1. Volume 5, Issue 6, Pages 1242-1262 (November 2012)
Retrograde Signaling and Photoprotection in a gun4 Mutant of Chlamydomonas reinhardtii 
Cinzia Formighieri, Mauro Ceol, Giulia Bonente, Jean-David Rochaix, Roberto Bassi 
Molecular Plant 
Volume 5, Issue 6, Pages (November 2012)
DOI: /mp/sss051
Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

2. Figure 1
Mapping of the Insertion Site and Identification of the Mutation in gun4.
(A) Schematic representation of the GUN4 genomic region. Rectangles represent the two exons and lines non-coding regions. The site of insertion of the pSL18 vector is indicated (+589) and the deletion caused by the insertion is marked by dashed lines. The arrows indicate the position of the primers used for the RT–PCR in Figure 1B.
(B) RT–PCR with GUN4-specific primers and control primers (CBL) from gun4 and cw15 RNA. Asterisk indicates a non-specific amplification product, verified by sequencing of the excised band. Markers size from top to bottom: 1000, 800, 600, 400 pb.
(C) Alignment of the predicted C. reinhardtii GUN4 protein sequence with the corresponding sequences from Arabidopsis thaliana, Synechocystis sp. PCC 6803, and Thermosynechococcus elongatus. The secondary structure proposed for the Synechocystis GUN4 domain (Verdecia et al., 2005) is depicted: cylinders represent α-helices (α1 to α8) and lines represent unstructured regions. The loops putatively involved in porphyrin binding are indicated (α2/α3 and α6/α7). The location of the insertion in the C. reinhardtii gun4 mutant is shown by the open triangle and the dashed line marks the deletion.
Molecular Plant 2012 5, DOI: ( /mp/sss051)
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3. Figure 2
Random Progeny Analysis of the Cross between Cell Wall-Less gun4 mt- and Cell Wall-Containing Wild-Type S34 mt+. Progeny colonies were tested on TAP either in dim light (6 µmol photons m–2 s–1) in the presence of paromomycin (15 µg ml–1, left panel) or in high light (500 µmol photons m–2 s–1, right panel). Marked with an asterisk are colonies with the gun4 photosensitive phenotype, dying in high light (right panel). Only these colonies grew on TAP-paromomycin (left panel). This result shows co-segregation between the mutant phenotype and the paromomycin resistance carried by the insertion cassette, indicating that the gun4 mutant is tagged by the transforming DNA. Independent segregation of the cell wall phenotype was checked to verify that crossing had indeed occurred. Parental strains are marked with an arrow on the same plate as references.
Molecular Plant 2012 5, DOI: ( /mp/sss051)
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4. Figure 3
Complementation of the gun4 Mutant with a Recombinant GUN4–HA Construct.
(A) Immunoblot analysis of total proteins extracts of cw15 (wild-type), gun4, and two complemented clones (R1 and R2) with α-GUN4 and α-PRK antibodies. The GUN4-HA protein in R1 and R2 is visible at a higher molecular weight compared to the native GUN4 in cw15. GUN4 protein level in the different lines is normalized to the PRK signal and expressed as percentage with respect to the wild-type.
(B) Growth test of cw15, gun4, R1, and R2. Five µl containing 107, 106, 105, and 104 cells per ml of each strain were spotted on acetate-containing medium (TAP) or minimal medium (HSM) and grown for 2 weeks under the indicated light conditions (µE = µmol photons m–2 s–1).
Molecular Plant 2012 5, DOI: ( /mp/sss051)
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5. Figure 4 Immunoblot Analysis on a Per-Protein Basis.
(A) Immunoblot analysis of the indicated polypeptides in gun4 and cw15. D, dark acclimated cells; CL, after 24 h of illumination at 60 µmol photons m–2 s–1.
(B) Time course analysis of the changes in protein content of gun4 and cw15 after a shift of dark-adapted cells to 60 µmol photons m–2 s–1 of light. Samples were collected at time 0 (Dark) and after 20 min, 40 min, 1 h, 3 h, 6 h, 12 h, and 24 h of continuous illumination at 60 µmol photons m–2 s–1.
