Evolution of CONSTANS Transcriptional Regulation and Protein Function in the Brassicaceae Family

Plants respond to seasonal changes in day length to control developmental transitions such as flowering and bud dormancy. In the model plant Arabidopsis thaliana (A. thaliana), CONSTANS (CO) activates the expression of FLOWERING LOCUS T (FT) specifically under long days, and FT protein then moves from the leaf to the shoot apical meristem where it promotes the transition to flowering. CO transcription is precisely regulated so that its mRNA is repressed in the morning and rises in the evening of a long day allowing activation of FT. Therefore, the diurnal pattern of CO transcription is an essential component of the regulation of flowering time in A. thaliana. The two closest homologs of CO in A. thaliana, CONSTANS-like 1 (COL1) and COL2 have no function in photoperiodic flowering response and show different mRNA patterns. All three genes evolved from a common ancestor after the latest whole genome duplication (At-α) in the lineage leading to A. thaliana that is believed to have occurred ~43 mya. The transcriptional patterns of CO and COL2 are conserved in the basal Brassicaceae species Aethionema arabicum. Also the CO homolog of this species but not its COL2 homolog can complement the co mutation in A. thaliana. As the Aethionema lineage represents the earliest split in the evolution of the Brassicaceae these findings indicate that CO function was already present at an early stage of Brassicaceae evolution and its activity is likely conserved throughout the family. The promoter of CO contains conserved sequence blocks which are shared in orthologs examined in ten Brassicaceaen species. DOF binding sites, E-Boxes and TCP binding sites are the most abundant cis-elements identified in ten CO promoters. Furthermore, DOF and TCP binding sites are strongly enriched in the promoters of CO genes compared to COL gene promoters. DOF binding sites are reported to enable binding of repressive CDF Transcription factors and were among the most highly conserved motifs in the promoters of CO orthologs. E-Boxes are putative binding sites for FLOWERING bHLH (FBH) TFs that function as transcriptional activators of CO. No role has so far been assigned for TCP transcription factors in regulation of CO transcription. Promoter reporter studies in A. thaliana revealed that the CO promoter contains functional regulatory sequences up to 2.6kb from the TS and that a short conserved regulatory motif that comprises DOF binding sites and is present both distally and proximally is sufficient to recreate mRNA patterns highly similar to endogenous CO. A model for the evolution of CO promoter is proposed in which downstream conserved DOF binding sites appeared distally upstream to regulate CO after the gene duplication leading to its diversification from COL1. Tarenaya hassleriana, a member of the Cleomaceae family which is sister to the Brassicaceae, has only one ortholog of CO (ThCOL) which shares one last common ancestral gene with CO, COL1 and COL2. ThCOL protein sequence is most similar to COL2 homologs from the Brassicaceae family. Additionally mRNA expression pattern and lower ability to promote early flowering in A. thaliana are common characteristics of this gene with COL1 and COL2 but not with CO. Hence, the transcriptional regulation of CO as well as an increase in capacity to promote flowering when compared to its close homologs COL1 and COL2 evolved in the Brassicaceae family and represents a novelty that arose in this family. My evolutionary studies indicate that CO evolved by gene duplication at the root of the Brassicaceae family. This gene then evolved a function in photoperiodic flowering through changes to its transcriptional regulation and protein function. Such a history contradicts the current view that CO has an ancient history predating the divergence of monocotyledonous and dicotyledonous angiosperms, but rather suggests that the role of CO homologous genes in conferring photoperiodic flowering might have evolved independently in different families of higher plants.