Supplementary Figure S1: Phylogenetic tree of COX10 proteins from different organisms. Protein sequences corresponding to plant homologues of Cox10p from ...
Supplementary Table S1: Known cis regulatory motifs presents in the AtCOX10 promoter region.
Sequence
Number of copies
Function
Reference
-174/-117
TGGGCC/T
2
Present in genes expressed in meristematic regions. Involved in the coordination of the expression of nuclear genes encoding mitochondrial proteins
Welchen and Gonzalez 2005; 2006.
Lat52
-126
AGAAA
1
Expression in pollen in transgenic tomato, tobacco and Arabidopsis.
Twell et al., 1991. Muschietti etl al., 1994
Lat52/56
-413/-152
TGTGGTT
2
Pollen-specific element
Eyal et al., 1995.
Prolamin-box
-232/-144
TGTAAAG
2
Highly conserved element found in promoters of many cereal seed storage protein genes
Vicente-Carbajosa et al., 1997. Mena et al., 1998
GCN4
-5
GAGTCA
1
Essential element determining endosperm-specific expression
Wu et al., 2000
Element
Location in AtCOX10
Site II
Supplementary Table S2: List of oligonucleotides used in this paper
Primer Name
Sequence (5´-> 3´)
pDAP-RB3
TAACAATTTCACACAGGAAACAGCTATAC
cox10F-Sail
CGCTCGTTGTTATTGGGAGCTTTCTAA
cox10R-Sail
CAAGGAATTAGCAGATGCAGCAAT
COX10P-FHind
GGGAAGCTTCAATGATGAACATCTTCTTC
COX10P-RBam
CCCGGATCCGTTAAGCCGTCAGAATTTT
Description
Analysis of T-DNA insertion mutant SAIL_1283_D03.V1 (AtCOX10)
Amplification of the AtCOX10 promoter to obtain a GUS fusion
COX10c-FBam
CTTGGATCCCGATGTGGCGAAGATCTG
Amplification of the AtCOX10 coding sequence for complementation in plants and yeast and RFP intracellular localization.
COX10-RSac
GGTGAGCTCCCAAAAATTCAATAGCTTG
Amplification of the AtCOX10 coding sequence for complementation in plants
COX10R-RFP
GCGCTCGAG GGAGAGTAGAAGGAAG
Amplification of the AtCOX10 coding sequence for RFP intracellular localization.
AHL1
TAGTTAGTTACTTAAGCTCGGGC
AHL2
CAGAGCTGCAGCTGGATGGC
35S-1b
GCTCCTACAAATGCCATCATTGC
RFP R
CCTTGGTCACCTTCAGCTTG
AtRFP B1F
GGGGACAAGTTTGTACAAAAAAGCAGGCTA CCATGGCCTCCTCCGAGGAC
AtRFP B2R
GGGGACCACTTTGTACAAGAAAGCTGGGTT TAGGCGCCGGTGGAGTG
10aqrhind
GCGAAGCTTTCATGGAGAGTAGAAGGAA
Addition of recombination sites for cloning using the Gateway technology
The underlined sequences are mRFPspecific.
Amplification of the AtCOX10 coding sequence for complementation in yeast.
