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

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.

A Dead Gene Walking: Convergent Degeneration of a Clade of MADS-Box Genes in Crucifers.

Genes are "born," and eventually they "die." These processes shape the phenotypic evolution of organisms and are hence of great biological interest. If genes die in plants, they generally do so quite rapidly. Here, we describe the fate of GOA-like genes that evolve in a dramatically different manner. GOA-like genes belong to the subfamily of Bsister genes of MIKC-type MADS-box genes. Typical MIKC-type genes encode conserved transcription factors controlling plant development. We show that ABS-like genes, a clade of Bsister genes, are indeed highly conserved in crucifers (Brassicaceae) maintaining the ancestral function of Bsister genes in ovule and seed development. In contrast, their closest paralogs, the GOA-like genes, have been undergoing convergent gene death in Brassicaceae. Intriguingly, erosion of GOA-like genes occurred after millions of years of coexistence with ABS-like genes. We thus describe Delayed Convergent Asymmetric Degeneration, a so far neglected but possibly frequent pattern of duplicate gene evolution that does not fit classical scenarios. Delayed Convergent Asymmetric Degeneration of GOA-like genes may have been initiated by a reduction in the expression of an ancestral GOA-like gene in the stem group of Brassicaceae and driven by dosage subfunctionalization. Our findings have profound implications for gene annotations in genomics, interpreting patterns of gene evolution and using genes in phylogeny reconstructions of species.

Non-canonical structure, function and phylogeny of the Bsister MADS-box gene OsMADS30 of rice (Oryza sativa).

Bsister MADS-box genes play key roles in female reproductive organ and seed development throughout seed plants. This view is supported by their high conservation in terms of sequence, expression and function. In grasses, there are three subclades of Bsister genes: the OsMADS29-, the OsMADS30- and the OsMADS31-like genes. Here, we report on the evolution of the OsMADS30-like genes. Our analyses indicate that these genes evolved under relaxed purifying selection and are rather weakly expressed. OsMADS30, the representative of the OsMADS30-like genes from rice (Oryza sativa), shows strong sequence deviations in its 3' region when compared to orthologues from other grass species. We show that this is due to a 2.4-kbp insertion, possibly of a hitherto unknown helitron, which confers a heterologous C-terminal domain to OsMADS30. This putative helitron is not present in the OsMADS30 orthologues from closely related wild rice species, pointing to a relatively recent insertion event. Unlike other Bsister mutants O. sativa plants carrying a T-DNA insertion in the OsMADS30 gene do not show aberrant seed phenotypes, indicating that OsMADS30 likely does not have a canonical 'Bsister function'. However, imaging-based phenotyping of the T-DNA carrying plants revealed alterations in shoot size and architecture. We hypothesize that sequence deviations that accumulated during a period of relaxed selection in the gene lineage that led to OsMADS30 and the alteration of the C-terminal domain might have been a precondition for a potential neo-functionalization of OsMADS30 in O. sativa.

Unusual evolution of a catalytic core element in CCA-adding enzymes.

CCA-adding enzymes are polymerases existing in two distinct enzyme classes that both synthesize the C-C-A triplet at tRNA 3'-ends. Class II enzymes (found in bacteria and eukaryotes) carry a flexible loop in their catalytic core required for switching the specificity of the nucleotide binding pocket from CTP- to ATP-recognition. Despite this important function, the loop sequence varies strongly between individual class II CCA-adding enzymes. To investigate whether this loop operates as a discrete functional entity or whether it depends on the sequence context of the enzyme, we introduced reciprocal loop replacements in several enzymes. Surprisingly, many of these replacements are incompatible with enzymatic activity and inhibit ATP-incorporation. A phylogenetic analysis revealed the existence of conserved loop families. Loop replacements within families did not interfere with enzymatic activity, indicating that the loop function depends on a sequence context specific for individual enzyme families. Accordingly, modeling experiments suggest specific interactions of loop positions with important elements of the protein, forming a lever-like structure. Hence, although being part of the enzyme's catalytic core, the loop region follows an extraordinary evolutionary path, independent of other highly conserved catalytic core elements, but depending on specific sequence features in the context of the individual enzymes.