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Figure S1. Overview of evaluation of gene expression and metabolic responses to low oxygen stress and reoxygenation treatments. Flow diagram of experimental strategy.

Figure S2. Regulation of mRNA association with polysomes in response to low oxygen stress and reoxygenation treatments. Venn diagram of genes with significant changes (FDR cut off = 0.001) in immunopurified mRNA levels, (a) Increase in response to 2 h (2HS) or 9 h (9HS) and decrease after 1 h of reoxygenation (1R) and (b) Decrease in response to HS and increase after 1 h of reoxygenation. mRNAs detected with a Present call in all total mRNA samples and showing call changes in the IP fraction (as predicted by mas 5.0) as a result of 2HS and 9HS are represented in panels (c) All Present to All Absent calls and (d) All Absent to All Present call.

Figure S3. Multivariate statistical evaluation of 1H NMR spectra of metabolites from control and low oxygen stressed seedlings. In the PCA plots samples are represented with individual symbols with the treatments color-coded as follows, 2NS(&#UF075;), 9NS (&#UF0A2;), 2HS (&#UF0C5;), 9HS (&#UF0B3;), and 1R (&#UF072;). (a) PCA scores plot for the first two principal components. (b) Loadings for the scores plot in (a). (c) PCA scores plot using only the 12 most significant integral regions (variables) determined from the loadings plot in (b). The PCA model was cross-validated by alternately including and omitting the 12 most significant spectral regions (Figure S3c, d). In (c) the sample groupings observed in (a) are retained, although they appear in a different order. (d) PCA scores plot for all of the integral regions except for the 12 most significant regions in (b). (e) Explained variance plot. Principal component (PC) 1 and 2 explain more than 67% of the variance in the data, whereas PC3 primarily describes the biological variance in sugars.

Figure S4. Metabolic reconfiguration under low oxygen stress. Integrated metabolic map of pathways affected by short and prolonged HS, as predicted by profiling metabolites. Predicted change in enzyme abundance/activity is based on changes in steady-state and polysomal mRNA. Metabolites showing increased or decreased levels in response to HS are represented in bold green and narrow red, respectively. Increases in transcript abundance for the depicted enzymes, based on expression of one or more gene family member. Glycolytic enzymes correspond to cytosolic forms. An enzyme indicated in bold with a box outlined with a dashed-gray line indicates a significant increase in steady-state mRNA level; boxes outlined in blue indicate a significant increase in both steady-state and polysomal mRNA. A significant decrease in transcript abundance is depicted with an orange boxes and purple font. Blue arrows indicate favored reaction or, when in bold, induced process. Abbreviations Starch Pase, starch phosphorylase; HXK, hexokinase; PGM, phosphoglucomutase; PGI, phosphoglucoisomerase; PEPC, phosphenolpyruvate carboxylase; PCK, phosphenolpyruvate carboxylase kinase; PDH, pyruvate dehydrogenase; PPDK, pyruvate Pi dikinase, PDK, pyruvate dehydrogenase kinase; ICDH, isocitrate dehydrogenase; 2-OGDH, 2-oxyglutarate dehydrogenase; SDH, succinate dehydrogenase; MDH, malate dehydrogenase; CS, citrate synthase; GS, glutamine synthase; GOGAT, NADPH-dependent glutamine: 2-oxoglutarate aminotransferase; GDH, glutamate dehydrogenase; GDC, glutamate decarboxylase; GABA-T, GABA transaminase; GHBDH, γ-aminobutyrase dehydrogenase; SSADH, succinate semialdehyde dehydrogenase; CoASH, coenzyme A; SUS, sucrose synthase; UGPPase, UDP-glucose pyrophosphorylase; NDP kinase, nucleoside diphosphate kinase; FK, fructokinase; PFK, ATP-dependent phosphofructokinase; PFP, PPi-dependent phosphofructokinase; PK, pyruvate kinase; ALDH, acetaldehyde dehydrogenase; PDC, pyruvate decarboxylase; LDH, lactate dehydrogenase; AlaAT, alanine aminotransferase; AspAT, aspartate aminotransferase; ADH, alcohol dehydrogenase; NR, nitrate reductase; NiR, nitrite reductase; FedrRED, reduced ferridoxin; FedOX, oxidized ferridoxin.

Figure S5. Dynamics in mRNA accumulation and translation controls proteins involved in cellular processes. (a) Heat map display of 109 mRNAs encoding proteins involved in light reactions and the Calvin cycle. (b) Heat map display of 143 mRNAs encoding enzymes involved in cell wall biogenesis, modification and degradation. (c) Heat map display of 769 mRNAs encoding proteins involved in gene transcription. (d) Heat map display of 382 mRNAs encoding proteins involved in signal transduction. In (a) to (d), genes organized by process and then sorted by signal log2 ratio (SLR) for IP mRNA at 2HS; columns represent mean SLR comparisons of total and immunopurified polysomal (IP) mRNA levels of transcripts of selected genes (all detected with Present call in all 30 hybridizations); and colors indicated increase (yellow), decrease (blue) or no-change (black) in mRNA abundance. Data used are compiled in Table S6.

Appendix S1. Supplementary experimental procedures.

Table S1. Evaluation of reproducibility between biological replicates. Total and Polysomal mRNA samples extracted from Arabidopsis 7-day-old seedlings were prepared from three independent biological replicates and hybridized to Affymetrix ATH1 chips. Reproducibility between replicates was evaluated by Pearson correlation of microarray data obtained and is expressed in R2 values.

