Supplemental Figure S1. RT-PCR analysis of At4g28010 (grey box) in SALK 113142 (5-1) and Col wild
type (Wt). PCRs (1 to 3, indicated under image) were performed with primer pairs 1: At4g28010.H
(P1)/At4g28010.R (P4), 2: At4g28010.H (P1)/At4g28010-KO.R (P2) and 3: At4g28010-KO.H
(P3)/At4g28010.R (P4). Products of expected sizes (920, 230 and 350 bp) were obtained with Col
wild-type cDNA as template, while no (reactions 1 and 3) or only weak products (reaction 2) were
amplified from cDNA from the SALK 113142 mutant. These results demonstrate the knockout of the
At4g28010 gene in the SALK 113142 mutant. The approximate locations of the T-DNA in SALK 113142
(triangle) and primers (horizontal arrows) are indicated. Schema not drawn to scale. Lengths of size
marker fragments (lanes M1 and M2) are given in [bp].
Supplemental Figure S2. CR-RT-PCR analyses of nad6, 26S and atp9 RNAs from Col wild-type (Wt),
the rpf5-1 mutant (5-1) and 4 rpf5-1 plants transformed with the wild-type allele of RPF5 (5-1 + RPF5,
1 to 4). The introduction of the RPF5 gene rescues the corresponding wild-type phenotype. Primer
pairs and sizes of the expected products are given on the right hand side. Annealing sites of the
primers are given in Figs. 1 to 3. Lengths of size marker fragments (lanes M1 and M2) are given in [bp].
Supplemental Figure S3. Diagram obtained after direct sequencing of the CR-RT-PCR product
amplified with primers At26S-Adapter.R3/Atmt26S2-F from the 26S rRNA precursor in the rpf5-1
mutant. The deduced sequence is given above the diagram. The sequence is unambiguous up to an
adenosine indicated by an arrow. A clear assignment of the exact 5’ end of the 26S rRNA precursor
(open blue box) is not possible since adenosines are also present at the 3’ terminus of this molecule.
The corresponding sequence encoded in the mtDNA is given below the diagram. The 3’ terminal
sequence of a potential small RNA (Ath-59-like) that is highly similar to the previously identified noncoding small RNA Ath-59 (Marker et al., 2002) is given in a black open box. The 5’ terminal sequence
of the 26S rRNA precursor is given in a blue open box.
Supplemental Figure S4. Diagram obtained after direct sequencing of the 420 bp CR-RT-PCR product
amplified on cDNA from rpf5-1 mutant (Fig. 2A). Automatically read sequence is given above the
diagram. Up to position 323 the sequence is without background. This sequence corresponds to 5’
terminal part of the 26S rRNA precursor (reverse complementary sequence shown below the
diagram). Although background peaks increase beyond position 323 (due to the ligation of RNA
molecules with heterogeneous ends) the sequence can still be read. This part corresponds to 3’
terminal part of the 26S rRNA. The position marked by an arrow corresponds either to a T at the 3’
end of a potential 26S rRNA degradation product (position 9044 in nc_001284) or to an A at the -60
5’ end of the 26S rRNA precursor (position 12147 in nc_001284).
Supplemental Figure S5. Diagram obtained after direct sequencing of the CR-RT-PCR product
amplified with oligonucleotides Atatp9-6/Atatp9-5 from the atp9 precursor RNA in the rpf5-1
mutant. The deduced sequence is given above the diagram. An arrow marks the last unambiguous
nucleotide of the sequence. This position corresponds to a G (red box), a frequently observed
transcription initiation site within a promoter motif (underlined) conserved in plant mitochondria
from dicotyledonous plants (Binder and Brennicke, 1993; Kühn et al., 2005). This end corresponds to
transcription initiation site Patp9-239.
Supplemental Figure S6. CR-RT-PCR analysis of mitochondrial transcripts from rpf5-1 and wild type
(Col). Oligonucleotides are given below the images. Sizes of DNA marker molecules are given in
nucleotides [bp]. Minor differences were observed in the patterns of atp6-2 and nad5 (see also
Supplemental Fig. S7).
Supplemental Figure S7. (a) CR-RT-PCR analysis of atp6-2 and nad5 transcripts from wild type, rpf5-1
and rpf5-1 plants complemented with RPF5 (rpf5-1 + RPF5). The atp6-2 (left panel) and nad5 (right
panel) CR-RT-PCR wild-type product pattern are rescued by the reintroduction of the RPF5 gene into
the rpf5-1 mutant (left panel). This result demonstrates that the altered patterns observed in the
rpf5-1 mutant are indeed due to the knockout of RPF5. The product a marked by an arrow (atp6-2,
about 350 bp) was directly sequenced. (b) Diagram of the sequence analysis identified a 5’ end at
position -176 (vertical arrow) with respect to the ATG of the atp6-2 reading frame. The
corresponding 5’ terminal sequence of the RNA is given below the diagram. No new termini could be
identified in the further analysis of nad5 transcripts in rpf5-1.
Supplemental Figure S8. Blue Native (BN)/SDS polyacrylamide gel electrophoresis of total
mitochondrial protein from Col wild type (Wt) and the rpf5-1 mutant (rpf5-1). Approximately 1.0 mg
protein extracted from mitochondria isolated from cell suspension cultures were separated by BN
PAGE in the first dimension. Lanes from the first dimension were subsequently separated by SDS
PAGE in the second dimension. A comparison between the protein patterns from Col wild type and
from the rpf5-1 mutant did not reveal any substantial differences in the steady state abundance of
any of the proteins. This result is consistent with the immunodetection analysis of the nad6 and nad9
polypeptides (Fig. 5). Positions of different respiratory chain complexes are indicated above the
images.
Supplemental Figure S9. Alignment of Arabidopsis sequences conserved at nad6, 26S rRNA and atp9
processing sites with corresponding sequences from other species. Nucleotide identities that differ
from the nad6 sequence from Arabidopsis are given in red. Sequences were identified by blastN
search with the -52 to +9 sequences from the nad6, 26S rRNA and atp9 genes from Arabidopsis.
Sequences are obtained from the following data bank entries: AtC24: Arabidopsis thaliana accession
C24 (NC_001284), Bj: Brassica juncea (JF920288.1), Bc: Brassica carinata (JF920287.1), Br: Brassica
rapa (JF920285.1), Bn (pol): Brassica napus polima (FR715249.1), Bn: Brassica napus wt
(AP006444.1), Bo: Brassica oleracea (JF920286.1), Ll: Lupinus luteus (AF279439.2), Cl: Citrulus lanatus
(water melon, GQ856147.1), Bv: Beta vulgaris (BA000009.3), Vr: Vigna radiata (HM367685.1), Sl:
Silene latifolia (HM562727.1), Cp: Cucurbita pepo (GQ856148.1), Cpa: Carica papaya (EU431224.1),
Nt: Nicotiana tabacum (BA000042.1), Ob (rrn26): X02559, Ob (atp9): X15765. Numbering is given
with respect to the 5’ terminal nucleotide (underlined) of the nad6 mRNA from Arabidopsis.
Nucleotide identities differing from the Arabidopsis nad6 sequence are given in red. The putative
binding sites for RPF5 are boxed (see also Fig. 7).