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Supplementary Information (Nature02516)
1. Supplementary Data
2. Supplementary Methods
3. Supplementary Note
4. Supplementary Figure Legend
1. Supplementary Data
To investigate if ETO1 is in the same genetic pathway as ETO2, we generated a double
mutant by crossing eto1-4 to a loss-of-function allele of ACS5, eto2-2 (originally called
cin5-1). We found that the Ctr- phenotype of eto1-4 etiolated seedlings is partially
suppressed in the eto1-4 eto2-2 double mutant, suggesting that a functional ACS5 is
required for eto1 phenotype. The implication of the genetic interaction between ETO1
and ACS5 supports the notion that ETO1 negatively regulates the activity of ACS5 in
etiolated seedlings and ETO1 may also regulate other ACS isozymes because of the
partial genetic suppression.
2. Supplementary Methods
Map-based cloning of ETO1
ETO1 was genetically mapped to the bottom of chromosome 3 using visible markers.
Using SSLP and CAPS markers, we fine-mapped ETO1 to a 58kb region including the
AtEm1 locus (GenBank accession no. AF049236). We then searched for possible deletion
by PCR in the 15 predicted ORFs (open reading frame) in this region, and found a 50
base-pair deletion affecting the second ORF (At3g51770) in two X-ray generated alleles,
eto1-2 and eto1-3. Further sequencing and PCR analysis of 7 other eto1 alleles enabled us
to identify single nucleotide changes, deletions, or genomic rearrangements, which would
result in nonsense or missense mutations of ETO1 in each. DNA sequences of mutant
alleles of the ETO1 gene were determined directly by genomic sequencing.
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Growth conditions of Arabidopsis suspension-cultured cells and transient expression
The Arabidopsis suspension-cultured cells were maintained at 24oC in the dark with
constant shaking (110 rpm). Growth medium (pH 5.7) contains Murashige and Skoog
basal salts, Gamborg’s B5 vitamins (Sigma), 3% sucrose, and 1mg/L 2,4dichlorophenoxyacetic acid (2,4-D). The cells were sub-cultured weekly by transferring
15 ml of cells to 35 ml fresh medium. Transient expression of ETO1 and ACS5 fusion
proteins were performed by inoculating 10-20 OD600 of Agrobacterial cells containing
desired expression constructs to 50 ml of suspension-cultured cells in the presence of 50
mg/L of acetosyringone (Sigma). Cells were collected and protein extracts were prepared
2-3 days after transformation.
Plasmid construction
Fusion constructs of ETO1, EOL1, EOL2, and ACS5 for overexpression plant cells and
for yeast two-hybrid system were generated as the follows. The binary vectors, pCHF1GFP or pCHF3-ProteinA with the fusion tag at the C-terminus (gifts from Y. Yin) were
used to construct pCHF1-HA-ACS5-GFP (designated as HA-ACS5 for simplicity in the
text and figures) and pCHF3-RGS-ETO1-ProteinA (designated as RGS-ETO1),
respectively. The 6xHis tag (derived from RGS tag in pQE, Qiagen) and HA tag were
engineered to the N-terminus of ETO1 and ACS5, respectively, by PCR amplification.
RT-PCR was used to clone the coding sequence of EOL1, EOL2, and ACS5 into twohybrid vectors (pACT2 for ACS5 and pAS2 for EOL1 and EOL2). The coding sequence
of ETO1 was amplified from the full-length clone by PCR and subcloned to pAS2. The
expression of the above hybrid clones in yeast (Y190) was verified by immunoblot
analysis. Details of plasmid constructions are available upon request.
3. Supplementary Note
Origins and accession numbers of each protein used in Figure 1d are listed below. ETO1,
EOL1, EOL2: Arabidopsis (this study), OsEOL1, OsEOL2, OsEOL3: O. sativa
(AC137072, AP003826, AC104847, respectively), mel-26: C. elegans (AAC63596),
kelch: D. melanogaster (A45773), PLZF: H. sapiens (AAD03619).
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4. Supplementary Figure Legend
Supplementary Figure 1.
Alignment of ETO1 and EOL amino acid sequences.
BTB (dashed line), TPR (solid line) and CC (dash-dotted line) motifs are indicated. All
the alignments were generated using CLUSTALW program. Identical and similar amino
acids are reversed and shaded, respectively.
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