Table S2. (a) Differential expression data obtained for all genes on the ATH1 GeneChip.

Expression data obtained in CEL files was extracted by use of the Bioconductor package of the R statistical software and normalized using Robust Multichip Average (RMA). The signal data obtained from the RMA analyses of the immunopurified polysomal RNA was further adjusted to account for variation in global polysome level under the different treatment conditions (normalization factors used for polysomal RNA samples were 2NS, 0.7485; 9NS, 0.7463; 2HS, 0.4253; 9HS, 0.3753; 1R, 0.6723). Data presented are the log2 signal values obtained for each probe-pair from each replicate hybridization onto Affymetrix ATH1 chips. The area shaded in gray includes comparisons between treatments expressed as signal log2 ratio (SLR) and FDR cut-offs. The area shaded in blue provides the mas 5.0 detection calls for each gene on each chip. 2NS, 2 h non-stress; 2HS, 2 h hypoxia-stress; 9NS, 9 h non-stress; 9HS, 9 h hypoxia stress; 1R, 1 h reoxygenation after 9 h hypoxia-stress; T, Total mRNA; IP, Immunopurified polysomal mRNA. (b) Detection call of all genes on the ATH1 GeneChip. Expression data obtained in CEL files was extracted by use of the Bioconductor package of the R statistical software and normalized using Robust Multichip Average (RMA). The signal data obtained from the RMA analyses of the immunopurified polysomal RNA was further adjusted to account for variation in global polysome level under the different treatment conditions (normalization factors used for polysomal RNA samples were 2NS, 0.7485; 9NS, 0.7463; 2HS, 0.4253; 9HS, 0.3753; 1R, 0.6723). Data presented are the log2 Signal values obtained for each probe-pair from each replicate hybridization onto Affymetrix ATH1 chips. The area shaded in gray includes comparisons between treatments expressed as signal log2 ratio (SLR) and FDR cut-offs. The area shaded in blue provides the mas 5.0 detection calls for each gene on each chip. 2NS, 2 h non-stress; 2HS, 2 h hypoxia-stress; 9NS, 9 h non-stress; 9HS, 9 h hypoxia stress; 1R, 1 h reoxygenation after 9 h hypoxia-stress; T, Total mRNA; IP, Immunopurified polysomal mRNA. (c) Signal obtained for all genes on the ATH1 GeneChip. Expression data obtained in CEL files was extracted by use of the Bioconductor package of the r statistical software and normalized using Robust Multichip Average (RMA). The signal data obtained from the RMA analyses of the immunopurified polysomal RNA was further adjusted to account for variation in global polysome level under the different treatment conditions (normalization factors used for polysomal RNA samples were 2NS, 0.7485; 9NS, 0.7463; 2HS, 0.4253; 9HS, 0.3753; 1R, 0.6723). Data presented are the log2 Signal values obtained for each probe-pair from each replicate hybridization onto Affymetrix ATH1 chips. The area shaded in gray includes comparisons between treatments expressed as signal log2 ratio (SLR) and FDR cut-offs. The area shaded in blue provides the mas 5.0 detection calls for each gene on each chip. 2NS, 2 h non-stress; 2HS, 2 h hypoxia-stress; 9NS, 9 h non-stress; 9HS, 9 h hypoxia stress; 1R, 1 h reoxygenation after 9 h hypoxia-stress; T, Total mRNA; IP, Immunopurified polysomal mRNA.

Table S3. Relative porportion of mRNA present in each group of genes when compared to total RNA detected for the whole data set (Present Calls). Relative proportion of mRNA present in each cluster when compared to total RNA detected for the whole data set (Present Calls): An estimate of the proportion of cellular mRNA represented in each cluster was obtained by calculating the sum of signal data, a measure of the amount of RNA detected, as compared to the sum of the signal data of the whole set of genes (n = 8863 genes). Values are percent mRNA of Total mRNA in the data set. RPs, ribosomal  proteins (= 174 probe pairs).

Table S4. Functional categorization of genes with increased association with polysomes in response to HS. Transcripts detected as Present in all 30 chips and showing significant increase in IP RNA levels (FDR cutoff = 0.001) exclusively after 2 h or after 9 h HS were compared and evaluated for the predicted biological process for the encoded gene product. This analysis was performed using the TAIR gene ontology retrieval tool http://www.arabidopsis.org/tools/bulk/index.jsp.

Table S5 (b) Annotation and functional category of gene cluster members, as designated by the MapMan software. Some loci are annotated in more than one functional category. A statisitical analysis of representation of categories for each cluster is presented in the sheets labelled Cluster 1 to 10.

Table S6. Dynamics in mRNA accumulation and translation of mRNAs encoding proteins with related function or processes. Data in this table are SLR data used for the heat map displays in Figure 4b, A2.

Table S7. Quantification of metabolites measured by coupled enzyme spectrophotometricand assay. Pulverized seedling tissue was extracted in perchloric acid, neutralized and an aliquot used to assay specific metabolites in the presence of the metabolite specific converting enzyme and co-factor (NAD+ or NADH) and reactions were driven to completion. Values presented are absolute concentrations (average of 3 or more bioreplicates) and standard deviation for the quatifiable metabolites. This data was used to calculate and evaluate statistical significance of differences between treatments (fold change). Lactate values for 2NS and 9NS samples may be underestimates because of low content in non-stressed samples.

Appendix S1. Supplemental experimental procedures.

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