A cornucopia of diversity-Ranunculales as a model lineage.

The Ranunculales are a hyperdiverse lineage in many aspects of their phenotype, including growth habit, floral and leaf morphology, reproductive mode, and specialized metabolism. Many Ranunculales species, such as opium poppy and goldenseal, have a high medicinal value. In addition, the order includes a large number of commercially important ornamental plants, such as columbines and larkspurs. The phylogenetic position of the order with respect to monocots and core eudicots and the diversity within this lineage make the Ranunculales an excellent group for studying evolutionary processes by comparative studies. Lately, the phylogeny of Ranunculales was revised, and genetic and genomic resources were developed for many species, allowing comparative analyses at the molecular scale. Here, we review the literature on the resources for genetic manipulation and genome sequencing, the recent phylogeny reconstruction of this order, and its fossil record. Further, we explain their habitat range and delve into the diversity in their floral morphology, focusing on perianth organ identity, floral symmetry, occurrences of spurs and nectaries, sexual and pollination systems, and fruit and dehiscence types. The Ranunculales order offers a wealth of opportunities for scientific exploration across various disciplines and scales, to gain novel insights into plant biology for researchers and plant enthusiasts alike.

A MITE insertion abolishes the AP3-3 self-maintenance regulatory loop in apetalous flowers of Nigella damascena.

MADS-box transcription factors are important regulators of floral organ identity through their binding to specific motifs, termed CArG, in the promoter of their target genes. Petal initiation and development depend on class A and B genes, but MADS-box genes of the APETALA3 (AP3) clade are key regulators of this process. In the early diverging eudicot Nigella damascena, an apetalous [T] morph is characterized by the lack of expression of the NdAP3-3 gene, with its expression being petal-specific in the wild-type [P] morph. All [T] morph plants are homozygous for an NdAP3-3 allele with a Miniature Inverted-repeat Transposable Element (MITE) insertion in the second intron of the gene. Here, we investigated to which extent the MITE insertion impairs regulation of the NdAP3-3 gene. We found that expression of NdAP3-3 is initiated in the [T] morph, but the MITE insertion prevents its positive self-maintenance by affecting the correct splicing of the mRNA. We also found specific CArG features in the promoter of the NdAP3-3 genes with petal-specific expression. However, they are not sufficient to drive expression only in petals of transgenic Arabidopsis, highlighting the existence of Nigella-specific cis/trans-acting factors in regulating AP3 paralogs.

Evolutionary analyses and expression patterns of TCP genes in Ranunculales.

TCP transcription factors play a role in a large number of developmental processes and are at the crossroads of numerous hormonal biosynthetic and signaling pathways. The complete repertoire of TCP genes has already been characterized in several plant species, but not in any species of early diverging eudicots. We focused on the order Ranunculales because of its phylogenetic position as sister group to all other eudicots and its important morphological diversity. Results show that all the TCP genes expressed in the floral transcriptome of Nigella damascena (Ranunculaceae) are the orthologs of the TCP genes previously identified from the fully sequenced genome of Aquilegia coerulea. Phylogenetic analyses combined with the identification of conserved amino acid motifs suggest that six paralogous genes of class I TCP transcription factors were present in the common ancestor of angiosperms. We highlight independent duplications in core eudicots and Ranunculales within the class I and class II subfamilies, resulting in different numbers of paralogs within the main subclasses of TCP genes. This has most probably major consequences on the functional diversification of these genes in different plant clades. The expression patterns of TCP genes in Nigella damascena were consistent with the general suggestion that CIN and class I TCP genes may have redundant roles or take part in same pathways, while CYC/TB1 genes have more specific actions. Our findings open the way for future studies at the tissue level, and for investigating redundancy and subfunctionalisation in TCP genes and their role in the evolution of morphological novelties.

Transcriptome Analysis Reveals Putative Target Genes of APETALA3-3 During Early Floral Development in Nigella damascena L.

Even though petals are homoplastic structures, their identity consistently involves genes of the APETALA3 (AP3) lineage. However, the extent to which the networks downstream of AP3 are conserved in species with petals of different evolutionary origins is unknown. In Ranunculaceae, the specificity of the AP3-III lineage offers a great opportunity to identify the petal gene regulatory network in a comparative framework. Using a transcriptomic approach, we investigated putative target genes of the AP3-III ortholog NdAP3-3 in Nigella damascena at early developmental stages when petal identity is determined, and we compared our data with that from selected eudicot species. We generated a de novo reference transcriptome to carry out a differential gene expression analysis between the wild-type and mutant NdAP3-3 genotypes differing by the presence vs. absence of petals at early stages of floral development. Among the 1,620 genes that were significantly differentially expressed between the two genotypes, functional annotation suggested a large involvement of nuclear activities, including regulation of transcription, and enrichment in processes linked to cell proliferation. Comparing with Arabidopsis data, we found that highly conserved genes between the two species are enriched in homologs of direct targets of the AtAP3 protein. Integrating AP3-3 binding site data from another Ranunculaceae species, Aquilegia coerulea, allowed us to identify a set of 18 putative target genes that were conserved between the three species. Our results suggest that, despite the independent evolutionary origin of petals in core eudicots and Ranunculaceae, a small conserved set of genes determines petal identity and early development in these taxa.

