamydomonas reinhardtii wild type strain 4A+ (137c genetic background). 5A7 lacks detectable chlorophyll, appears yellowishbrown in color and grows only under heterotrophic conditions in the dark or in the dim light in the presence of acetate in the growth media (Figure 1)
Summary
The green micro-alga Chlamydomonas reinhardtii possesses a photosynthetic apparatus very similar to that of higher plants, can grow photo-autotrophically and heterotrophically (it can metabolize exogenous acetate as a carbon source) and possesses a completely sequenced genome[1]. These attributes make it an elegant model organism to study oxygenic photosynthesis and chloroplast biogenesis[2,3]. Biosynthesis of Chl and heme occur via a common branched pathway that involves both nuclear- and chloroplast-encoded enzymes in most photosynthetic organisms[4]. CHLI2 can fully rescue an Arabidopsis chli1chli[2] double mutant[12]
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