Identification of a Novel Gene,CIA6, Required for Normal Pyrenoid Formation inChlamydomonas reinhardtii

Chlamydomonas reinhardtii possesses a CO(2)-concentrating mechanism (CCM) that allows the alga to grow at low CO(2) concentrations. One common feature seen in photosynthetic organisms possessing a CCM is the tight packaging of Rubisco within the cell. In many eukaryotic algae, Rubisco is localized to the pyrenoid, an electron-dense structure within the chloroplast. In order to identify genes required for a functional CCM, insertional Bleomycin resistance (Ble(R)) mutants were generated and screened for growth on minimal medium under high CO(2) conditions (5% CO(2) in air) but only slow or no growth under very low CO(2) conditions (0.01% CO(2) in air). One mutant identified from this screen was named cia6. Physiological studies established that cia6 grows poorly on low levels of CO(2) and has an impaired ability to accumulate inorganic carbon. The inserted Ble(R) disrupted a gene encoding a protein with sequence similarity to proteins containing SET domain methyltransferase, although experiments using overexpressed CIA6 failed to demonstrate the methyltransferase activity. Electron microscopy revealed that the pyrenoid of cia6 mutant cells is highly disorganized. Complementation of the mutant restored the pyrenoid, the ability to grow under low-CO(2) conditions, and the ability to concentrate inorganic carbon. Quantitative reverse transcription-polymerase chain reaction data from a low-CO(2) induction time-course experiment demonstrated that the up-regulation of several CCM components is slower in cia6 compared with the wild type. This slow induction was further confirmed at the protein level using western blots. These results indicated that CIA6 is required for the formation of the pyrenoid and further supported the notion that the pyrenoid is required for a functional CCM in C. reinhardtii.