Isolation and characterization of high-CO2 requiring mutants from Chlamydomonas reinhardtii by gene tagging

To isolate genes essential to the carbon concentrating mechanism and the CO2-signal transduction mechanisms, seven high-CO2 requiring mutants were isolated from Chlamydomonas reinhardtii by the insertion of nit1 gene encoding nitrate reductase. These mutants were grouped into two classes. Class I contains mutants that show a severe CO2-requiring phenotype (C1 and C3). Class II includes those with a moderate CO2-requiring phenotype (C2, C5, C7, C15, and C16). One of the class II mutants, C5, appeared to be defective in the utilization of intracellularly accumulated inorganic carbon. On the other hand, the mutant C16 appeared to be defective not only in the uptake of carbon but also in the induction of the periplasmic carbonic anhydrase gene, CAH1, as well as in the development of pyrenoid structure under low-CO2 conditions. These mutations in C5 and C16 mutants appeared to be caused by single insertional events of the introduced DNA fragment into the genomic DNA, suggesting that their corresponding genes could be tagged using the nit1 gene.