Figure 5.
Overview of the life cycle of Chlamydomonas and differential accumulation of pCRY in vegetative cells (V), prepregametes (pPG), pregametes (PG), G1 gametes (G1), and gametes (G). A, Life cycle of Chlamydomonas cells (modified from Huang and Beck, 2003; Goodenough et al., 2007), including the asexual and the sexual cycle with the different mating types (mt+, mating type plus; mt−, mating type minus). Light-dependent steps are indicated by the sun symbol. B, Treatment and harvesting schemes for the experiments shown in C, D, F, and G. Black bars indicate darkness and white bars indicate light. Application of a proteasome inhibitor is indicated by solid lines (1-h treatment) or dotted lines (2-h treatment), with the vertical arrows depicting the time of harvest and the horizontal tails depicting the length of the inhibitor treatment. Part 1, Scheme representing the harvesting conditions of V grown in nitrogen-containing medium (+N) under different light/dark regimes. Part 2, Scheme representing the harvesting conditions of pPG, PG, G1, and G induced by transfer into nitrogen-deprived medium (−N) under different light/dark regimes. C, Accumulation of pCRY in PG and G. Equal amounts of proteins from crude extracts (70 µg per lane) of CC-124 wild-type cells were resolved by SDS-PAGE on a 7% polyacrylamide gel and used for immunoblots with anti-pCRY antibodies. V were grown under an LD12:12 cycle in TAP medium (+N) and harvested in the night (LD22). For the induction of gametogenesis, cells were transferred to TAP medium without nitrogen (−N) at the end of the light phase (LD12) and kept in darkness for 14 h. By this time, PG were harvested. Afterward, the cells were illuminated with white light for 6 h, and G were harvested. Cells were treated with 10 µm proteasome inhibitor MG-132 (PI; carbobenzoxy-leucyl-leucyl-leucinal) for 1 h before harvesting (+PI) and compared with untreated cells (−PI). As a loading control, the PVDF membrane was stained with Coomassie Brilliant Blue R 250 (Cm) after immunochemical detection. From this stain, selected, unspecified protein bands are shown (middle). The quantified pCRY protein levels of three biological replicates are shown in the diagram at bottom along with the sd. D, Accumulation of pCRY in V during early subjective day. Immunoblotting and quantifications were carried out as described in C along with proteins of V harvested at CT2 (VCT2; see B, part 1), followed in one case by a 1-h light treatment (VCT2+L). Proteasome inhibitor was added as indicated. The black arrowhead indicates pCRY, and the white arrowhead indicates the Coomassie Brilliant Blue R 250-stained loading control. E, pCRY transcript accumulation analyzed by reverse transcription-quantitative PCR (RT-qPCR) for SAG73.72 wild-type cells. Cells were grown under an LD12:12 cycle in TAP medium, then transferred to TAP medium without nitrogen at the end of the light phase and kept in darkness for 14 h. The cells were then illuminated with blue light for 5 h to induce gametogenesis. Light-emitting diode (LEDs) with an energy fluence rate of 2.6 W m−2 were used. Total RNA was isolated, and equal amounts of RNA were used for the RT-qPCR with RACK1 as a reference gene. Changes in transcript levels after blue light illumination of gametes in comparison with dark-adapted pregametes are shown (n = 3 biological replicates; error bars represent sd). The asterisk indicates a significant difference estimated by Student’s t test (*, P < 0.05). F, Accumulation of pCRY in G1. Equal amounts of proteins from crude extracts (70 µg per lane) of CC-124 wild-type cells were resolved by SDS-PAGE on a 7% polyacrylamide gel and used for immunoblots with anti-pCRY antibodies. V were treated as described in C. Moreover, G1 were produced by exposing PG for 1 h to light. In some cases, a proteasome inhibitor was added in the dark (+PI) 2 h before harvesting the cells in the light. Quantification based on three biological replicates was done as in C. The black arrowhead indicates pCRY, and the white arrowhead indicates the Coomassie Brilliant Blue R 250-stained loading control. G, Immunoblots and quantifications were carried out as described in F along with proteins of pPG, PG, and G1 (see B, part 2). Proteasome inhibitor was added as indicated.