My postdoctoral research was focused on identification of novel genes and proteins that are involved in the signal transduction pathway involved in these acclimation mechanisms to fluctuations in irradiance in the unicellular green alga Chlamydomonas reinhardtii. My postdoctoral research project specifically involved identifying and characterizing novel genes that define the chlorophyll antenna size in the model green alga C. reinhardtii employing biochemical and molecular approaches. Up to 600 chlorophyll (Chl) molecules associated with the two photosystems in the chloroplast of C. reinhardtii, are needed to increase the photon absorption cross-section of the photosynthetic apparatus. This large Chl antenna size affords the cells a competitive advantage in the wild, where sunlight is often limiting. However, only about 130 Chl molecules are absolutely needed for the proper assembly and function of the photosystems. The Chl antenna size of the photosynthetic apparatus in chloroplasts is defined genetically by the nucleus via an unknown control mechanism. Specifically, there are unknown nuclear genes that regulate the development and define the size of the Chl antenna in all photosynthetic organisms.
tla1, (truncated light harvesting chlorophyll antenna size) a chlorophyll deficient insertional mutant with a smaller Chl antenna size compared to that of the wild type was identified upon screening of an insertional mutant library. TAIL/Inverse PCR analysis revealed that in the mutant the 5’ UTR and the promoter of a novel TLA1 gene was misplaced from its original location to a location upstream of the inserted vector that was used for insertional mutagenesis. RT-PCR and 5’RACE analysis using the cDNA of the mutant and the wild type revealed that TLA1 transcript is expressed in the mutant and that the 3’end of the inserted vector was acting as the new 5’UTR and promoter in the mutant. Moreover, I identified a novel gene RDP1 that overlaps at its 3’end with the 5’end of the TLA1 gene. The TLA1 gene encodes a novel protein of 213 amino acids and shows substantial homology to proteins of unknown function in diverse eukaryotic organisms, ranging from higher plants to invertebrates and mammals. RDP1 codes for a protein with zinc RING finger domain that are common in protein involved in protein turnover and regulators of transcription and translation.
TLA1 protein was overexpressed as a His-tagged recombinant fusion protein and purified it to generate an antibody for immunolocalization studies. Western blotting experiment indicated that the TLA1 protein is drastically reduced in the mutant. Complementation of the tla1 mutant strain with the full length TLA1 gene restored the wild type phenotype. Immunolocalization studies using a TLA1 specific antibody showed that TLA1 protein is localized in the chloroplast. Since the TLA1 protein belongs to a novel yet uncharacterized protein family, predicting a broad functional role of TLA1 is difficult. Through application of computational-based bioinformatics tools I have shown that the conserved domains of TLA1 like proteins have a remote homology with the plain MPN/MOV34 domains, which are domains present in proteins involved in protein degradation, translation factors and transcription regulators like subunits of COP9 signalosome (CSN6), eukaryotic translation initiation factor (eIF3f and eIF3h) etc. I performed over-expression and down-regulation of the TLA1 gene (using RNA interference) to alter the optical properties of green algae by conferring a larger or truncated, Chl antenna size. The over-expression and down regulation of the TLA1 gene affected the concentration of some of the major photosynthetic proteins and altered the organization of the thylakoid membrane structure in the chloroplast. This research has important application in algal biotechnology for improving photosynthetic productivity and solar conversion efficiency of commercially important photosynthetic microalgae, biomass accumulation and carbon sequestration (see CV for research patent).