Growth Phenotype and Biochemical Characterization of a Chlamydomonas reinhardtii (Green Micro-Alga) Pigment Deficient Mutant F3 (14)

Abstract

The green micro alga Chlamydomonas reinhardtii represents an elegant model for studying photosynthesis. Attributes such as a haploid genome, quick replication time of 8 to 10 hours, autotrophic and heterotrophic growth ability, amenability to nuclear and chloroplast transformation, and possession of a completely sequenced genome all add to its versatility for photosynthesis study. Furthermore, unlike angiosperms that possess a single biosynthetic pathway for chlorophyll biosynthesis, C. reinhardtii has two such pathways: a light-dependent pathway that operates strictly under light (common to all photosynthetic organisms) and a light-independent pathway that functions under both dark and light conditions (found in some gymnosperms and cyanobacteria). In an effort to identify genes crucial to the biosynthesis of photosynthetic pigments, the Chlamydomonas strain 4A+ of 137c genetic background was chosen for its consistent growth pattern and chlorophyll biosynthesis under both dark and light conditions for generation of a DNA insertional mutant library. Random insertional mutagenesis was carried out using the vector pBC1 conferring paromomycin resistance to naturally paromomycin sensitive Chlamydomonas. The paromomycin- resistant transformants were then subjected to growth analysis under different light intensities, as well as visual and spectrophotometric based screening, to identify those deficient in chlorophyll pigment synthesis. We isolated a chlorophyll deficient mutant, F3 (14). F3(14) is yellow-green under dim light conditions (25 μmole photons m-2s-1) and turns yellow to white-brown under increasing light conditions (50-100 μmole photons m-2s-1) when taken from dark-adapted cells. Furthermore, it cannot grow heterotrophically above 100 μmole photons m-2s-1 when taken from dark-adapted stock nor autotrophically at any light condition. F3 (14) also has 25% less chlorophyll under dark compared to its parental strain 4A+. Under light conditions, the chlorophyll level decreases tenfold in F3 (14) compared to 4A+. This data indicates that while both the light-independent and light-dependent chlorophyll biosynthetic pathways are affected in F3 (14), the light-dependent pathway is more severely affected than the light- independent pathway. Currently, genetic crossing is being performed to confirm linkage of F3 (14)’s phenotype to paromomycin resistance. High performance liquid chromatography (HPLC) analysis is also being employed in order to perform comparative studies of steady state tetrapyrrole precursor levels in F3(14) and 4A+. HPLC data will allow us to identify which tetrapyrrole precursors are over accumulating due to the mutation. This information will be important in identification of key steps defective in the chlorophyll biosynthetic pathway in the mutant.