Toward the identification and regulation of the Arabidopsis thaliana ABI3 regulon

Abstract

The plant-specific, B3 domain-containing transcription factor ABSCISIC ACID INSENSITIVE3 (ABI3) is an essential component of the regulatory network controlling the development and maturation of the Arabidopsis thaliana seed. Genome-wide chromatin immunoprecipitation (ChIP-chip), transcriptome analysis, quantitative reverse transcriptase-polymerase chain reaction and a transient promoter activation assay have been combined to identify a set of 98 ABI3 target genes. Most of these presumptive ABI3 targets require the presence of abscisic acid for their activation and are specifically expressed during seed maturation. ABI3 target promoters are enriched for G-box-like and RY-like elements. The general occurrence of these cis motifs in non-ABI3 target promoters suggests the existence of as yet unidentified regulatory signals, some of which may be associated with epigenetic control. Several members of the ABI3 regulon are also regulated by other transcription factors, including the seed-specific, B3 domain-containing FUS3 and LEC2. The data strengthen and extend the notion that ABI3 is essential for the protection of embryonic structures from desiccation and raise pertinent questions regarding the specificity of promoter recognition.

Figure 1.

Numbers of ABI3 target gene candidates identified by ChIP-chip (bound promoters) and transcriptome analysis (regulated genes). The candidate genes were subdivided according to their expression in seeds: Genes with seed MI > 31 exhibit a strong seed-specific expression (definition of MI see below); genes with MI < 31 are here considered as non-seed-specific genes; NA, MI not available. The overlapping 42 genes are called core regulon. ChIP-chip was performed with chromatin isolated from wild-type seeds; transcript analysis was performed with transgenic seedlings transformed with an ABI3-inducible gene construct.

Figure 2.

Promoter activation by ABI3 in transient assay. Protoplasts of an embryogenic Arabidopsis cell culture were transformed with promoter–reporter constructs and GUS activity was determined in the absence and presence of an ABI3 activator construct. Fold activation of individual experiments are given as dots.

Figure 3.

ABI3-mediated activation of transcription of divergent, overlapping promoters. Intergenic regions of less than 1500 bp flanked by a pair of genes in opposite directions are depicted with their relevant features (filled triangles, translation start; vertical lines, proposed TSS; open circle, GBL motif; filled circles, RYL motif). Promoter regions cloned in front of GUS and assayed for activation by ABI3 in transiently transformed protoplasts of an embryogenic cell culture are shown above and below the intergenic regions (solid: ABI3 activated; open: not activated). Corresponding log2 transformed activation ratios as well as the MI and the gene ID are given on the left; the distance between the two proposed TSSs are noted on the right.

Figure 4.

Members of the ABI3 regulon are involved in a range of biological processes. Over-representation for specific GO categories was assessed using the BiNGO plugin for Cytoscape. The color coding represents the significance of enrichment (P-value). The node size represents the number of ABI3 target genes in each particular category.

Figure 5.

Over-represented motifs and their position within the promoters of ABI3 regulon genes. GBL and RYL sequence motifs were detected in the region −750 to +250 bp relative to the TSS. Density refers to the frequency of motif occurrence in that region.

Figure 6.

The ABI3 regulon in the context of the AGRIS gene regulatory network. The network shows the overlap of the ABI3 regulon with the regulons of the B3 domain TFs FUS3 (AT3G26790) and LEC2 (AT1G28300) and other TF classes such as LEC1 (AT1G21970), HY5 (AT5G11260), AGL15 (AT5T13790), PIF1/PIL5 (AT2G20180), AP2 (AT4G36920), GL3 (AT5G41315), AGL9/SEP3 (AT1G24260), AtbZIP10 (AT4G02640), EEL (AT2G41070), ABI5 (AT2G36270), MYB88 (AT2G02820) and FLP/MYB124 (AT1G14350). Genes of the ABI3 regulon are arranged within a circle and categorized according to the occurrence of regulatory motifs in their corresponding promoters (RY+G-box: blue; RY: red; G-box: green and no motif: yellow). Additional information is given in Supplementary Table S5.