Foot notes
Figure 1. Motif discovery and identification of functional amino acid positions in CGEs. Sequence motifs found enriched in type 1 (A) and type 2 (B) CGEs by analysis with DREME tool (
http://meme-suite.org/doc/dreme.html?man_type=web). The input sequences were randomized to generate a control set, the E-value threshold for motif discovery was set to 0.05. Alignment of
E. coli GrpE and the consensus sequences of type 1 and type 2 CGEs (C), the numbers and arrows over positions 73,74,82,122,183, and 192 point to the amino acids that are functionally relevant in
E. coli.
Figure 2. Bacterial complementation assay. The growth of the thermo-sensitive E. coli OD212 strain, transformed with either the empty vector (pDEST14ΔccdB) or the expression vectors pDEST14::ΔCGE1 or pDEST14::ΔCGE2, was tested at different temperature conditions: 25 °C (A), 37 °C (B), and 43 °C (C). The bacterial samples were tested in LB-agar plates as 5 μl drops of the corresponding dilutions.
Figure 3. Subcellular localization of CGE1, CGE2 and cpHsc70-1 proteins. Mesophyll protoplasts from N. benthamiana leaves expressing GFP (A) and the translational fusions CGE1:GFP (B), CGE2:GFP (C), and cpHsc70-1:GFP (D). Images corresponding to the Bright field, Chlorophyll fluorescence, GFP fluorescence, and the Merge between the two fluorescence channels are shown. As stated in the Material and Methods section, protoplasts were prepared 96 hours after the infiltration of the N. benthamiana leaves. Scale bars corresponding to 10 μm are shown in the bottom-right corner.
Figure 4. Suborganellar localization of CGE1, CGE2 and cpHsc70-1 proteins. Western blot analysis of protein extracted from purified chloroplast envelope (left section) and stroma (right section) from not infiltrated leaves (lane 1) and leaves infiltrated with pNE::CPHSC701 (lanes 2 and 4), pNE::CGE1 (lane 3), and pNE::CGE2 (lane 5). Immunological detection of the c-Myc tagged protein fusions (αc-Myc), RbcS (αRbcS), and Tic40 (αTic40) proteins in the suborganellar samples is shown in the respective panels. Ponceau S staining of the protein-containing membranes is shown as loading control. Molecular weight marker (kDa) is shown at the left.
Figure 5. In vivo interaction between CGEs and cpHsc70-1 proteins. Mesophyll protoplasts from N. benthamiana leaves co-expressing the translational fusions CGE1-cYFP and cpHsc70-1-nYFP (A), CGE1-nYFP and cpHsc70-1-cYFP (B), CGE2-cYFP and cpHsc70-1-nYFP (C), and CGE2-nYFP and cpHsc70-1-cYFP (D).Images corresponding to the Bright field, Chlorophyll fluorescence, reconstituted YFP fluorescence, and the Merge between the two fluorescence channels are shown. As stated in the Material and Methods section, protoplasts were prepared 96 hours after the infiltration of the N. benthamiana leaves. Scale bars corresponding to 10 μm are shown in the bottom-right corner.
Figure 6. In vivo determination of CGE1 and CGE dimer formation. Mesophyll protoplasts from N. benthamiana leaves co-expressing the translational fusions cpHsc70-1-cYFP and cpHsc70-1-nYFP (A) as a negative control for dimer formation, CGE1-cYFP and CGE1-nYFP (B), CGE2-cYFP and CGE2-nYFP (C), CGE1-cYFP and CGE2-nYFP (D), and CGE2-cYFP and CGE1-nYFP (E).Images corresponding to the Bright field, Chlorophyll fluorescence, reconstituted YFP fluorescence, and the Merge between the two fluorescence channels are shown. As stated in the Material and Methods section, protoplasts were prepared 96 hours after the infiltration of the N. benthamiana leaves. Scale bars corresponding to 10 μm are shown in the bottom-right corner.
Figure 7. Expression analysis of CGE1, CGE2 and cpHsc70-1 under heat stress. Northern blot analysis of total mRNA extracted from Wild-type seedlings after 0, 30, 60, and 90 minutes of heat stress. Specific hybridization of probes against CGE1 (A), CGE2 (B), CPHSC70-1 (C), and DXS1 (D) genes is shown. Ethidium bromide staining of rRNA 28S is shown as a loading control for each experiment. Three biologically independent experiments were carried out, representative results are shown.
