Article Figures & Data
Figures
- Figure 1.
Single-cell colonies of C. reinhardtii wild type and tla2 mutant grown on agar. Note the dark-green coloration of the wild-type strains, as compared to the light-green coloration of the tla2 mutant. [See online article for color version of this figure.]
- Figure 2.
Light-saturation curves of photosynthesis obtained with the C. reinhardtii wild type (black squares) and the tla2 mutant (white circles). The initial slopes of both curves are similar, suggesting equal quantum yield of the photosynthesis. The light-saturated rate Pmax was greater in the tla2 mutant than in the wild type, suggesting a greater productivity on a per-Chl basis in the tla2 than in the wild type.
- Figure 3.
Map of the tla2 and wild-type C. reinhardtii genomic DNA in the pJD67 insertion site. Plasmid insertion in the tla2 mutant caused deletion of a 12.5-kb segment comprising three genes, namely Cre05.g241450, Cre05.g241500, and Cre05.g241550. In addition, a 358-kb segment of the tla2 genomic DNA flipped by 180° in orientation (5′ to 3′), disrupting the continuity of two additional genes at each end of the rearrangement site, namely Cre05.g239000 and Cre05.g241400. Thus a total of five genes were affected by the pJD67 insertion in the tla2 mutant. Also shown is the C. reinhardtii genomic DNA region covered in BAC clones 28L06, 21D17, 08N24, and 36L15. The plasmid insertion site and the identity of the TLA2 gene (Cre05.g241450 in red font) are also indicated. [See online article for color version of this figure.]
- Figure 4.
Genetic cross analysis of tla2 with AG1x3.24 (arg2) strain. One representative tetrad from a single cross is shown (a total of 10 tetrads were analyzed), plated on nonselective TAP + ARG media (top section), or selective TAP-only media (middle section). The Chl a/b ratio of these progeny is shown at the top of the sections. The bottom section shows the result of PCR reactions, two lanes per progeny: The PCR reaction using an insertion-specific primer set was loaded on lanes 1, 3, 5, 7, and a positive control PCR on lanes 2, 4, 6, and 8. [See online article for color version of this figure.]
- Figure 5.
Top, Amino acid sequence of the C. reinhardtii FTSY protein. Domains of the CrCpFtsY protein are defined as follows: amino acids 1 to 36, transit peptide (green font). Amino acids 66 to 147, helical bundle domain (Pfam), SRP54-type protein (blue font). Amino acids 162 to 370, GTPase domain (Pfam), SRP54-type protein (red font). Amino acids 164 to 183, P-loop nucleotide binding motif, (pre; red, gray-shaded font). Amino acids 170 to 176, 258 to 262, and 322 to 325, homologous nucleotide binding (red, black-background font). Bottom, Domain presentation of the CrCpFTSY protein. CpTP, Chloroplast transit peptide; HB, helical bundle domain; GTPase, GTPase domain. [See online article for color version of this figure.]
- Figure 6.
CLUSTAL 2.1 multiple sequence alignment of AtCpFTSY, ZmCpFTSY, and CrCpFTSY. The comparative amino acid sequence analysis showed 46.1% identity and 61.9% similarity of the CrCpFTSY to AtCpFTSY and 50.5% identity and 65.5% similarity to ZmCpFTSY, strengthening the notion of a chloroplast localization and function for the CrCpFTSY identified in this work.
- Figure 7.
. Western-blot analysis of the light-harvesting antenna proteins of PSII in C. reinhardtii wild type and the tla2 mutant. A, Immunodetection of proteins with specific polyclonal antibodies raised against the light-harvesting proteins Lhcb1/Lhcb2, Lhcb3, Lhcb4, and Lhcb5 of Arabidopsis, the PSII reaction center protein D2, the PSI reaction center protein PsaL, Rubisco, and the β-subunit of the ATP synthase are shown. B, Coomassie-Blue-stained SDS-PAGE analysis of the samples shown in A.
- Figure 8.
. Western-blot analysis of C. reinhardtii total cell protein extracts isolated from wild type, the tla2 mutant strain, and tla2 lines C1, C2, C3, and C4 complemented with a wild-type copy of the CrCpFTSY gene. A, Immunodetection of CrCpFTSY and other thylakoid membrane proteins was performed with polyclonal antibodies against PSII subunits CP43 and PsbO, PSI subunit PsaL, and against the LHCII subunit Lhcb1 of Arabidopsis. Loading of lanes was based on Chl and corrected for the Chl content per cell so as to load proteins on an equal cells basis. B, Coomassie-Blue-stained SDS-PAGE analysis of the samples shown in A.
- Figure 9.
Cell fractionation and localization of the CpFTSY protein. A, Immunoblot analysis of wild-type total cell protein extract (1.5 μg Chl loaded), total membrane extract (1.5 μg Chl loaded), total soluble fraction (75 μg of protein loaded), and isolated chloroplast extract (1.5 μg Chl loaded). Western-blot analysis was conducted with specific polyclonal antibodies raised against the CrCpFTSY, CrCpSRP54, PsbO, or D2 proteins. B, Coomassie-Blue-stained SDS-PAGE analysis of the samples shown in A.
- Figure 10.