Unraveling the Developmental and Genetic Mechanisms Underpinning Floral Architecture in Proteaceae.

Proteaceae are a basal eudicot family with a highly conserved floral groundplan but which displays considerable variation in other aspects of floral and inflorescence morphology. Their morphological diversity and phylogenetic position make them good candidates for understanding the evolution of floral architecture, in particular the question of the homology of the undifferentiated perianth with the differentiated perianth of core eudicots, and the mechanisms underlying the repeated evolution of zygomorphy. In this paper, we combine a morphological approach to explore floral ontogenesis and a transcriptomic approach to access the genes involved in floral organ identity and development, focusing on Grevillea juniperina, a species from subfamily Grevilleoideae. We present developmental data for Grevillea juniperina and three additional species that differ in their floral symmetry using stereomicroscopy, SEM and High Resolution X-Ray Computed Tomography. We find that the adnation of stamens to tepals takes place at early developmental stages, and that the establishment of bilateral symmetry coincides with the asymmetrical growth of the single carpel. To set a framework for understanding the genetic basis of floral development in Proteaceae, we generated and annotated de novo a reference leaf/flower transcriptome from Grevillea juniperina. We found Grevillea homologs of all lineages of MADS-box genes involved in floral organ identity. Using Arabidopsis thaliana gene expression data as a reference, we found homologs of other genes involved in floral development in the transcriptome of G. juniperina. We also found at least 21 class I and class II TCP genes, a gene family involved in the regulation of growth processes, including floral symmetry. The expression patterns of a set of floral genes obtained from the transcriptome were characterized during floral development to assess their organ specificity and asymmetry of expression.

WOX14 promotes bioactive gibberellin synthesis and vascular cell differentiation in Arabidopsis.

Procambial and cambial stem cells provide the initial cells that allow the formation of vascular tissues. WOX4 and WOX14 have been shown to act redundantly to promote procambial cell proliferation and differentiation. Gibberellins (GAs), which have an important role in wood formation, also stimulate cambial cell division. Here we show that the loss of WOX14 function phenocopies some traits of GA-deficient mutants that can be complemented by exogenous GA application, whereas WOX14 overexpression stimulates the expression of GA3ox anabolism genes and represses GA2ox catabolism genes, promoting the accumulation of bioactive GA. More importantly, our data clearly indicate that WOX14 but not WOX4 promotes vascular cell differentiation and lignification in inflorescence stems of Arabidopsis.

Genes of the most conserved WOX clade in plants affect root and flower development in Arabidopsis.

BACKGROUND: The Wuschel related homeobox (WOX) family proteins are key regulators implicated in the determination of cell fate in plants by preventing cell differentiation. A recent WOX phylogeny, based on WOX homeodomains, showed that all of the Physcomitrella patens and Selaginella moellendorffii WOX proteins clustered into a single orthologous group. We hypothesized that members of this group might preferentially share a significant part of their function in phylogenetically distant organisms. Hence, we first validated the limits of the WOX13 orthologous group (WOX13 OG) using the occurrence of other clade specific signatures and conserved intron insertion sites. Secondly, a functional analysis using expression data and mutants was undertaken. RESULTS: The WOX13 OG contained the most conserved plant WOX proteins including the only WOX detected in the highly proliferating basal unicellular and photosynthetic organism Ostreococcus tauri. A large expansion of the WOX family was observed after the separation of mosses from other land plants and before monocots and dicots have arisen. In Arabidopsis thaliana, AtWOX13 was dynamically expressed during primary and lateral root initiation and development, in gynoecium and during embryo development. AtWOX13 appeared to affect the floral transition. An intriguing clade, represented by the functional AtWOX14 gene inside the WOX13 OG, was only found in the Brassicaceae. Compared to AtWOX13, the gene expression profile of AtWOX14 was restricted to the early stages of lateral root formation and specific to developing anthers. A mutational insertion upstream of the AtWOX14 homeodomain sequence led to abnormal root development, a delay in the floral transition and premature anther differentiation. CONCLUSION: Our data provide evidence in favor of the WOX13 OG as the clade containing the most conserved WOX genes and established a functional link to organ initiation and development in Arabidopsis, most likely by preventing premature differentiation. The future use of Ostreococcus tauri and Physcomitrella patens as biological models should allow us to obtain a better insight into the functional importance of WOX13 OG genes.

The ethanol switch: a tool for tissue-specific gene induction during plant development.

Controlled gene expression in time and space is a powerful tool for the analysis of gene function during plant development. Here, we report ethanol inducible gene expression in defined sub-domains of the shoot apical and floral meristems. For this, expression of an ethanol-regulated transcription factor, ALCR, is restricted to precise domains using specific promoters. Gene expression activation is followed using reporters under the control of the alcA promoter, which responds to ALCR only in the presence of the ethanol. We demonstrate that precise control of spatially limited gene expression can be achieved. The kinetics of reporter gene activation and inactivation following a pulse of ethanol induction shows that the system is dynamic and suitable for precise temporal control of expression. The system is both flexible and robust, permitting simultaneous expression of two genes in a given domain or, conversely, the expression of a gene in two separate domains. We also show that this strategy can be applied to mis-express genes with developmental roles, by manipulating expression of the SHOOT MERISTEMLESS (STM) and CYCLIN D3;1 (CYCD3;1) genes during plant development.