Supporting Information – legends
Figure S1. Phylogenetic analysis of ATP4 relatives. Full length protein alignments were created utilizing
MUSCLE (Edgar, 2004) using default parameters. A maximum likelihood tree was calculated with PhyML
3.0 (Guindon et al., 2010) at Phylogeny.fr (http://www.phylogeny.fr/) using default parameters.
Numbers indicate branch support by bootstrap analysis (100 iterations, the maximum permissible at
Phylogeny.fr). Os and At indicate rice and Arabidopsis proteins, respectively.
Figure S2. Alignment of ATP4 orthologs from Arabidopsis (At), Rice (Os), and Maize (Zm). The position of
the peptide used for antibody production is indicated by yellow arrows. At-ATP4 corresponds to locus
At4g16390; Os ATP4 corresponds to TIGR locus Os03g0215900. Zm-ATP4 corresponds to maize locus
GRMZM2G128665 (B73 RefGen version 2).
Figure S3. Light-response curves of chlorophyll-a fluorescence and P700 difference absorbance
parameters of wild-type plants and atp4 mutants. (A) The accelerated onset of photoprotective nonphotochemical quenching (qN) indicates a strong thylakoid lumen acidification in atp4 mutants already
at low light. (B) The quantitative reduction of the PSII acceptor side (more rapidly decreased values of
chlorophyll-a fluorescence parameter qL) already in low light indicates a strong restriction of linear
electron flux behind PSII in atp4 mutants. (C) The accelerated accumulation of oxidized, donor-side
limited PSI in atp4 mutants indicates that the limitation in linear electron flux is located between PSII and
PSI at the cytochrome-b6f complex. (D) Slower reduction kinetics of photo-oxidised P700 after the end of a
saturating light pulse confirm down-regulation of linear electron flux. Saturating light was switched-off at
time point zero, and the P700 reduction kinetics of the wild type and atp4 mutants were normalized for
better comparability. The fully oxidized state of P700 at the end of the saturating illumination was
normalized to one, and the fully reduced state in darkness was normalized to zero.
Figure S4. Quantitative immunoblot analysis in atp4 mutants. To support the quantification of Western
analyses shown in Fig. 2, we analyzed dilution series of wild-type protein extract side-by-side with a
mutant sample. Blots were probed with the indicated antibodies. PonceauS stains are shown to
demonstrate successful transfer and desired loading of protein. While the AtpB protein is stongly
reduced, other proteins do show little if any differences to wild-type samples.
Figure S5. Immunoblot analysis of a representative ATP4 immunoprecipitation used for RIP-chip analysis.
Left: Aliquots of proteins from supernatants (S) or pellets (P) of immunoprecipitations using either the
ATP4 antiserum or the corresponding pre-immune serum were separated by SDS-PAGE and blotted to a
nitrocellulose membrane. The aliquots represent 1/10th of the material used for the RIP-chip assays. The
filter was probed with the same antibody used for immunoprecipitation. IgGs in the immunoprecipitates
are detected by the secondary antibody used to probe the immunoblot. The arrow indicates the band
corresponding to ATP4. The asterisks denote the heavy and light chain of the antibody used to
immunoprecipitate ATP4, which is visible only in pellet fractions, as expected. Right: staining of the blot
with Ponceau S demonstrates equal amounts of proteins in supernatant fractions.
Figure S6. Control analyses to support the RIP-chip experiments. (A) Differential enrichment of specific
RNAs in RIP-chip experiments using ATP4 antibodies versus experiments using pre-immune sera or an
anti-OEC23 sera. These oligonucleotides correspond to RNAs not enriched in the whole-genome RIP-chip
experiment shown in figure 3A. The oligonucleotides were present on the same array as those presented
in figure 3B and 3D and were thus hybridized in parallel. As expected, the enrichment values for these
probes are low. Panels B and D are identical to Fig. 3B and C with the exception that the Y-axes represent
the GC-content of the oligonucleotides. Maxima of enrichment identified in Fig. 3B and C do not coincide
with maxima or minima in GC-content, thus excluding an artifactual signal pattern caused by base
composition differences between oligonucleotides. In panel C, GC content of control oligonucleotides is
shown. (E) Slot-blot hybridization analysis to verify that atpB RNA is enriched in ATP4
coimmunoprecipitates. Coimmunoprecipitations and RNA extractions were performed as for the RIP-chip
assays. An equal proportion of RNA purified from the supernatants (S) and pellets (P) were applied to
nylon membrane with a slot-blot manifold, and hybridized with a radiolabeled PCR product spanning the
atpB 5’ UTR. In a parallel experiment, we assessed co/precipitation of rps2 as a negative control. An
immunoprecipitation with antibody to OEC23 was included as a control for non-specific precipitation of
atpB RNA in our assay.
Figure S7. RNA gel blot hybridization of atpF intron-containing RNAs in atp4 mutants. Total leaf RNA (5
µg/lane) was analyzed by RNA gel blot hybridization using a radiolabelled atpF intron DNA probe. Mutant
seedlings from two different allelism crosses (atp4-1/+ x +/atp4-3, atp4-2/+ x +/atp4-3), and a
phenotypically-normal sibling from each cross were analyzed. Transcripts reduced in atp4-mutants are
marked with an asterisk. The positions of RNA markers (knt-kilonucleotides) are shown. Numbers next to
the atpF intron autoradiogram refer to transcripts in figure 4B. rRNAs on the same filters were detected
by staining with methylene blue and are shown below.
Figure S8: RNA gel blot hybridization of chloroplast rRNAs in atp4 mutants. 2 µg of total leaf RNA was
separated on a denaturing agarose gel, blotted onto a nylon membrane and hybridized with
radiolabelled DNA probes specific for the indicated genes. RNA samples of hcf7 mutants have been
analysed because they are known to be generally impaired in translation and display rRNA processing
defects (Barkan, 1993). Precursor rRNAs that may slightly over-accumulate in these mutants are
indicated by asterisks.
Table S1. RIP-chip data for Fig. 3A.
Table S2. RIP-chip data for Fig. 3B/C and Figure S5A.
Table S3. RIP-chip data for Fig. 3B/C and Fig. S5A.
Appendix S1. Supplemental methods.
Supplemental References
Barkan, A. (1993). Nuclear Mutants of Maize with Defects in Chloroplast Polysome Assembly Have
Altered Chloroplast RNA Metabolism. Plant Cell 5, 389-402.
Edgar, R.C. (2004). MUSCLE: multiple sequence alignment with high accuracy and high throughput.
Nucleic Acids Res 32, 1792-1797.
Guindon, S., Dufayard, J.F., Lefort, V., Anisimova, M., Hordijk, W., and Gascuel, O. (2010). New
algorithms and methods to estimate maximum-likelihood phylogenies: assessing the
performance of PhyML 3.0. Syst Biol 59, 307-321.