Modulation of CHT7 Complexes during Light/Dark- and Nitrogen-Mediated Life Cycle Transitions of Chlamydomonas

Chlamydomonas CHT7 is involved in repressing DNA replication and mitotic genes during synchronous growth

“…Our previous observations showed largely synchronized growth of the cht7 mutant and WT cells in photobioreactors in the presence of N while the cultures were bubbled with CO 2 ( Takeuchi et al 2020a ). To investigate the role of CHT7 in cell cycle regulation, 3 strains, wild type (WT), the cht7 mutant, and the cht7 complemented line CHT7-HA::cht7 , were cultured under 3 sequential growth conditions determined by the availability of N: N-replete (N+), N-deprived (ND), and N resupplied (NR).…”

“…5b . However, it remains unclear whether mitosis of cht7 cells was directly affected given that previous studies did not show obvious abnormal cell division phenotypes in the cht7 mutant under N+ conditions ( Takeuchi et al 2020a,b ). Together, these findings suggest that CHT7 is required for downregulating the transcription of genes involved in S/M phase-related processes during both the regular cell division cycle and quiescence, and that in cht7 DNA replication seems to be activated across all conditions.…”

“…Initial characterization of the 3 mating-type minus, cell-walled C. reinhardtii strains used in this study, 6145c (WT), cht7 , and the cht7 complementation line CHT7-HA::cht7 , was reported previously ( Takeuchi et al 2020b ). Synchronization of Chlamydomonas cells in HS medium with an environmental photobioreactor system (ePBR) using turbidostatic conditions ( Lucker et al 2014 ) has been described in detail by Takeuchi et al (2020a ). Three cell-walled, mating-type minus strains were used in this study: WT (6145c), the cht7 mutant, and the cht7 complemented line CHT7-HA::cht7 .…”

“…The complemented line was included here as an additional control to detect any possible effects caused by the deletion of genes other than CHT7 and other possible background mutations in the cht7 mutant ( Takeuchi et al 2020b ). For synchronization of Chlamydomonas cells in ePBRs ( Lucker et al 2014 ; Takeuchi et al 2020a ), cells were grown autotrophically in 330 ml of nitrogen containing high salt (HS+N) medium ( Sueoka 1960 ) in ePBR under 12 h light:12 h dark cycles with a light intensity of 2,000 µmol m −2 s −1 , and 5% CO 2 was pumped into the cell culture regularly (30 s per 15 min). To maintain cell growth in mid-log phase, OD 940 of the cell culture was monitored and diluted automatically by the ePBR using its turbidostat function.…”

“…One reason is that previous studies were done using asynchronous cell cultures ( Tsai et al 2014 ; Takeuchi et al 2020b ). To address this issue, we recently used cell cultures grown under conditions optimized for synchronization of cell division to investigate the impact of CHT7 on cell cycle progression, and we found that CHT7 proteins are more abundant during the S/M phase and may interact with the RB protein, suggesting that CHT7 might be a part of the cell cycle regulatory network ( Takeuchi et al 2020a ). Here, to better understand the role of CHT7 in cell cycle regulation and to globally characterize the effect of CHT7 on gene expression under various conditions, we performed an RNA-seq of cht7 and 2 control strains grown under light–dark conditions to achieve synchronized division.…”