Article Figures & Data
Figures
- Figure 1.
Glucose Has Profound Effects on Gene Expression Compared with Inorganic Nitrogen in 6-d-Old Arabidopsis Seedlings Predominantly Consisting of Shoot Tissue.
To remove inconsistent replicates, log10 normalized signal scores were subjected to RCBD analysis (P ≤ 0.001) before twofold filtering.
- Figure 2.
Regulation of Gene Expression Orchestrated by Glucose and Nitrogen.
Cluster analysis was conducted using GeneCluster2 (Golub et al., 1999) using the genes identified in Figure 1, except those showing significant regulation by 3-OMG were removed from consideration. A self-organizing map (SOM) was generated for genes showing greater than a twofold change with expression above background/noise levels. Blue lines represent the mean expression, and the area between red lines represents the range of values within the cluster. This SOM explained 95.1% of the variance occurring in the data set. Value associated with each cluster represents the number of genes with similar behavior.
- Figure 3.
Microarray Data Validation by RNA Gel Blot and RT-PCR Analyses.
Genes chosen for analysis include glucose downregulated genes (A), glucose upregulated genes (B), a gene upregulated specifically by glucose and nitrogen (C), nitrate upregulated genes (D), and two unregulated genes (E).
- Figure 4.
Glucose Regulates Genes with Diverse Functions.
Shown are genes responding to glucose with at least threefold change after normalizing data and conducting RCBD analysis at P ≤ 0.001. Putative functions were determined using spot annotations (The Arabidopsis Information Resource; http://arabidopsis.org), gene ontology searches (http://www.geneontology.org), pathway analyses, and literature review.
- Figure 5.
Glucose Induction Often Requires de Novo Protein Synthesis.
Frequency of glucose induction versus glucose repression in the presence of CHX. Expression patterns with and without CHX were determined for the genes identified in Figure 2 using SOM software.
- Figure 6.
Expression Patterns of Carbohydrate-Related Genes Identified in Figure 4 with or without CHX.
Hierarchical average linkage clustering with correlation measure–based distance (uncentered) was used for the analysis. Red or green represents upregulation or downregulation, respectively, and gray represents either genes at background/noise levels or changes below the fold-change cutoff.
- Figure 7.
Transcription Factors Are Differentially Regulated by Glucose.
(A) Number of all genes versus transcription factors upregulated or downregulated by glucose with a twofold or threefold cutoff.
(B) Distribution comparison of glucose-regulated transcription factors with all transcription factors in the Arabidopsis genome (Jiao et al., 2003). Percentage of glucose responsive TFs is derived from the number of each category versus total number of glucose responsive TFs.
- Figure 8.
Glucose Affects Expression of Ethylene and Stress Associated Genes.
Shown are hierarchical average linkage clustering analyses. Red or green represents upregulation or downregulation, respectively, and gray represents either genes at background/noise levels or no changes with specified cutoff.
(A) Nutrient response of genes implicated in sugar signaling based on genetic studies (León and Sheen, 2003; Gibson, 2004). ABI4 is not included because expression levels were near background/noise levels. None of these genes showed a more than twofold change in the presence of CHX; however, CTR1, EIN3, and EIL1 were repressed by glucose more than 1.5-fold in the presence of CHX (Table 2).
(B) Nutrient response of hormone biosynthetic genes. Filtering criteria were relaxed to twofold for CHX-treated plants.
(C) Numerous heat shock proteins are affected by glucose.
(D) Other stress-associated genes are highly glucose-responsive.
- Figure 9.
Nutrient Response of Various Transporters.
Shown are hierarchical average linkage clustering analyses. Red or green represents upregulation or downregulation, respectively, and gray represents either genes at background/noise levels or no changes with specified cutoff.
- Figure 10.
Multiple Sugar Signaling Pathways Revealed by the Regulation of Sugar Transporters.
Glucose has profound effects on the expression of monosaccharide transporters. By contrast, only one disaccharide transporter is affected by glucose, consistent with the idea that disaccharide transporters are uniquely regulated by disaccharides (Choiu and Bush, 1998).
- Figure 11.
Genes Associated with Nitrogen Metabolism Are Predominantly Regulated by Glucose.
The selected genes were normalized and subjected to RCBD analysis (P ≤ 0.001) and showed a more than twofold transcriptional change.
- Figure 12.
A Proposed Model Summarizes the Metabolic and Signaling Roles of Glucose.
Arrows pointing upward are induction and those pointing downward are repression.
Tables
- Table 1.
