Cédric Finet

Evolution of the ARF Gene Family in Land Plants: Old Domains, New Tricks

Auxin response factors (ARF) are key players in plant development. They mediate the cellular response to the plant hormone auxin by activating or repressing the expression of downstream developmental genes. The pivotal activation function of ARF proteins is enabled by their four-domain architecture, which includes both DNA-binding and protein dimerization motifs. To determine the evolutionary origin of this characteristic architecture, we built a comprehensive data set of 224 ARF-related protein sequences that represents all major living divisions of land plants, except hornworts. We found that ARFs are split into three subfamilies that could be traced back to the origin of the land plants. We also show that repeated events of extensive gene duplication contributed to the expansion of those three original subfamilies. Further examination of our data set uncovered a broad diversity in the structure of ARF transcripts and allowed us to identify an additional conserved motif in ARF proteins. We found that additional structural diversity in ARF proteins is mainly generated by two mechanisms: genomic truncation and alternative splicing. We propose that the loss of domains from the canonical, four-domain ARF structure has promoted functional shifts within the ARF family by disrupting either dimerization or DNA-binding capabilities. For instance, the loss of dimerization domains in some ARFs from moss and spikemoss genomes leads to proteins that are reminiscent of Aux/IAA proteins, possibly providing a clue on the evolution of these modulators of ARF function. We also assessed the functional impact of alternative splicing in the case of ARF4, for which we have identified a novel isoform in Arabidopsis thaliana. Genetic analysis showed that these two transcripts exhibit markedly different developmental roles in A. thaliana. Gene duplications, domain rearrangement, and post-transcriptional regulation have thus enabled a subtle control of auxin signaling through ARF proteins that may have contributed to the critical importance of these regulators in plant development and evolution.

Parallel structural evolution of auxin response factors in the angiosperms.

Here we analyze the structural evolution of the paralogous transcription factors ETTIN (ETT/ARF3) and AUXIN RESPONSE FACTOR 4 (ARF4), which control the development of floral organs and leaves in the model angiosperm Arabidopsis. ETT is truncated at its C terminus, and consequently lacks two regulatory domains present in most other ARFs, including ARF4. Our analysis indicates ETT and ARF4 to have been generated by the duplication of a non-truncated ARF gene prior to the radiation of the extant angiosperms. We furthermore show that either ETT or ARF4 orthologs have become modified to encode truncated ARF proteins, lacking C-terminal regulatory domains, in representatives of three groups that separated early in angiosperm evolution: Amborellales, Nymphaeales and the remaining angiosperm clade. Interestingly, the production of truncated ARF4 transcripts in Amborellales occurs through an alternative splicing mechanism, rather than through a permanent truncation, as in the other groups studied. To gain insight into the potential functional significance of truncations to ETT and ARF4, we tested the capacity of native, truncated and chimeric coding sequences of these genes to restore a wild-type phenotype to Arabidopsis ett mutants. We discuss the results of this analysis in the context of the structural evolution of ARF genes in the angiosperms.

Insights from ANA-grade angiosperms into the early evolution of CUP-SHAPED COTYLEDON genes

BACKGROUND AND AIMS The closely related NAC family genes NO APICAL MERISTEM (NAM) and CUP-SHAPED COTYLEDON3 (CUC3) regulate the formation of boundaries within and between plant organs. NAM is post-transcriptionally regulated by miR164, whereas CUC3 is not. To gain insight into the evolution of NAM and CUC3 in the angiosperms, we analysed orthologous genes in early-diverging ANA-grade angiosperms and gymnosperms. METHODS We obtained NAM- and CUC3-like sequences from diverse angiosperms and gymnosperms by a combination of reverse transcriptase PCR, cDNA library screening and database searching, and then investigated their phylogenetic relationships by performing maximum-likelihood reconstructions. We also studied the spatial expression patterns of NAM, CUC3 and MIR164 orthologues in female reproductive tissues of Amborella trichopoda, the probable sister to all other flowering plants. KEY RESULTS Separate NAM and CUC3 orthologues were found in early-diverging angiosperms, but not in gymnosperms, which contained putative orthologues of the entire NAM + CUC3 clade that possessed sites of regulation by miR164. Multiple paralogues of NAM or CUC3 genes were noted in certain taxa, including Brassicaceae. Expression of NAM, CUC3 and MIR164 orthologues from Am. trichopoda was found to co-localize in ovules at the developmental boundary between the chalaza and nucellus. CONCLUSIONS The NAM and CUC3 lineages were generated by duplication, and CUC3 was subsequently lost regulation by miR164, prior to the last common ancestor of the extant angiosperms. However, the paralogous NAM clade genes CUC1 and CUC2 were generated by a more recent duplication, near the base of Brassicaceae. The function of NAM and CUC3 in defining a developmental boundary in the ovule appears to have been conserved since the last common ancestor of the flowering plants, as does the post-transcriptional regulation in ovule tissues of NAM by miR164.

Cabomba as a model for studies of early angiosperm evolution

BACKGROUND The angiosperms, or flowering plants, diversified in the Cretaceous to dominate almost all terrestrial environments. Molecular phylogenetic studies indicate that the orders Amborellales, Nymphaeales and Austrobaileyales, collectively termed the ANA grade, diverged as separate lineages from a remaining angiosperm clade at a very early stage in flowering plant evolution. By comparing these early diverging lineages, it is possible to infer the possible morphology and ecology of the last common ancestor of the extant angiosperms, and this analysis can now be extended to try to deduce the developmental mechanisms that were present in early flowering plants. However, not all species in the ANA grade form convenient molecular-genetic models. SCOPE The present study reviews the genus Cabomba (Nymphaeales), which shows a range of features that make it potentially useful as a genetic model. We focus on characters that have probably been conserved since the last common ancestor of the extant flowering plants. To facilitate the use of Cabomba as a molecular model, we describe methods for its cultivation to flowering in the laboratory, a novel Cabomba flower expressed sequence tag database, a well-adapted in situ hybridization protocol and a measurement of the nuclear genome size of C. caroliniana. We discuss the features required for species to become tractable models, and discuss the relative merits of Cabomba and other ANA-grade angiosperms in molecular-genetic studies aimed at understanding the origin of the flowering plants.

Evolution of the YABBY gene family in seed plants.

Members of the YABBY gene family of transcription factors in angiosperms have been shown to be involved in the initiation of outgrowth of the lamina, the maintenance of polarity, and establishment of the leaf margin. Although most of the dorsal‐ventral polarity genes in seed plants have homologs in non‐spermatophyte lineages, the presence of YABBY genes is restricted to seed plants. To gain insight into the origin and diversification of this gene family, we reconstructed the evolutionary history of YABBY gene lineages in seed plants. Our findings suggest that either one or two YABBY genes were present in the last common ancestor of extant seed plants. We also examined the expression of YABBY genes in the gymnosperms Ephedra distachya (Gnetales), Ginkgo biloba (Ginkgoales), and Pseudotsuga menziesii (Coniferales). Our data indicate that some YABBY genes are expressed in a polar (abaxial) manner in leaves and female cones in gymnosperms. We propose that YABBY genes already acted as polarity genes in the last common ancestor of extant seed plants.