Isolation and characterization of novel high-CO2-requiring mutants of Chlamydomonas reinhardtii
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Aquatic microalgae induce a carbon-concentrating mechanism (CCM) to maintain photosynthetic activity in low-CO2 (LC) conditions. Although the molecular mechanism of the CCM has been investigated using the single-cell green alga Chlamydomonas reinhardtii, and several CCM-related genes have been identified by analyzing high-CO2 (HC)-requiring mutants, many aspects of the CO2-signal transduction pathways remain to be elucidated. In this study, we report the isolation of novel HC-requiring mutants defective in the induction of CCM by DNA tagging. Growth rates of 20,000 transformants grown under HC and LC conditions were compared, and three HC-requiring mutants (H24, H82, and P103) were isolated. The photosynthetic CO2-exchange activities of these mutants were significantly decreased compared with that of wild-type cells, and accumulation of HLA3 and both LCIA and HLA3 were absent in mutants H24 and H82, respectively. Although the insertion of the marker gene and the HC-requiring phenotype were linked in the tetrad progeny of H82, and a calcium-sensing receptor CAS was disrupted by the insertion, exogenous expression of CAS alone could not complement the HC-requiring phenotype.
KeywordsChlamydomonas reinhardtii Carbon-concentrating mechanism CO2-signal transduction High-CO2-requiring mutants
We thank Dr. Yoshiki Nishimura for technical help and assistance with the tetrad analysis. We also thank Dr. Haru-aki Yanagisawa for providing the expression vectors, pGenD-aphVIII and pGenD-HA. We also thank Dr. Tan Inoue and Dr. Yoshihiko Fujita for letting us use the quantitative real-time PCR system, LightCycler 480. This work was supported by the Promotion of Science (JSPS) KAKENHI (Grant Nos. 23120514, 22380059, and 25120714 to H.F., and 25840109 to T.Y.) and the Japan Science and Technology Agency (JST), Advanced Low Carbon Technology Research and Development Program (ALCA).
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