Figure 8. Molecular phenotype of emb1241 (+/-) and Dcphsc70-1 mutants. (A) The ratio of chlorophyll a to chlorophyll b (Chlorophyll a/b) of Wild-type, emb1241-1 (+/-), emb1241-2 (+/-), cge2-1 (+/-), and Dcphsc70-1 mutants is represented. The error bars represent the standard error, the symbol (*) over the bars represent statistically significant differences calculated in a one-way analysis of variance with a significance level of 0.01. (B) Western blot analysis of total protein extracted from Wt (lane 1), emb1241-2 (+/-) (lane 2), and Dcphsc70-1 (lane 3) adult plants, immunodetection of psaD and D1 proteins using specific antibodies is shown. Ponceau S staining of the protein-containing membranes is shown as a loading control, molecular weight marker (kDa) is shown at the left. (C) Blue native-PAGE of leaf protein from Wild-type (lane 1), emb1241-2 (+/-) (lane 2), and Dcphsc70-1 (lane 3) plants. Arrows indicate the bands corresponding to the supramolecular complexes visible in the samples.
Figure S1. Molecular Phylogenetic analysis by Maximum Likelihood method of CGE proteins. The evolutionary history was inferred by using the Maximum Likelihood method based on the JTT matrix-based model [1]. The tree with the highest log likelihood (-17896.1536) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. The three main clades algae type (blue), type 1 (green), and type 2 (yellow) CGEs are shown. Branch labels are in the format: UniprotID (Species name).
Figure S2. Genetic constructs used in the study. The expression vectors for the expression of GFP (pBN::GFP), CGE1-GFP (pEG::CGE1), CGE2-GFP (pEG::CGE2), cpHsc70-1-GFP (pEG::CPHSC70-1), CGE1-c-Myc-nYFP (pNE::CGE1), CGE2-c-Myc-nYFP (pNE::CGE2), cpHsc701-c-Myc-nYFP (pNE::CPHSC70-1), CGE1-HA-cYFP (pCE::CGE1), CGE2-HA-cYFP (pCE::CGE2), cpHsc701-HA-cYFP (pCE::CPHSC70-1), ΔCGE1 (pDEST14::ΔCGE1), and ΔCGE2 (pDEST14::ΔCGE2). The regulatory elements included are the Cauliflower mosaic virus promoter (p35S), RNA polymerase T7-dependent promoter (pT7), Nopaline synthase terminator (nos 3'), OCS terminator (ocs 3'), transcription terminator T7 (T7), Shine-Dalgarno sequence (SD), recombination sequences (attB1 and attB2).
Figure S3. Genotyping of the mutant lines used in the study. Representation of the genomic regions comprising the CGE1 (A), CGE2 (B), and CPHSC70-1 (C) genes. The position of the T-DNA insertion in each of the mutant lines analyzed is displayed in empty triangles. The position of the specific oligonucleotides used for the genotyping of the T-DNA insertion mutants is shown as arrows over the corresponding exon (gray arrow) or noncoding region (straight line). The size of the PCR fragments expected from each pair of oligonucleotides is indicated below the corresponding diagram. The genomic regions used to generate probes for the northern blot assays are underlined. PCR of amplification of the DNA regions corresponding to the wild-type (D) and the mutated (E) alleles of CGE1, CGE2, and CPHSC70-1 genes in genomic DNA from wild-type plants and the mutant lines emb1241-1 (+/-), emb1241-2(+/-), cge2-1(+/-), and Dcphsc70-1. Arrow heads indicate the specific bands obtained in the PCR reactions, molecular weight marker is provided at both ends. Oligonucleotide sequences can be found in supplemental table S1.
Figure S4. Negative controls for BiFC assay. Mesophyll protoplasts from N. benthamiana leaves expressing the translational fusions CGE1-cYFP (A), CGE1-nYFP (B), CGE2-cYFP(C), CGE2-nYFP, cpHsc70-1-cYFP (D), and cpHsc70-1-nYFP (E).Images corresponding to the Bright field, Chlorophyll fluorescence, and YFP fluorescence channels are shown. As stated in the Material and Methods section, protoplasts were prepared 96 hours after the infiltration of the N. benthamiana leaves. Scale bars corresponding to 10 μm are shown in the bottom-right corner.
Table S1. Oligonucleotides used in the study.
Table S2. Conservation of functionally relevant amino acid residues in type 1 and type 2 CGEs.
Table S3. Pigment quantification.
*Number of independent replicates.
Data shown includes standard error calculated from the total population tested.