Fig 1.
Organization of the ATP synthase operon of cyanobacteria and co-expression of genes of the ATP synthase operon with CGL160 in Synechocystis sp PCC 6803.
A. The genes encoding the ATP synthase are organized in a single operon together with atp1 that is related to CGL160 (previously CGLD22 in Chlamydomonas). The genes are depicted as arrows, with the orientation indicated by the direction of the arrow. This information was obtained from The SEED viewer (http://pubseed.theseed.org) B. The genes of the ATP synthase are co-expressed with atp1 as described in [41]. The atp1 gene encodes the putative ortholog of CGL160; the curve showing the expression profile of atp1 is in red. The microarray data used to generate the expression curves were obtained from the Gene Expression Omnibus. Microarray values normalized against the median of the ratio of each sample against the reference, and log-transformed. The plotted data include 24 experiments. C-D. Affymetrix (ATH1 Gene Chip) Gene expression data in different Arabidopsis organs for CGL160 and for select nucleus-encoded genes. Genes for chloroplast localized ATP synthase subunits C, or D, mitochondrial ATP synthase. Data were retrieved from the Arabidopsis Electronic Fluorescent Pictograph (eFP) Browser. E. Expression profile of CGL160 and mitochondrial ATP synthase genes in maize (Zea mays) (ATP3, RMZM2G321725; ATP5, GRMZM2G156068) and chloroplast ATP synthase genes (ATPC, GRMZM2G048907; ATPD: GRMZM2G025171) during maize leaf development. Representative images for CGL160 and ATP synthase encoding genes are taken from http://bar.utoronto.ca/efp_maize/cgi-bin/efpWeb.cgi. Proteins that are involved in photosynthesis typically exhibit peak expression in mature chloroplasts corresponding to leaf sections 9 and 14.
Fig 2.
Isolation and characterization of Arabidopsis cgl160 mutants.
A. DNA insertion sites in CGL160 gene of Arabidopsis. DNA insertion sites (black triangles) are shown in relation to the CGL160 gene structure. The two cgl160 alleles analyzed in this study are denoted as cgl160-1 and cgl160-2. The CGL160 coding region is indicated by the translational start (ATG). The CGL160 genomic locus contains nine exons but only the first four are shown in the fig (grey boxes), shown are also the first four introns (black thin connecting lines). Before ATG is the promoter region in light gray. The region used for CGL160 specific antibody is shown as antigen. B. Characterization of CGL160 amount in Arabidopsis cgl160-1 mutant from isolated chloroplasts. The CGL160 antibody was used for immunoblotting and 10 μg protein was loaded in each lane. The LHCB1 antibody was used as a loading control. C. Characterization of CGL160 amount in Arabidopsis cgl160-2 mutant from isolated chloroplasts. The CGL160 antibody was used for immunoblotting and 10 μg protein was loaded in each lane. The LHCB1 antibody was used as a loading control.
Fig 3.
CGL160 is a membrane-integral chloroplast protein located in non-appressed thylakoid membranes.
A. Subcellular localization of CGL160. Immunoblot analysis performed on initial plant extract, isolated chloroplasts or isolated mitochondria. TOM40 is a marker for the mitochondrion. B. Suborganellar localization determined by immunoblot analysis of chloroplast proteins diagnostic for photosystem I (PsaA), photosystem II (D1), Rubisco, LHCII (LHCB1.2) and CGL160. Protein extracts were separated by SDS-PAGE and probed with specific antibodies directed against PsaA, (PSI reaction center subunit), D1 (PSII reaction center subunits), LHCB1 (outer PSII antenna protein), the large subunit of Rubisco, a soluble protein in the stroma, and CGL160. C. Immunoblot analysis show that CG160 is an integral membrane protein associated with the thylakoid membranes. Isolated thylakoid membranes were washed with 0.4 M NaCl, and the thylakoid membranes and the supernatant were probed by immunoblotting with antibodies against CGL160 and the PSII reaction center protein D1.
Fig 4.
Phenotypic characterization of Arabidopsis cgl160 mutant plants.
A. Top panel shows, from left to right, a wild type plant, cgl160-1 and wild type, cgl160-2 plants grown for 3 weeks under normal light conditions (120 μmol m-2 s-1) at long day (16 hours light and 8 hours dark). B. Wild type, cgl160-1 and cgl160-2 plants grown under short day conditions (120 μmol m-2 s-1; 8 hours light and 16 hours dark) for 4 weeks. C. Shows, from left to right, a wild type plant, cgl160-1 and wild type, cgl160-2 plants grown for 1 week under normal light conditions (120 μmol m-2 s-1) at long day (16 hours light and 8 hours dark) and then shifted to fluctuating low/normal light conditions (5 min 120 μmol m-2 s-1, 5 min 20 μmol m-2 s-1 changing every 5 min for 16 hours light and then 8 hours dark) for two weeks. D. Bar graph of Fv/Fm as a measure of photosynthetic performance under the indicated conditions.
