Comparative morphoanatomy and transcriptomic analyses reveal key factors controlling floral trichome development in Aristolochia (Aristolochiaceae).

Trichomes are specialized epidermal cells in aerial plant parts. Trichome development proceeds in three stages, determination of cell fate, specification, and morphogenesis. Most genes responsible for these processes have been identified in the unicellular branched leaf trichomes from the model Arabidopsis thaliana. Less is known about the molecular basis of multicellular trichome formation across flowering plants, especially those formed in floral organs of early diverging angiosperms. Here, we aim to identify the genetic regulatory network (GRN) underlying multicellular trichome development in the kettle-shaped trap flowers of Aristolochia (Aristolochiaceae). We selected two taxa for comparison, A. fimbriata, with trichomes inside the perianth, which play critical roles in pollination, and A. macrophylla, lacking specialized trichomes in the perianth. A detailed morphoanatomical characterization of floral epidermis is presented for the two species. We compared transcriptomic profiling at two different developmental stages in the different perianth portions (limb, tube, and utricle) of the two species. Moreover, we present a comprehensive expression map for positive regulators and repressors of trichome development, as well as cell cycle regulators. Our data point to extensive modifications in gene composition, expression, and putative roles in all functional categories when compared with model species. We also record novel differentially expressed genes (DEGs) linked to epidermis patterning and trichome development. We thus propose the first hypothetical genetic regulatory network (GRN) underlying floral multicellular trichome development in Aristolochia, and pinpoint key factors responsible for the presence and specialization of floral trichomes in phylogenetically distant species of the genus.

Evolution and expression of LEAFY genes in ferns and lycophytes.

BACKGROUND: The LEAFY (LFY) transcription factors are present in algae and across land plants. The available expression and functional data of these genes in embryophytes suggest that LFY genes control a plethora of processes including the first zygotic cell division in bryophytes, shoot cell divisions of the gametophyte and sporophyte in ferns, cone differentiation in gymnosperms and floral meristem identity in flowering plants. However, their putative plesiomorphic role in plant reproductive transition in vascular plants remains untested. RESULTS: We perform Maximum Likelihood (ML) phylogenetic analyses for the LFY gene lineage in embryophytes with expanded sampling in lycophytes and ferns. We recover the previously identified seed plant duplication that results in LEAFY and NEEDLY paralogs. In addition, we recover multiple species-specific duplications in ferns and lycophytes and large-scale duplications possibly correlated with the occurrence of whole genome duplication (WGD) events in Equisetales and Salviniales. To test putative roles in diverse ferns and lycophytes we perform LFY expression analyses in Adiantum raddianum, Equisetum giganteum and Selaginella moellendorffii. Our results show that LFY genes are active in vegetative and reproductive tissues, with higher expression in early fertile developmental stages and during sporangia differentiation. CONCLUSIONS: Our data point to previously unrecognized roles of LFY genes in sporangia differentiation in lycophytes and ferns and suggests that functions linked to reproductive structure development are not exclusive to seed plant LFY homologs.

Gene expression underlying floral epidermal specialization in Aristolochia fimbriata (Aristolochiaceae).

BACKGROUND AND AIMS: The epidermis constitutes the outermost tissue of the plant body. Although it plays major structural, physiological and ecological roles in embryophytes, the molecular mechanisms controlling epidermal cell fate, differentiation and trichome development have been scarcely studied across angiosperms, and remain almost unexplored in floral organs. METHODS: In this study, we assess the spatio-temporal expression patterns of GL2, GL3, TTG1, TRY, MYB5, MYB6, HDG2, MYB106-like, WIN1 and RAV1-like homologues in the magnoliid Aristolochia fimbriata (Aristolochiaceae) by using comparative RNA-sequencing and in situ hybridization assays. KEY RESULTS: Genes involved in Aristolochia fimbriata trichome development vary depending on the organ where they are formed. Stem, leaf and pedicel trichomes recruit most of the transcription factors (TFs) described above. Conversely, floral trichomes only use a small subset of genes including AfimGL2, AfimRAV1-like, AfimWIN1, AfimMYB106-like and AfimHDG2. The remaining TFs, AfimTTG1, AfimGL3, AfimTRY, AfimMYB5 and AfimMYB6, are restricted to the abaxial (outer) and the adaxial (inner) pavement epidermal cells. CONCLUSIONS: We re-evaluate the core genetic network shaping trichome fate in flowers of an early-divergent angiosperm lineage and show a morphologically diverse output with a simpler genetic mechanism in place when compared to the models Arabidopsis thaliana and Cucumis sativus. In turn, our results strongly suggest that the canonical trichome gene expression appears to be more conserved in vegetative than in floral tissues across angiosperms.

Evolution of Class II TCP genes in perianth bearing Piperales and their contribution to the bilateral calyx in Aristolochia.