RT-qPCR oligonucleotides Gene
Locus
Forward (5' to 3')
Reverse (5' to 3')
PP2AA3
At1g13320
CCTGCGGTAATAACTGCATCT
CTTCACTTAGCTCCACCAAGCA
COX10
At2g44520
ACATGATCCCTCTCGGTTTCATCGCCT
TTGCACATCTCTGCCGCAAT
SAG12
At5g45890
AAGCGACTGGCGGCTTGACA
ACCGGTTGGTGTGCCACTGC
CAB1
AT1G29930
TGGGCCTCGCTACTGACCCC
GCCAAGTGGTCCGCGAGGTT
CHS
AT5G13930
ACATGTCGAGCGCGTGCGTT
AGGAACGCTGTGCAAGACGACT
Supplementary Figure Legends: Supplementary Figures
Supplementary Figure S1: Phylogenetic tree of COX10 proteins from different organisms. Protein sequences corresponding to plant homologues of Cox10p from S. cerevisiae were downloaded from Phytozome 9.1 (http://www.phytozome.net/) and sequences from other organisms were searched using the standard protein blast (BlastP®) from NCBI. Sequence alignment was made using default parameters established in the WebPRANK alignment server (Löytynoja and Goldman, 2012). Maximum likelihood (ML) tree reconstruction with the best model and 1000 bootstrap pseudoreplicates were run using the software Seaview 4.5.0 and the algorithm PhyML-aLRT(SHLIKE) with 100 random starts. The tree was represented with the FigTree v1.4.0 software (Gouy et al., 2010). The AtCOX10 protein is highlighted with a grey arrow. The presence of two gene copies encoding COX10 in Zea mays, Glycine max, Brassica rapa and Panicum virgatum is marked with stars.
Supplementary Figure S2: Characterization of an AtCOX10 T-DNA insertion mutant (A) Proportion of AtCOX10/atcox10 (grey bar) and wild-type plants (black bar) in the progeny of AtCOX10/atcox10 plants through three consecutive generations (F1 to F3), as deduced from PCR analysis of genomic DNA. No homozygous atcox10/atcox10 plant could be identified. (B) Proportion of normal and abnormal seeds in AtCOX10/atcox10 and wild-type plants. (C) (AtCOX10/atcox10) mutant plants were complemented with a construct that expresses AtCOX10 under the control of its own promoter (pCOX10:AtCOX10). Siliques corresponding to an AtCOX10/atcox10 plant and two homozygous (atcox10/atcox10) complemented lines are shown. (D) PCR analysis of genomic DNA from an atcox10/atcox10 complemented line showing the presence of the T-DNA inserted in the endogenous AtCOX10 gene and the absence of the wild-type allele. Primers Cox10FS and Cox10RS, which detect the presence of a wild-type copy of AtCOX10, were used in lanes 1, 3 and 5, while pDAP-RB3 and Cox10RS, which detect the presence of the T-DNA insertion, were used in lanes 2, 4 and 6 (see Figure 4A for details). Bands of 166 pb, 250 pb and 350 pb correspond to the presence in the genome of the AtCOX10 cDNA, the AtCOX10 wild-type gene or the mutant allele, respectively
Supplementary Figure S3: Complementation of the atcox10 mutation restores CcO activity levels in plants. CcO activity staining of mitochondrial protein extracts from wild-type, heterozygous mutant (AtCOX10/atcox10) and complemented (atcox10/atcox10, pCOX10/AtCOX10) plants after separation through BN-PAGE. Mitochondria-enriched protein extracts corresponding to equal
amounts of citrate synthase activity, used as a standard of the amount of mitochondria, were loaded in each lane. MWM: Molecular Weight Marker. The brown precipitate, due to DAB oxidation, corresponds to the activity of CcO.
Supplementary Figure S4: Phenotypic parameters in wild-type, AtCOX10/atcox10 and complemented plants under normal growth conditions. Rosette diameter (A), transition to the reproductive phase (B) and inflorescence stem height (C) were measured in wild-type, AtCOX10/atcox10 and complemented plants.
Supplementary Figure S5: Characterization of senescence progression in AtCOX10/atcox10 plants. (A) Senescence progression was analyzed in wild-type (black bars), heterozygous (light-grey bars) and complemented homozygous mutant plants (dark-grey bars) by measuring Fv/Fm in all rosette leaves at days 33, 40, 45 and 54 after sowing. (B) Total, CcO-dependent and alternative (AOX) respiration rates were measured in rosette leaves 5 and 6 weeks after sowing. Asterisks indicate significant differences (P < 0.05).
Supplementary Figure S6: Dark-induced senescence in wild-type and AtCOX10/atcox10 plants. (A) Phenotype of detached 4-week-old rosette leaves incubated in darkness for 2 days. (B) Total chlorophyll content of leaves measured after 2, 4 and 7 days of dark treatment.
Supplementary Figures