Molecular Plant 2012 5, DOI: ( /mp/sss051)
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6. Figure 5
Native Electrophoretic Separation of Chlorophyll-Containing Photosynthetic Complexes.
(A) DERIPHAT–PAGE, gradient 4.5–12%. Unstacked thylakoids were extracted from cultures grown at 30 µmol photons m–2 s–1. Thylakoids were then solubilized with different amounts of αDM (n-dodecyl-α-D-maltoside). 30 µg of chlorophylls are loaded in each lane. Thylakoids from Arabidopsis thaliana are shown in the first lane for comparison.
(B) PsaA subunit of photosystem I core was detected in the green gel-eluted bands with a specific antibody (1 µg of thylakoid chlorophylls is loaded as a positive control).
(C) Densitometric analysis of green bands. The reported values are in arbitrary units (A.U.) and refer to the sum of the bands in the lane, the sum of monomeric and trimeric antennae, PSI/PSII core, PSI/PSII supercomplexes.
(D) Chlorophyll fluorescence emission at 77 K. Spectra were recorded both in cells dark-adapted in state I before the measurement (dark) and in samples dark-adapted and then pre-illuminated at 760 µmol photons m–2 s–1 (light). Chlorophylls were excited at 475 nm (width of the beam 17 nm). The peaks at 686 and 694 nm originate from photosystem II, whereas the emission around 710 nm is attributed to photosystem I. The spectrum is normalized with respect to the 686-nm peak.
Molecular Plant 2012 5, DOI: ( /mp/sss051)
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7. Figure 6 Analysis of P700 Photo-Oxidation In Vivo.
Cells in state I were pre-illuminated for 20 min at 400 µmol photons m–2 s–1 before the measurement. Data were normalized with respect to the value at the end of the actinic light phase (60 s, 940 µmol photons m–2 s–1).
(A) P700 oxidation in cw15 without (untreated) or with 10 µM DCMU.
(B) P700 oxidation in gun4 without (untreated) or with 10 µM DCMU.
Molecular Plant 2012 5, DOI: ( /mp/sss051)
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8. Figure 7 Photochemical Yield of Photosystem II (ϕPSII).
It was calculated as (Fm’ – Fs)/Fm’ with an actinic light of 100 µmol photons m–2 s–1 in the absence (white bars) or after addition (gray bars) of pgal.
(A) Cells grown at 30 µmol photons m–2 s–1 and dark-adapted before the measurement.
(B) Cells grown as in (A) but submitted to a hyper-osmotic stress (0.5 M sucrose).
Molecular Plant 2012 5, DOI: ( /mp/sss051)
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9. Figure 8
Analysis of PTOX Accumulation by Immunoblot from Total Protein Extracts or Thylakoids.
The amount (μg) of chlorophyll loaded in each lane is indicated. PRK is used as internal control in total protein extracts, while it is not present in thylakoids since it is a soluble protein. Numbers 1 and 2 on the left indicate the putative PTOX1 and PTOX2 signals.
Molecular Plant 2012 5, DOI: ( /mp/sss051)
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10. Figure 9 Transcript Levels in cw15 and gun4.
(A) RNA blot analysis during light acclimation of gun4 and cw15. Samples were collected at time 0 (Dark) and after 30 min, 1 h, 2 h, 8 h, and 24 h of continuous illumination at 60 µmol photons m–2 s–1.
(B) Real-time RT–PCR analysis of the transcript content of LHCBM1 and CP29 in cw15 and gun4. In the histogram are reported the differences between the cycles in which the SyBR Green fluorescence crosses a threshold (–ΔΔCT). The error bars refer to different biological replicates for each genotype and condition. Values around zero mean that the expression of the gene is the same in the samples analyzed. Positive values indicate an up-regulation and negative values a down-regulation. Data are normalized with respect to the constitutive expression of 18S rRNA. gun4/cw15 LL, comparison of transcription in gun4 with respect to cw15 at 30 µmol photons m–2 s–1. cw15 HL/LL and gun4 HL/LL, comparison of transcription in cw15 and gun4, respectively, after exposure to 400 µmol photons m–2 s–1 for 1 h relative to the sample in low light.
Molecular Plant 2012 5, DOI: ( /mp/sss051)
Copyright © 2012 The Authors. All rights reserved. Terms and Conditions