Table 1.
Photosynthetic parameters in wild-type plants and the two cgl160 T-DNA-insertion lines were determined for plants grown under long-day and plants grown under short-day conditions.
Fig 5.
Linear electron transport, non-photochemical quenching (qN) and redox state of the PSII acceptor side are affected in cgl160 mutants both under long- and short-day conditions.
Measurements were done in long (A-C)—and short-day (D-F) adapted plants (16 hours light and 8 hours light, respectively). A and D. Light response curve of linear electron flux as calculated from PSII yield measurements. In both cgl160 T-DNA insertion lines, linear electron flux capacity is reduced. B and E. Light response curves of non-photochemical quenching (qN). In both T-DNA insertion lines, the induction of qN is shifted towards lower light intensities, but the maximum light-saturated qN is unaltered C and F. Light response curves of the redox state of the PSII acceptor side, determined as qL. When qL is one, QA is fully oxidized; when qL is zero, QA is fully reduced. With increasing light intensity, the PSII acceptor side becomes more rapidly reduced in both T-DNA insertion lines.
Fig 6.
Thylakoid membrane energization, pmf partitioning, and chloroplast ATP synthase activity in Arabidopsis cgl160 mutant plants.
A. The maximum pmf across the thylakoid membrane, as determined from the maximum amplitude of the electrochromic shift signal during a dark-interval relaxation kinetic (ECST), is increased in cgl160 mutants under long- and short-day conditions. B. pmf partitioning, deduced from the slow relaxation phase of the electrochromic shift signal, is slightly shifted in favor of ΔΨ in Arabidopsis cgl160 mutant plants under short-day conditions. C. ATP synthase activity (gH+), determined from rapid dark-interval relaxation kinetics of the electrochromic shift signal, is significantly decreased in Arabidopsis cgl160 mutant plants under both long-day and short-day conditions.
Fig 7.
Altered protein accumulation and stability of the chloroplast ATP synthase in the cgl160 mutant visualized by immunoblotting.
A. Immunoblots with antibodies against essential subunits of the photosynthetic protein complexes of wild-type (Col-4) Arabidopsis and the two cgl160 T-DNA insertion lines grown under long-day and short-day conditions. Isolated thylakoid membranes were used, and equal amounts of chlorophyll were loaded onto the SDS-PAGE gel. For approximate quantification, wild-type samples from long-day plants were diluted to 10%, 25% and 50%, respectively. Accumulation of PSII was probed with antibodies against PsbB and PSBO. Additionally, the PSBS protein involved in NPQ and the minor PSII antenna protein LHCB4 were probed. Accumulation of the cytochrome b6f complex was probed with antibodies against the essential subunits PetA (cytochrome f), PetB (cytochrome b6), and PETC (Rieske protein). Accumulation of PSI was probed with antibodies against the reaction center subunit PsaB and the stromal ridge subunit PsaD. ATP synthase accumulation was probed with antibodies against the CF1 subunits AtpA (CF1α), AtpB (CF1β) and AtpD (CF1δ) and antibodies against the CF0 subunits AtpF (CF0b) and AtpI (CF0a). B. Loading difference estimation for immunoblotting CF1 between wild type and cgl160-1. To obtain similar immunoblotting signal three times more (15 μg protein) was needed for cgl160-1 compared to wild type (5 μg protein). C. Maintenance of CF1 was measured by incubating leaves from wild type and cgl160-1 in solution containing the plastid protein synthesis inhibitor chloramphenicol for the indicated time points. Protein extract was isolated and separated by SDS-PAGE, immunoblotted and probed with specific antibodies against CF1 and LHCB2.1. Three times more protein was loaded from the mutant to obtain equal level of CF1 immunoblotting signal, as specified in B.
Fig 8.
2D native / SDS-PAGE gel immunoblotting and crosslinking indicate interaction between CGL160 and the chloroplast ATP synthase complex.
A. Blue-native gel electrophoresis analysis of thylakoidal protein complexes in wild type and mutant plants. PSII-SC photosystem II supercomplex, PSII-D photosystem II dimer, PSII-M photosystem II monomer, LHCII-T light harvesting complex II trimer. The Blue-native gel was used for immunoblotting against D1, CF1 and CGL160 antibodies as indicated. B. Solubilized and cross-linked thylakoid membranes were separated by SDS-PAGE and probed with specific antibodies against CGL160 and CF1. C. Detection of CGL160 and ATP synthase subcomplexes by immunoblot analyses of 2D BN/SDS gels as in panel A, employing antibodies specific for CGL160 and individual ATPase subunits as indicated in the figure.