SUPPLEMENTAL MATERIAL
Supplemental Figure S1. Determination of the 5’ end of the ccmC transcript in Sorbo.
The sequence analysis of the low abundant PCR product derived from CR-RT-PCR analysis of
the ccmC transcripts in Sorbo. An increase of background peaks in the sequence diagram
indicates a 5’ end at position -392 (marked by a dotted line) identical to what is seen in C24.
Supplemental Figure S2: Complementation tests by crossing Col genotype accessions.
ccmC transcripts were analyzed by CRRT-PCR with primer pair Atccb3-Mega5‘nah/Atccb3-3.
Product a (arrow) with a size of 470 bp indicates a ccmC mRNA with a mature 5’ end
around position -484. Accessions are indicated above the corresponding lanes. Each two
different F1 plants are analyzed ( #1 and #2).
Supplemental Figure S3: Alignment of RPF3 amino acid sequences from different Arabidopsis
accessions and RPF6 from Col. Dotted black lines indicate the beginning and end of the repeats
(Numbered R1 to R15), which are consecutively numbered. The 1st, the 3rd and the 6th amino acid
positions are marked in yellow, grey and green, respectively.
Supplemental Figure S4: The RPF3 allele from C24 (RPF3(C24)) supports processing
of the ccmC transcripts derived from the Col ccmC genotype. The RPF3 allele (At1g62930)
from C24 was stably introduced in to rpf3-1 plants. The analysis of transformants both by
CRRT-PCR (A) and northern blot analyses (B) confirmed the function of the allele in
processing of ccmC transcripts transcribed from the Col ccmC genotype. Loading of the
RNA gel was examined by methylene blue staining, here shown for the cytoplasmic 26S
rRNA.
Supplemental Figure S5: Semi qRT-PCR analysis of RPF3 transcripts from different
accessions with defective ccmC processing. A 600 bp cDNA product is amplified with
primers At1g62930.D and At1g62930.I. In comparison to Col, the amount of amplification
product indicates a slight reduction of the RPF3 mRNA only in Lip-0. No product is
amplified in the rpf3-1 knockout mutant.
Supplemental Figure S6: Mapping of RPF6. (A) The rough mapping indicates that the
RPF6 locus is encoded on the lower arm of chromosome 1. At1g62670 (RPF2) can be
used as an insertion/deletion marker as found in a previous analysis (Jonietz et al. 2010). (B)
Fine mapping identified the RPF6 gene within a 1 Mbp region. This region contains a
gene cluster encoding several PPR proteins (given by their AGI code) including RPF2 and
RPF3. Arabidopsis chromosomes (chr1-5) are given as black and grey bars. Positions and
designations of markers used for mapping are indicated. The percentage of the No-0 alleles
found at these markers is indicated.
Supplemental Figure S7: (A) Alignment of the amino acid sequences (given in the single
letter code) deduced from the RPF6 alleles from Col and No-0. Letters given in red highlight
differences at positions indicated to be crucial for the interaction of the PPR motifs (R1 to
R15, confined by dashed lines) with distinct nucleotide identities (positions 1’, 3 and 6
indicated in blue). The sequences share 94% identical amino acids (*). (:) indicates amino
acids with strongly similar properties, whereas (.) indicates amino acids with weakly similar
properties. (B) Alignment of the amino acid sequences (given in the single letter code)
deduced from the RPF6 and RPF3 genes from Col. The proteins share a sequence identity of
86 %. Designations see Supplemental Fig. S7A.
Supplemental Figure S8: PCR analysis of the RPF6 allele in No-0. (A) Schema of the
RPF6 gene (At1g63130) and the upstream located gene (At1g63120). The approximate
locations of primers (horizontal arrows, listed in Table S4) are given. An insertion and a
deletion are indicated and their approximate sizes given. Schema not drawn to scale. (B)
Amplification reactions were performed with different primer pairs (given below the images)
covering different parts of the RPF6 gene as well as 5' and 3' flanking regions.
Supplemental Figure S9: Complementation tests by crossing different accessions with
the C24 ccmC genotype. The ccmC transcripts in F1 hybrids (#1 to #4) obtained from
reciprocal crossings (indicated above the images) were investigated by CRRT-PCR analysis. A
380 bp product (b) confirmed ccmC processing in the C24 control, while no such product
was amplified in one of the F1 plants demonstrating that all of the tested accessions contain
defective RPF6 alleles.
Supplemental Figure S10: Investigation of RPF6 alleles in different accession with
defective processing of ccmC transcripts. (A) PCR analysis of the RPF6 alleles.
Amplification reactions were done with primer pairs (P64)At1g63130.H/(P9)RPF3_6Chimär.R2 (upper panel) and (P6-8)At1g63130.C/(P611)At1g63130kompl.R1 (lower panel). Locations of the oligonucleotides are given in Fig. S8.
Products expected for Col are 1,200 bp and 2,000 bp, respectively. (B) Semi qRT-PCR of
RPF6 mRNAs. The amounts of 200 bp cDNA products obtained with primer pair (P66)At1g63130.A/(P6-7)At1g63130.B indicate reduced transcript levels in accessions No-0 and
Rube and elevated levels in Ms-0.
Supplemental Figure S11: CR-RT-PCR analysis of F1 hybrids obtained by crossing the
ccmC maturation defective Col-type with C24-type accessions. (A) ccmC Col-type
accessions Lip-0 and Ta with the C24–type accessions Ms-0, Kas-1 and Rube or (B) crossing
the C24–type accessions Ms-0 and Rube with Col-type accessions Lip-0 and Ta. Prominent
products of 470 bp (A) and 380 bp (B) indicate the generation of mature ccmC transcripts.
PCR was done with primers Atccb3-Mega5’nah and Atccb3-3.
Supplemental Fig. S12: RPF3/RPF6 composite proteins did not support ccmC mRNA
processing (A) Binding predictions of composite proteins RPF3N/6C combining the Nterminal part of RPF3 (grey) with the C-terminal part of RPF6 (black) and RPF6N/3C
combining the N-terminal part of RPF6 with the C-terminal part of RPF3. Binding is shown
for Col and C24 ccmC binding sites. (B) CR-RT-PCR analysis of No-0 and rpf3-1 plants (#1,
#2 or #4) transformed with different chimeric constructs (given above the image). None of
the chimeric construct restores ccmC processing. Control reactions are shown in the right.
Supplemental Table 1: Transcript phenotype and mitochondrial DNA genotype of the
ccmC gene in 51 Arabidopsis accessions. The ccmC phenotype was analyzed by a CR-RT-PCR
analysis. The ccmC genotype was analyzed by using a Kpn2I digestion of a PCR product
spanning the genotype discriminating region. Here all accessions showed either the C24 or
the Col specific pattern (see Fig.1B).
Supplemental Table S2: Genes analyzed in complementation tests in No-0. Several PPR
genes encoded in gene cluster on the lower arm of chromosome I are tested for their ability
to restore the ccmC 5‘ processing in No-0. The candidate genes including the 5’ and 3’
flanking regions where stably transformed into No-0. None of the here listed genes was able
to restore the ccmC 5’ processing in this accession.
Supplemental Table S3: Genomic markers.
Supplemental Table S4: Oligonucleotide sequences.
Supplemental Methods S1: Complementation tests.