Real-Time Monitoring of Chloroplast Gene Expression by a Luciferase Reporter: Evidence for Nuclear Regulation of Chloroplast Circadian Period

Abstract

Chloroplast-encoded genes, like nucleus-encoded genes, exhibit circadian expression. How the circadian clock exerts its control over chloroplast gene expression, however, is poorly understood. To facilitate the study of chloroplast circadian gene expression, we developed a codon-optimized firefly luciferase gene for the chloroplast of Chlamydomonas reinhardtii as a real-time bioluminescence reporter and introduced it into the chloroplast genome. The bioluminescence of the reporter strain correlated well with the circadian expression pattern of the introduced gene and satisfied all three criteria for circadian rhythms. Moreover, the period of the rhythm was lengthened in per mutants, which are phototactic rhythm mutants carrying a long-period gene in their nuclear genome. These results demonstrate that chloroplast gene expression rhythm is a bona fide circadian rhythm and that the nucleus-encoded circadian oscillator determines the period length of the chloroplast rhythm. Our reporter strains can serve as a powerful tool not only for analysis of the circadian regulation mechanisms of chloroplast gene expression but also for a genetic approach to the molecular oscillator of the algal circadian clock.

We thank Kosuke Shimogawara (Teikyo University) for providing the wild-type 137c strain, Hiroshi Hashimoto and Genzo Ito (Hokkaido System Science Co., Ltd.) for synthesis of the lucCP gene, Shingen Yamamoto (Nagoya University) for assistance in the early phase of this project, Syuzo Ishikawa and Chiyomi Miwa (Nagoya University) for advice on automated continuous culture, and Miriam Bloom (SciWrite Biomedical Writing and Editing Services) for professional editing.

This work was supported by grants to M.I. from the Japanese Ministry of Education, Culture, Sports, Science and Technology (MEXT); the Program for Promotion of Basic Research Activities for Innovative Biosciences (BRAIN); Research for the Future Novel Gene Function Involved in Higher-Order Regulation of Nutrition-Storage in Plants (Japan Society for the Promotion of Science); Ground-Based Research for Space Utilization (Japan Space Forum); and the Promoting Cooperative Research Project (Aichi Science and Technology Foundation) and by grants to T.M. from Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists. The Division of Biological Science, Graduate School of Science, Nagoya University, was supported by a 21st Century COE grant from MEXT.