Figure 1.
Analysis of thylakoid carbonic anhydrase (Cah3) activity and expression during the acclimation of high-CO2-grown C. reinhardtii cells to low CO2 conditions.
(A) CA activity (WA units (mg Chl)−1) was measured in thylakoid membranes isolated from high-CO2-grown cells or acclimated to low CO2 for 2, 4 and 8 h. Values are means ± SE (n = 5). (B) Semiquantitative RT-PCR analysis of Cah3 gene expression. Total RNA to be used for RT was isolated by using Trizol™ reagent according to the manufacturers protocol (Life Technologies, US). Aliquots of the reaction mix were loaded and ethidium bromide stained in 1% agarose gels. (C) Immunoblot analysis of total cell extracts from cells of C. reinhardtii with antibodies raised against over-expressed Cah3 polypeptide. The lanes were loaded with 10 µg protein.
Figure 2.
Staurosporine, a protein kinase inhibitor, partially inhibits the activation of Cah3 activity.
High-CO2-grown C. reinhardtii cells were acclimated to low CO2 conditions for 2 h in the absence or in the presence of 0.1 µM Staurosporine. CA activity was measured in thylakoid membranes isolated from control and treated cells. As a control, periplasmic CA activity was measured using intact cells of the same cultures. Values are means ± SE (n = 5).
Figure 3.
Phosphorylation of LHCIIP and PSII polypeptides during acclimation to low CO2 conditions.
(A) Immunoblot analysis of thylakoid membrane proteins isolated from high-CO2-grown cells (H), and cells acclimated to low CO2 for 1 (1 h) and 2 h (2 h) probed with antibodies against phosphothreonine (Thr(P)). (B) Immunoblot analysis of extrinsic thylakoid proteins isolated from thylakoids of high-CO2-grown cells (H), and cells acclimated to low CO2 for 1 (1 h) and 2 h (2 h), probed with affinity-purified antibodies against Cah3. (C) Changes in the immunoresponse of Thr(P) antibody to a 30-kDa phosphoprotein during the acclimation to low CO2. The inset shows immunoblot analysis of extrinsic thylakoid proteins isolated from thylakoids of high-CO2-grown cells (H), and cells acclimated to low CO2 for 1 (1 h) and 2 h (2 h), probed with Thr(P) antibodies. The lanes were loaded with 10 µg protein.
Figure 4.
Immunoprecipitation and dephosphorylation experiments of extrinsic thylakoid polypeptides.
Extraction of extrinsic thylakoid proteins was accomplished by washing the thylakoid membranes with a medium containing low concentrations (0.05%) of Triton X-100. (A) The 30-kDa extrinsic phosphoprotein immunoprecipitates with Cah3. Extrinsic thylakoid proteins released from thylakoid membranes of C. reinhardtii cells acclimated to low CO2 for 2 h (C) were immunoprecipitated with affinity-purified antibodies against Cah3 and protein A-Sepharose CL-4-B beads. The Sepharose beads were washed and the immunoprecipitate (I) and the supernatant (SN) obtained after centrifugation were analysed by SDS-PAGE and immunoblot and probed with antibodies against Cah3 (left) and Thr(P) (right). (B) Effect of Alkaline phosphatase (AP) treatment on extrinsic proteins released from thylakoid membranes isolated from both high-CO2-grown cells (High) or cells acclimated to low CO2 for 2 h (Low). All lanes were loaded with 10 µg protein.
Figure 5.
Immunogold labelling of C. reinhardtii cells grown on high-CO2 or acclimated to low CO2 conditions for 2 and 5 h.
(A) and (B) High-CO2-grown cells probed with affinity-purified antibodies against Cah3. (C) and (D) Cells acclimated to low CO2 conditions for 3 h and (E) pyrenoid of a cell acclimated to low CO2 conditions for 5 h, probed with affinity-purified antibodies against Cah3. Bars indicated 0.5 µm. C, chloroplast; P, pyrenoid; Ssh, starch sheath; and St, stroma chloroplast.
Table 1.
Calculation of Cah3 fraction in both the pyrenoid and the stroma of the chloroplast of both high-CO2 cells and cells acclimating to low CO2 conditions for 3 and 5 h.
Figure 6.
Immunoblot analysis of total cell extracts and isolated pyrenoid fractions from high- (H) and low-CO2-grown (L) Chlamydomonas cells probed with antibodies against D1 protein of PSII, Cah3 and Rubisco large subunit.
All lanes were loaded with 10 µg protein.
Table 2.
Light-saturated O2 evolution rates in both BBY preparations and PSII core complex isolated from high- and low-CO2 C. reinhardtii cells.
Figure 7.
Differential extraction of Cah3 polypeptide from PSII core complexes isolated from C. reinhardtii cells grown under high-CO2 (A) or acclimated to low CO2 conditions for 4 h (B).
PSII hydrophobic and hydrophilic proteins were extracted from PSII core complexes using a chloroform/methanol (2∶1, v/v) mixture (see Materials and Methods). Immunoblot analysis of integral (I) and peripheral (P) protein fractions from PSII core complexes were probed with antibodies against Cah3 (Cah3) and PsbO protein (PsbO). The lanes were loaded with 10 µg protein.