A Comparison of Nitrate-Regulated Gene Expression between Wang et al. (2003) and This Study
Wang et al. (2003) Fold-Change Ratios Probe Set ID Gene Description Nitrate/Control Ratio Glc/Control N/Control Glc and N/Control 260623_at Nitrate transporter (NRT2.1) 19.6a NC NC NC 259681_at Nitrate reductase (NIA1) 3.2 1.1 19.5 19.6 261979_at Nitrate reductase (NIA2) 2.4 −3.0 1.8 1.3 265475_at Nitrite reductase (NiR) 24.3 8.0 7.3 30.0 249325_at Urophorphyrin III methylase 13.5 2.3 2.6 14.2 255230_at Ferredoxin NADP reductase 4.2 5.3 1.5 21.9 261806_at Ferredoxin NADP reductase 4.8 1.9 −1.1 8.8 265649_at Putative ferredoxin 2.8 1.9 1.3 5.0 264859_at Glucose-6-phosphate 1-dehydrogenase 36.3 4.0 1.1 62.0 245977_at Glucose-6-phosphate 1-dehydrogenase 5.1 1.6 1.1 7.6 249266_at 6-Phosphogluconate dehydrogenase 5.2 3.5 −1.0 12.3 262323_at 6-Phosphogluconate dehydrogenase 2.6 1.4 −1.0 3.0 248267_at Glu synthase (GOGAT NADH) 1.6 1.8 2.3 4.6 247218_at Asn synthetase (ASN2) 2.0 2.6 9.7 30.6 262180_at Phosphoglycerate mutase 32.3 8.2 1.5 35.2 264246_at Trehalose-6-phosphate synthase NC −5.1 1.4 −3.4 263019_at Trehalose-6-phosphate synthase NC −19.7 1.4 −10.9 257217_at Phosphoenolpyruvate carboxylase (PPC) 2.1 1.6 −1.0 2.0 252407_at Chloroplast malate dehydrogenase 2.1 2.0 −1.0 3.6 Shoot data rather than root data were used (Wang et al., 2003) for comparison because shoot tissue was overrepresented in our whole plant samples collected for analysis. NC, no change.
↵a Expression signal near background levels.
- Table 2.
The Effects of Glucose on the Expression of Genes Associated with Ethylene Biosynthesis or Signal Transduction
Fold Change Spot ID Description CHX: − +a 250911_at CTR1 −2.5 −1.5 257981_at EIN3 −2.2 −1.8 266302_at EIL1 −2.5 −1.7 249125_at 2-Oxoglutarate-dependent dioxygenase, similar to tomato ethylene synthesis regulated protein E8 −3.2 −1.3 247774_at Oxidoreductase, similar to ACC oxidase −3.7 −1.9 253999_at ACC synthase, putative −3.8 −2.0 246843_at 2-Oxoglutarate-dependent dioxygenase, similar to tomato ethylene synthesis regulated protein E8 −4.3 −3.5 264346_at ACC oxidase, putative −3.4 −1.1 ↵a Fold-change values for glucose treatment with CHX are compared relative to the CHX control.
Supplemental Data
Files in this Data Supplement:
- Supplemental Figure 1A - Assessment of microarray reproducibility. (A) Scatter plot of control versus control. Spots with absent, marginal, or present detection signals are displayed in yellow, blue, or red, respectively. (B) Plot of log2 average versus log2 difference from two control normalized replicates, suggesting that scalar normalization is appropriate. Results from all other possible pairwise comparisons with normalized control data yielded plots with very similar appearance (not shown).
- Supplemental Figure 1B
- Supplemental Figure 2 and 3 - Supplemental Figure 2. Determination of false discovery rate (FDR) comparing treatment versus control for p <_ _0.001.="_0.001." the="the" fdr="fdr" was="was" calculated="calculated" using="using" software="software" q="q" storey="storey" and="and" tibshirani="tibshirani" _2003.="_2003." pisub="pisub">0 is an estimate for the overall proportion of truly null values for p less than or equal to a selected threshold t using tuning parameter λ = 1, and q is the minimum FDR attainable when calling an event significant. Like p, q is used to determine the proportion of events likely to give a false event rather than the probability that an event is false. (A) FDR determination for 3% glc versus control. (B) FDR for 60 mM N versus control. (C) FDR for 3% glc and 60 mM N versus control. (D) FDR for 3% 3-O-methylglucose versus control. Supplemental Figure 3. FDR determination for treatments containing CHX. (A) 3% glc & CHX versus CHX control. (B) 60 mM N & CHX versus CHX control. (C) 3% glc, 60 mM N, & CHX versus CHX control. (D) 3% 3-O-methylglucose & CHX versus CHX control.
- Supplemental Table 1 - Expression level of genes described in Figure 1.
- Supplemental Table 2 - Glucose-regulated genes described in Figure 4.
- Supplemental Table 3 - Genes associated with carbohydrate metabolism described in Figure 6.
- Supplemental Table 4A - Expression data for genes described in Figures 8A through 8D.
- Supplemental Table 4B
- Supplemental Table 4C
- Supplemental Table 4D
- Supplemental Table 5 - Transport-associated genes described in Figure 8.
- Supplemental Table 6 - Summary of nitrogen metabolism genes described in Figure 11.
