Consensus by democracy. Using meta-analyses of microarray and genomic data to model the cold acclimation signaling pathway in Arabidopsis

Abstract

The whole-genome response of Arabidopsis (Arabidopsis thaliana) exposed to different types and durations of abiotic stress has now been described by a wealth of publicly available microarray data. When combined with studies of how gene expression is affected in mutant and transgenic Arabidopsis with altered ability to transduce the low temperature signal, these data can be used to test the interactions between various low temperature-associated transcription factors and their regulons. We quantized a collection of Affymetrix microarray data so that each gene in a particular regulon could vote on whether a cis-element found in its promoter conferred induction (+1), repression (-1), or no transcriptional change (0) during cold stress. By statistically comparing these election results with the voting behavior of all genes on the same gene chip, we verified the bioactivity of novel cis-elements and defined whether they were inductive or repressive. Using in silico mutagenesis we identified functional binding consensus variants for the transcription factors studied. Our results suggest that the previously identified ICEr1 (induction of CBF expression region 1) consensus does not correlate with cold gene induction, while the ICEr3/ICEr4 consensuses identified using our algorithms are present in regulons of genes that were induced coordinate with observed ICE1 transcript accumulation and temporally preceding genes containing the dehydration response element. Statistical analysis of overlap and cis-element enrichment in the ICE1, CBF2, ZAT12, HOS9, and PHYA regulons enabled us to construct a regulatory network supported by multiple lines of evidence that can be used for future hypothesis testing.

Figure 1.

Schematic representation of low temperature signal transduction assembled from reviews published by Chinnusamy et al. (2006) and Nakashima and Yamaguchi-Shinozaki (2006). Protein TFs are represented by ovals while TF regulons are represented by “REG” boxes. Where reported, the consensus sequences (cis-elements) bound by the upstream TF are shown as color-coded boxes within the promoter space (solid line) preceding the REG box or TF gene coding sequence (blue box, DRE; green box, ICEr1; pink box, ICEr2; and yellow box, EP2). Regulatory connections are mapped using triangular arrowheads to represent gene activation and flattened arrowheads to represent gene repression. Dashed arrows represent regulatory connections with experimental support but accomplished by unknown (and possibly posttranscriptional) mechanisms. Protein phosphorylation events, where known, are indicated by circles attached to the protein TFs. Genes modeled (and corresponding Arabidopsis Genome Initiative no.) are ICE1, AT3G26744; ZAT12, AT5G59820; HOS9, AT2G01500; CBF2, AT4G25470; CBF1, AT4G25490; CBF3, AT4G25480; ZAT10, AT1G27730; RAP2.1, AT1G46768; and RAP2.6, AT1G43160.

Figure 2.

Observed transcriptional response of genes containing the ICEr3 (HACACGT), ICEr4 (HCCACGT), ABRE (GACACGT), HOS9r1 (ACGCGT), DRE (RCCGAC), ICEr1 (CACATG), and ZAT12r1 (CATTGA) elements in their 500 bp promoters to cold (2°C) treatment (using microarray data from WeigelWorld AtGenExpress Database). The number of genes induced (left column) and repressed (right column) by the treatment (black bars) was compared to the expected frequency (white bars), based on the percentage of all present cold-responding genes on the chip. Statistically significant difference was assessed by χ² test: ** = P < 0.01. A, ICEr3-containing induced genes. B, ICEr4-containing induced genes. C, ABRE-containing induced genes. D, HOS9r1-containing induced genes. E, DRE-containing induced genes. F, ICEr1-containing induced genes. G, ZAT12r1-containing induced genes. H, ICEr3-containing repressed genes. I, ICEr4-containing repressed genes. J, ABRE-containing repressed genes. K, HOS9r1-containing repressed genes. L, DRE-containing repressed genes. M, ICEr1-containing repressed genes. N, ZAT12r1-containing repressed genes.

Figure 3.

Observed transcriptional response of genes containing the ICEr3 (HACACGT), ICEr4 (HCCACGT), HOS9r1 (ACGCGT), DRE (RCCGAC), ICEr1 (CACATG), and ABRE (GACACGT) elements in their 500 bp promoters to cold (0°C) treatment in wild-type and ice1 Arabidopsis backgrounds (using microarray data published in Lee et al., 2005). The number of genes induced by the treatment was compared to the expected frequency, based on the percentage of all present cold-responding genes on the chip. Statistically significant difference was assessed by χ² test: ** = P < 0.01. A, ICEr3-containing induced genes. B, ICEr4-containing induced genes. C, HOS9r1-containing induced genes. D, DRE-containing induced genes. E, ICEr1-containing induced genes. F, ABRE-containing induced genes.

Figure 4.

In silico mutagenesis of the ICEr3 (HACACGT)-, HOS9r1 (ACGCGT)-, ICEr1 (CACATG)-, and DRE (RCCGAC)-binding consensus sequences. Identity and position of each nucleic acid mutation in the corresponding consensus sequence are indicated. The number of genes induced or repressed in the genetic background/treatment indicated (black bars) was compared to the expected frequency (white bars), based on the percentage of all present and responding genes on the chip. Statistically significant difference was assessed by χ² test: * = P < 0.05, ** = P < 0.01. A, ICEr3-containing genes repressed in 6 h cold-treated ice1 plants (versus wild type). B, HOS9r1-containing genes repressed in 24 h cold-treated hos9-1 plants (versus wild type). C, ICEr1-containing genes repressed in 6 h cold-treated ice1 plants (versus wild type). D, DRE-containing genes induced in 35S:CBF2 overexpressors at growth temperatures (versus wild type).

Figure 5.

Schematic representation of low temperature signal transduction. Solid arrows represent connections with at least two pieces of bioinformatic support (triangular arrowhead represents gene activation, flattened arrowhead represents gene repression). Dashed arrows represent regulatory connections with experimental/bioinformatic support but accomplished by unknown (and possibly posttranscriptional) mechanisms. Boxes represent cis-elements within the 500 bp promoters of the genes listed (ICE1, AT3G26744; ZAT12, AT5G59820; HOS9, AT2G01500; CBF2, AT4G25470; NAC072, AT4G27410; CBF1, AT4G25490; and CBF3, AT4G25480) and are color coded according to cis-element identity (red box, ICEr3; orange box, ICEr4; blue box, DRE; green box, ICEr1; purple box, I HOS9r1; and yellow box, EP2). Protein phosphorylation events, where known, are indicated by circles containing “P” attached to the protein TFs. Unknown types of posttranslational modification are indicated by circles containing “?” attached to the protein TFs. The ICEr4 element present in the CBF3 promoter contains the CCACGT core, but lacks an “H” in the first position.