Gibberellic acid and cGMP-dependent transcriptional regulation in Arabidopsis thaliana

Abstract

An ever increasing amount of transcriptomic data and analysis tools provide novel insight into complex responses of biological systems. Given these resources we have undertaken to review aspects of transcriptional regulation in response to the plant hormone gibberellic acid (GA) and its second messenger guanosine 3',5'-cyclic monophosphate (cGMP) in Arabidopsis thaliana, both wild type and selected mutants. Evidence suggests enrichment of GA-responsive (GARE) elements in promoters of genes that are transcriptionally upregulated in response to cGMP but downregulated in a GA insensitive mutant (ga1-3). In contrast, in the genes upregulated in the mutant, no enrichment in the GARE is observed suggesting that GARE motifs are diagnostic for GA-induced and cGMP-dependent transcriptional upregulation. Further, we review how expression studies of GA-dependent transcription factors and transcriptional networks based on common promoter signatures derived from ab initio analyses can contribute to our understanding of plant responses at the systems level.

Figure 1

Frequency of occurrence of the GARE (TAACAAR) core motif in artificial clusters generated in POBO (ekhidna.biocenter.helsinki.fi/poxo/pobo) for A. thaliana background promoters compared to the promoters (1 kb upstream of the transcription start site) of the 59 genes upregulated >2 fold after cGMP treatment was analyzed. The default settings were used (number of sequences to pick-out = 50, number of samples to generate = 1000, sequence length = 1000 bps) and a two-tailed p-value was calculated in the linked online GraphPad Web site using the generated t-value and degrees of freedom to determine the statistical differences between input sequences and background. This analysis determined that compared to the background (0.78 copies/promoter), there was a significant (t-test: p-value >0.0001) enrichment in the frequency of the TAACAAR motif in our dataset (1.08 copies/promoter).

Figure 2

Heatmap illustrating fold change (log2) in expression of cGMP-induced genes in the Arabidopsis GA-deficient mutants ga1-3 and penta. In order to reveal the transcriptional relationship between cGMP-induced genes and GA, expression of the top 59 cGMP upregulated genes was investigated in a publicly available microarray data set (Accession no: E-MEXP-849) containing the GA-deficient mutants ga1-3 and penta (a gal-3 della mutant lacking all four DELLA proteins). Gene expression M values [log2(mutant/wildtype)] were determined for seed and flower tissues in both mutants as previously described. The heatmap indicates clusters of up-and downregulated GA-responsive genes, most noticeably in the gal-3 mutant with evidence of tissue-specific expression between seeds and leaves.

Figure 3

Frequency of occurrence of the GARE (TAACAAR) core motif in artificial clusters generated in POBO for A. thaliana background promoters compared to the promoters of cGMP upregulated genes that were downregulated (A) or upregulated (B) in the ga1-3 mutant. (A) The 1 kb upstream promoter sequences of the selected genes were analyzed in POBO to determine the frequency of occurrence of the TAACAAR GARE core motif. The analysis determined that compared to the A. thaliana background (0.78 copies/promoter), there was a significant (t-test: p-value >0.0001) enrichment in the frequency of the TAACAAR motif in our dataset (1.33 copies/promoter). (B) The analysis determined that compared to the A. thaliana background (0.78 copies/promoter), there was no significant (t-test: p-value >0.0001) enrichment in the frequency of the TAACAAR motif in our dataset (0.77 copies/promoter).

Figure 4

Transcriptional regulatory network diagram of cGMP upregulated genes generated by promoter analysis in order to predict A. thaliana co-expressed genes. The 59 upregulated gene promoters of a length 2,200 nt covering 2,000 nt upstream and 200 nt downstream of the 5′ end of the respective genes. The data was created with the Dragon Motif Builder (apps.sanbi.ac.za/MotifBuilder/index.php) and displayed with Cytoscape. (Dragon Motif Builder parameters: Algorithm EM2, motif length 9, threshold 0.9, no background, number of motif families = 30). The red dot denotes a hub gene (AtCHX8) and signifies the cGMP upregulated gene that shares the highest number of common promoter motifs with other A. thaliana genes. The green dot indicates At5G36240, a zinc knuckle (CCHC-type) family protein which contains the highest number of shared motifs per edge. At5g38480 general regulatory factor, a 14-3-3 gene, is represented by the blue dot on the figure. This gene is only linked to one gene and thus shares notable common motifs with a single gene only.

Figure 5

Genevestigator analysis (www.genevestigator.com/gv/index.jsp) of GA mutant genes. Expression of zinc knuckle (CCHC-type) family protein (AT5G36240) node members, which shares 20 common elements, show a less varied response pattern (A) than in the case of the ATCHX8 linked genes (B and C), which only shares 13/14 motifs per edge. As a control, genes linked to AT4g18160, K⁺ channel ATTPK/KCO family of proteins were used (D), since all its node members only shared 13 common motifs. The results also showed a response pattern that was more varied than the one of the zinc knuckle (CCHC-type) family protein.