Figura 1- Complementacion de levaduras

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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