Conservation of the Restricted Expression of Brassicaceae Bsister-Like Genes in Seeds Requires a Transposable Element in Arabidopsis thaliana

Abstract

Changes in transcription factor binding sites (TFBSs) can alter the spatiotemporal expression pattern and transcript abundance of genes. Loss and gain of TFBSs were shown to cause shifts in expression patterns in numerous cases. However, we know little about the evolution of extended regulatory sequences incorporating many TFBSs. We compare, across the crucifers (Brassicaceae, cabbage family), the sequences between the translated regions of Arabidopsis Bsister (ABS)-like MADS-box genes (including paralogous GOA-like genes) and the next gene upstream, as an example of family-wide evolution of putative upstream regulatory regions (PURRs). ABS-like genes are essential for integument development of ovules and endothelium formation in seeds of Arabidopsis thaliana. A combination of motif-based gene ontology enrichment and reporter gene analysis using A. thaliana as common trans-regulatory environment allows analysis of selected Brassicaceae Bsister gene PURRs. Comparison of TFBS of transcriptionally active ABS-like genes with those of transcriptionally largely inactive GOA-like genes shows that the number of in silico predicted TFBS) is similar between paralogs, emphasizing the importance of experimental verification for in silico characterization of TFBS activity and analysis of their evolution. Further, our data show highly conserved expression of Brassicaceae ABS-like genes almost exclusively in the chalazal region of ovules. The Arabidopsis-specific insertion of a transposable element (TE) into the ABS PURRs is required for stabilizing this spatially restricted expression, while other Brassicaceae achieve chalaza-specific expression without TE insertion. We hypothesize that the chalaza-specific expression of ABS is regulated by cis-regulatory elements provided by the TE.

Keywords: Bsister gene; Brassicaceae; GUS assay; MADS-box gene; evolution of gene expression regulation; transcription factor binding site (TFBS); transposable element (TE).

Fig. 1.

Sequence conservation of Brassicaceae B_sister gene CREs. (A) Shadowing analysis of ABS-like s.s. PURRs and transcribed regions of C. rubella, A. alpina, and E. salsugineum in relation to the corresponding sequence of ABS. The position of protein coding exons and UTRs of ABS, the evolutionary conserved regions (ECR1 and ECR2), the TE insertion specific to A. thaliana are marked as boxes. Highlighted sequence segments show a conservation above 70% over 100 bp (cutoff at 50% nucleotide identity). (B) Shadowing analysis of GOA-like sequences for comparison. Dot blots visualizing pairwise alignments of ABS-like s.s. (C) and GOA-like (D) genomic sequences including the PURRs and the transcribed regions. Uninterrupted lines indicate sequence conservation, triangles mark start codons and the arrow indicates the direction of transcription. (E) Venn diagrams of in silico predicted shared and unique regulatory elements within the PURR based on A. thaliana CRE.

Fig. 2.

Comparison of GO terms of regulatory factors binding to CRSs in PRGs of B_sister genes. (A) Venn Diagrams of significantly enriched GO terms for regulatory factors binding to CRSs of ABS-like genes s.s. (left), GOA-like genes (right), and CREs shared among all ABS-like genes s.l. (middle). The GO terms are derived from single representations of each factor; multiple binding sites within the PURRs were not considered. (B) Comparison of enriched GO terms between regulatory factors of ABS-like gene s.s. CREs using semantic spaces. Each circle represents a single term (see figure legend); size and y-axis indicate enrichment strength. The eight GO terms with the strongest enrichment among predicted regulatory factors of A. thaliana as well as the only significantly enriched term related to the anthocyanin biosynthesis (GO:00311537) in all ABS-like gene s.s. data sets are highlighted by color and used for comparison to other ABS-like gene s.s. PURRs (y-axis, relative fold enrichment; x-axis, semantic similarity). (C) Distribution of binding sites for TFs related to anthocyanin biosynthesis that were predicted to bind. The presence of a binding site for a factor (column) in a certain sequence segment (row) is marked with x.

Fig. 3.

Readout of ABS-like regulatory sequences by A. thaliana trans-acting factors visualized by GUS staining. Activity of PURRs and transcribed regions of Brassicaceae ABS-like genes s.s. in transgenic A. thaliana lines (A) GUS staining pattern of A. thaliana lines containing the PURR and transcribed regions of Brassicaceae ABS-like genes s.s. translationally fused to the GUS gene. (B) Schematic drawing of the relationship of species and GUS staining pattern in four floral stages of the A. thaliana plants carrying the constructs in A. (C) GUS staining pattern of A. thaliana lines with only the PURR of Brassicaceae ABS-like genes s.s. driving GUS expression. (D) Schematic drawing of the relationship of species and GUS staining pattern in four floral stages of the A. thaliana plants carrying the constructs in C. (E) GUS staining of ovules at anthesis (S13). Stages according to Smyth et al. (1990): S5, carpel initiation; S8, carpel elongation; S10, ovule initiation; and S12, ovule development completed. Scale bar is 200 µm; black arrowheads emphasize stained tissue especially mentioned in the text, yellow arrowheads point to the chalazal end, and red arrowheads point to the micropylar end.

Fig. 4.

GUS expression driven by different deletion constructs of the A. thaliana ABS PURR. (A) Schematic representation of the GUS reporter constructs. (B) Ovules at anthesis of plants containing pABS:ABS:GUS and pABS:GUS. (C, D) GUS reporter staining is shown in overview (top) and close up (bottom) of ovules during early embryo development. GUS reporter lines carrying the PURR of ABS lacking (C) ECR1 or (D) the transposon insertion. (E) GUS expression driven by the iP lacking ECR1, ECR2, and the transposon insertion. (F) Schematic model of transcriptional activation and repression by ECR1 (orange), ECR2 (green), and the insertion (purple). Black arrows indicate staining; the scale bar is 200 µm (ii, inner integument; oi, outer integument; c, chalazal region; nc, nucellus; f, funinculus).