Analysis of photosynthetic complexes from thylakoid membranes, resolved by nondenaturing Deriphat-PAGE and denaturing second-dimension electrophoresis. Samples tested were from wild type, tla2 mutant, and tla2 lines C1, C2, C3, and C4 complemented with a wild-type copy of the CrCpFTSY gene. A, Pigment-protein complexes resolved by nondenaturing Deriphat-PAGE. Protein complexes were identified by their molecular mass of the first nondenaturing and second denaturing dimension. Masses of the marker on the left are given in kD. B, Silver-nitrate-stained second denaturing dimension from wild type and tla2. 1, PSI reaction center proteins PsaA and PsaB dimer; 2, LHCI proteins; 3, PSII reaction center proteins CP43 and CP47; 4, PSII reaction center proteins D1 and D2; 5, LCHII proteins; 6, α- and β-subunit of the ATP synthase. Molecular size markers are given in kD. [See online article for color version of this figure.]
- Figure 11.
Working model of the function of the CrCpSRP transmembrane complex assembly system in the model green algae C. reinhardtii. Precursor LHC proteins are targeted to the chloroplast via the transit peptide and the heat shock protein HSP70, which functions as a molecular chaperon to prevent aggregation of the preassembled proteins. Chloroplast protein import is facilitated by the envelope-localized TOC and TIC complexes, which catalyze protein import through the outer and inner envelope membranes of the chloroplast. The transit peptide is cleaved off and the molecular chaperon CpSRP43 binds to the incoming light-harvesting protein to prevent its aberrant misfolding and aggregation. CpSRP54 and CpFTSY guide this CpSRP43-LHC complex to the membrane-bound translocase ALB3. Upon integration of the light-harvesting protein into the nascent thylakoid membrane, the LHC-CpSRP43-CpSRP54-CpFTSY complex disassembles, making the SRP subunits available for another carry-and-assembly cycle. [See online article for color version of this figure.]
Tables
- Table I. Chl and Car content and pigment ratios for wild type, tla2 mutant, and tla2-complemented strains of C. reinhardtii (n = 3–5; means ± sd)
Strain Chl/Cell Chl a/b Car/Cell Car/Chl fmol fmol Low light (80 μmol photons m−2 s−1) 4A+ 2.57 ± 0.43 2.72 ± 0.07 1.07 ± 0.17 0.42 ± 0.00 CC-125 2.66 ± 0.13 3.00 ± 0.03 1.11 ± 0.06 0.42 ± 0.00 CC-504 2.36 ± 0.05 2.73 ± 0.05 0.93 ± 0.04 0.39 ± 0.01 CC-425 2.33 ± 0.10 2.86 ± 0.04 0.95 ± 0.04 0.41 ± 0.01 C1 1.93 ± 0.17 2.87 ± 0.02 0.67 ± 0.16 0.42 ± 0.00 C2 1.55 ± 0.04 3.01 ± 0.03 0.67 ± 0.00 0.43 ± 0.01 C3 1.06 ± 0.01 3.35 ± 0.16 0.54 ± 0.01 0.51 ± 0.00 C4 0.61 ± 0.09 3.92 ± 0.09 0.42 ± 0.05 0.68 ± 0.02 tla2 0.46 ± 0.04 9.60 ± 0.98 0.38 ± 0.00 0.82 ± 0.06 Medium light (450 μmol photons m−2 s−1) 4A+ 1.66 ± 0.37 2.45 ± 0.09 0.85 ± 0.17 0.51 ± 0.01 CC-125 1.85 ± 0.49 2.75 ± 0.14 1.01 ± 0.33 0.54 ± 0.04 CC-504 1.68 ± 0.33 3.08 ± 0.12 0.84 ± 0.13 0.50 ± 0.03 CC-425 1.35 ± 0.19 2.85 ± 0.04 0.74 ± 0.10 0.55 ± 0.01 C1 1.03 ± 0.04 2.71 ± 0.09 0.56 ± 0.03 0.54 ± 0.02 C2 0.71 ± 0.07 3.62 ± 0.11 0.52 ± 0.03 0.74 ± 0.03 C3 0.51 ± 0.07 4.36 ± 1.05 0.43 ± 0.07 0.85 ± 0.07 C4 0.35 ± 0.05 6.49 ± 0.56 0.34 ± 0.04 0.97 ± 0.02 tla2 0.33 ± 0.04 7.92 ± 0.83 0.30 ± 0.03 0.90 ± 0.01 - Table II. minus2 minus1Photosynthesis, respiration, and photochemical apparatus characteristics of wild type and the tla2 mutant of C. reinhardtii grown photoautotrophically under medium-light (450 μmol photons m−2 s−1) conditions
Photosystem Chl antenna size and reaction center concentrations were measured spectrophotometrically (Melis, 1989). WT, Wild type; n = 3; means ± sd.
Parameter Measured WT tla2 Respiration (mmol O2 [mol Chl]−1 s−1) 30.2 ± 11.9 49.1 ± 15.2 Respiration (amol O2 cell−1 s−1) 55.8 ± 26.3 16.2 ± 5.4 Quantum yield, relative units 100 ± 25 108 ± 17 Photosynthesis (mmol O2 [mol Chl]−1 s−1) 106.3 ± 12.8 152.3 ± 18.0 Photosynthesis (amol O2 cell−1 s−1) 196.2 ± 46.2 50.3 ± 7.3 Photosynthesis/respiration capacity ratio 3.5 ± 1.9 3.1 ± 1.1 Half-saturation intensity (μmol photons m−2 s−1) 210 380 Functional PSIIα Chl antenna size 249 ± 27 160 ± 7 Functional PSIIβ Chl antenna size 90 ± 30 90 ± 12 Fraction of PSIIα (%) 61 ± 1 46 ± 1 Average PSII Chl antenna size 190 ± 20 120 ± 9 Functional PSI Chl antenna size 180 ± 9 123 ± 5
Supplemental Data
Supplemental Data, Table, and Figures
Files in this Data Supplement:
- Supplemental Data - Supplemental Data, Supplemental Table, and Supplemental Figures 1-3
