Electronic supplementary material
Supplementary Table S1: Transcriptome dataset including signal log ratio values, cluster and Gene
Ontology analysis for treatment and genotype comparisons.
Supplementary Figure Legends
Supplementary fig. 1. Plasmid map for amino terminal-epitope tagged MPK3, MPK4 and MPK6.
The vector p35S:HF-MPK was used for ectopic overexpression of amino terminal-eptiope tagged MPK3,
MPK4 or MPK6 in Arabidopsis Col-0. The EcoRI-HindIII cassette was ligated with the backbone of the
binary T-DNA vector pPZP111, which contains the chloramphenicol resistance gene for bacterial
selection. Restriction sites are shown. Values in nucleotides (nt) of each segment of the vector are given.
Arrows indicate directions of transcription. RB and LB, right border and left border of the T-DNA.
NPTII, A. tumefaciens neomycin phosphotransferase gene that confers kanamycin resistance in plants.
35S, Cauliflower mosaic virus 35S constitutive promoter. , modified tobacco mosaic virus omega leader
used as a translational enhancer. HF, His6-FLAG at the amino terminus of MPK3, 4 or 6 open reading
frame (ORF) with 1113 nt, 1131 nt, or 1188 nt, respectively. OCS, 3’-terminator region of the A.
tumefaciens octopine synthase gene. The vectors were expressed in Col-0 plants through Agrobacteriummediated transformation.
Supplementary fig. 2. Evaluation of HF tagged MPK protein in Col-0, MPK OE and MPK KO
seedlings and HF-MPK protein levels of the oxygen deprivation time course.
Soluble proteins were extracted from 10-d-old Col-0, MPK OE and MPK KO seedlings, grown vertically
on solid 0.5x MS media (1% (w/v) sucrose). Protein was separated by 12% (w/v) SDS-PAGE, transferred
onto a nitrocellulose membrane, immunodetected with anti-FLAG-horseradish-peroxidase-conjugated
monoclonal antibody (-FLAG, 1:500), and visualized with an enhanced chemiluminescence system and
exposure to X-ray film. (A) Levels of HF-MPKs in seedlings deprived of oxygen for up to 8 h (15 µg
protein). Arrows indicate epitope-tagged MPKs. Asterisks indicate two non-specific cross-reacting
proteins observed in all genotypes. Values on the right are molecular mass markers for proteins in kDa.
(B) Levels of immunopurified HF-MPKs in seedlings. Samples in panels A and B are those used for Fig.
2 panels B and C, respectively.
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Supplementary fig. 3. MPK activation upon reoxygenation at two light levels. Ten-d-old Ler
seedlings subjected to oxygen deprivation under 4.0 (light) or 0.15 (dim light) µmol photons s-1 m-2 light
for up to 8 h. At each time point, whole seedlings were either harvested or re-oxygenated in air for up to
15 min. Mock-treated samples (M) were harvested at the 2, 4 or 8 h time point from Petri dishes
incubated under identical light and humidity conditions but without oxygen deprivation. MPK activity
was assayed in 15 µg of soluble protein from crude cell extracts of whole seedlings by the in gel kinase
assay described in Fig. 1. Exposure times for individual panels were similar. Arrows indicate MPK6 and
MPK3. Data are representative of at least two biological replicates.
Supplementary fig. 4. Pre-treatment with antimycin A or KCN but not rotenone or malonic acid
reduces MPK activation by oxygen deprivation.
Ten-d-old Ler seedlings, grown vertically on solid 0.5x MS media (1% (w/v) sucrose), were subjected to
15 min of chemical pre-treatment followed by oxygen deprivation for 2 h. The mock treatment (M) for
each compound was the concentration of solvent in the solution with the highest concentration of the
chemical. MPK activity was assayed in 15 µg of soluble protein from a whole seedling crude cell extract
as described in Fig. 1. Data are representative of three biological replicates with varying ranges of
chemical concentrations. Exposure times for individual panels were the same.
Supplementary fig. 5. Complete-linkage hierarchical clustering of expression datasets to determine
similarity. The individual hybridizations datasets (unlogged RMA-normalized expression values) were
compared using complete-linkage hierarchical clustering and Spearman correlation as a distance metric.
The individual samples clustered into two major groups, corresponding to the two organs samples.
Samples were further clustered by environmental condition: hypoxia versus mock control, with biological
replicates in close proximity to one another. Sample labels are genotype (O, MPK6_1 OE; KO, mpk6; C,
Col-0), treatment (M, mock control; H, hypoxia) and organ (R, root; S, shoot) followed by the
bioreplicate number (1-3).
Supplementary fig. 6.
Anaerobic response genes are similarly regulated in three genotypes. (A) Heat map analysis of the 49
core anaerobic response genes upregulated in polysomal mRNA across seedling root and shoot cell types
(Mustroph et al., 2009). (B) Heat map analysis of the 200 Arabidopsis genes that are most highly induced
across publically available experiments (Mustroph et al., 2010). Genes were manually grouped by
function, including designation of three categories of hypoxia-induced genes that encode Proteins of
2
Unknown Function (PUF). Expression values and additional gene information are in Supplementary
Table S1d.
Supplementary fig. 7. Cluster analysis to identify genes co-regulated in response to low oxygen
stress. Complete hierarchical clustering analysis on the normalized unlogged RMA expression values for
the 1695 probe sets that were differentially expressed in one or more treatment comparison
(hypoxia/control) for the three genotypes using the data from Supplemental Table 1d. The genes of each
of the ten clusters are plotted as a grey line for each of the treatment comparisons. The red line indicates
the average Signal log2 ratio (SLR) value for the cluster. Mean SLR values for each comparison are
shown in Fig. 7B.
Supplementary fig. 8. Cluster analysis to identify genes co-regulated in three genotypes under
mock or oxygen deprivation conditions. Complete hierarchical clustering analysis on the normalized
unlogged RMA expression values for the 706 probe sets that were differentially expressed in one or more
comparison based on genotype using the data from Supplemental Table 1b. The genotype comparisons
were mpk6/Col-0; MPK6_1/Col-0 ad MPK6_1/mpk6. The genes of each of the eight clusters are plotted as
a grey line for each of the genotype comparisons as shown for the first cluster diagram. The red line
indicates the average SLR value for the cluster. Mean SLR values for each comparison are shown in Fig.
7C.
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