The greatest difference between 35S ABF3 and control plant lines in the percentage of genes belonging to a particular functional category was 1. 3% for the other metabolic processes category at the 2 h time point. This suggests that the overall functional response of 35S ABF3 and control plant lines at the gene expression level was similar. In total, 1234 screening libraries genes were differentially expressed in Inhibitors,Modulators,Libraries at least one plant line during at least one time point. The overlap in genes expressed in the two plant lines at the two different time points is depicted with a four way Venn diagram in Figure 3. These genes can be subdi vided into three categories. There are 564 genes that were differentially expressed in both control and 35S ABF3 lines at the same time points suggesting that they were commonly regulated in both lines.

There Inhibitors,Modulators,Libraries are 407 genes that are regulated differently in control and 35S ABF3 plant lines that show an enhanced response in the 35S ABF3 line. Finally, there are 263 genes that are regulated differently in control and 35S ABF3 plant lines that show an attenuated response in the 35S ABF3 line. In the latter two categories, these genes are either uniquely differentially expressed in one line or the other, or they are differentially expressed in one line at one time point but not in the other line at that time point. Since only two time points were examined, it is difficult to determine if the observed differences in the two lines are due to differences in the timing of gene expression or in the magnitude of gene expression or some combination of both of these factors.

Inhibitors,Modulators,Libraries Additional file 3 contains a list of all of the differentially expressed genes found in each of the three categories. In order to confirm the microarray results, RT PCR was performed on 32 genes from the enhanced and atte nuated categories. Twenty nine of the examined genes exhibited expression patterns that were consistent with the microarray results, confirming the reliability of the microarray data. Inhibitors,Modulators,Libraries Amplification of one gene, At2g22760, produced two bands, one corresponding to the expected size of 526 bp. The primers for this gene were designed around an intron and the size of the sec ond band is similar to the 664 bp that would be expected if the intron were not spliced, suggesting that this band may represent a splice variant.

Genes commonly regulated in both 35S ABF3 and control lines There are 564 genes that are commonly regulated in both 35S ABF3 and control Inhibitors,Modulators,Libraries lines. Of these, 172 show differential expression in both lines at both time points, 120 are only differentially expressed at 2 h and 272 are only differentially expressed at 24 h. A number of genes CHIR99021 GSK-3 in this category are known to act in pathways that are upstream or independent of ABF3, which is consistent with their common pattern of regu lation in both 35S ABF3 and control plant lines. Since ABF3 is an ABA dependent transcrip tion factor, ABA biosynthesis should occur upstream of ABF3 activity.