Controlled spatiotemporal cell division and expansion are responsible for floral bilateral symmetry. Genetic studies have pointed to class II TCP genes as major regulators of cell division and floral patterning in model core eudicots. Here we study their evolution in perianth-bearing Piperales and their expression in Aristolochia, a rare occurrence of bilateral perianth outside eudicots and monocots. The evolution of class II TCP genes reveals single-copy CYCLOIDEA-like genes and three paralogs of CINCINNATA (CIN) in early diverging angiosperms. All class II TCP genes have independently duplicated in Aristolochia subgenus Siphisia. Also CIN2 genes duplicated before the diversification of Saruma and Asarum. Sequence analysis shows that CIN1 and CIN3 share motifs with Cyclin proteins and CIN2 genes have lost the miRNA319a binding site. Expression analyses of all paralogs of class II TCP genes in Aristolochia fimbriata point to a role of CYC and CIN genes in maintaining differential perianth expansion during mid- and late flower developmental stages by promoting cell division in the distal and ventral portion of the limb. It is likely that class II TCP genes also contribute to cell division in the leaf, the gynoecium and the ovules in A. fimbriata.

Class I KNOX Is Related to Determinacy during the Leaf Development of the Fern Mickelia scandens (Dryopteridaceae).

Unlike seed plants, ferns leaves are considered to be structures with delayed determinacy, with a leaf apical meristem similar to the shoot apical meristems. To better understand the meristematic organization during leaf development and determinacy control, we analyzed the cell divisions and expression of Class I KNOX genes in Mickelia scandens, a fern that produces larger leaves with more pinnae in its climbing form than in its terrestrial form. We performed anatomical, in situ hybridization, and qRT-PCR experiments with histone H4 (cell division marker) and Class I KNOX genes. We found that Class I KNOX genes are expressed in shoot apical meristems, leaf apical meristems, and pinnae primordia. During early development, cell divisions occur in the most distal regions of the analyzed structures, including pinnae, and are not restricted to apical cells. Fern leaves and pinnae bear apical meristems that may partially act as indeterminate shoots, supporting the hypothesis of homology between shoots and leaves. Class I KNOX expression is correlated with indeterminacy in the apex and leaf of ferns, suggesting a conserved function for these genes in euphyllophytes with compound leaves.

Floral MADS-box protein interactions in the early diverging angiosperm Aristolochia fimbriata Cham. (Aristolochiaceae: Piperales).

Floral identity MADS-box A, B, C, D, E, and AGL6 class genes are predominantly single copy in Magnoliids, and predate the whole genome duplication (WGD) events in monocots and eudicots. By comparison with the model species Arabidopsis thaliana, the expression patterns of B-, C-, and D-class genes in stamen, carpel, and ovules are conserved in Aristolochia fimbriata, whereas A-, E-class, and AGL6 genes have different expression patterns. Nevertheless, the interactions of these proteins that act through multimeric complexes remain poorly known in early divergent angiosperms. This study evaluates protein interactions among all floral MADS-box A. fimbriata proteins using the Yeast Two Hybrid System (Y2H). We found no homodimers and less heterodimers formed by AfimFUL when compared to AfimAGL6, which allowed us to suggest AGL6 homodimers in combination with AfimSEP2 as the most likely tetramer in sepal identity. We found AfimAP3-AfimPI obligate heterodimers and AfimAG-AfimSEP2 protein interactions intact suggesting conserved stamen and carpel tetrameric complexes in A. fimbriata. We observed a broader interaction partner set for AfimSEP2 than for its paralog AfimSEP1. We show conserved and exclusive MADS-box protein interactions in A. fimbriata in comparison with other eudicot and monocot model species in order to establish plesiomorphic MADS-box protein floral networks in angiosperms.

Deep into the Aristolochia Flower: Expression of C, D, and E-Class Genes in Aristolochia fimbriata (Aristolochiaceae).

Aristolochia fimbriata (Aristolochiaceae) is a member of an early diverging lineage of flowering plants and a promising candidate for evo-devo studies. Aristolochia flowers exhibit a unique floral synorganization that consists of a monosymmetric and petaloid calyx formed by three congenitally fused sepals, and a gynostemium formed by the congenital fusion between stamens and the stigmatic region of the carpels. This floral ground plan atypical in the magnoliids can be used to evaluate the role of floral organ identity MADS-box genes during early flower evolution. In this study, we present in situ hybridization experiments for the homologs of the canonical C-, D-, and E-class genes. Spatiotemporal expression of the C-class gene AfimAG is restricted to stamens, ovary, and ovules, suggesting a conserved stamen and carpel identity function, consistent with that reported in core-eudicots and monocots. The D-class gene AfimSTK is detected in the anthers, the stigmas, the ovary, the ovules, the fruit, and the seeds, suggesting conserved roles in ovule and seed identity and unique roles in stamens, ovary, and fruit development. In addition, AfimSTK expression patterns in areas of organ abscission and dehiscence zones suggest putative roles linked to senescence processes. We found that both E-class genes are expressed in the anthers and the ovary; however, AfimSEP2 exhibits higher expression compared to AfimSEP1. These findings provide a comprehensive picture of the ancestral expression patterns of the canonical MADS-box floral organ identity genes and the foundations for further comparative analyses in other magnoliids.

B-function expression in the flower center underlies the homeotic phenotype of Lacandonia schismatica (Triuridaceae).

Spontaneous homeotic transformations have been described in natural populations of both plants and animals, but little is known about the molecular-genetic mechanisms underlying these processes in plants. In the ABC model of floral organ identity in Arabidopsis thaliana, the B- and C-functions are necessary for stamen morphogenesis, and C alone is required for carpel identity. We provide ABC model-based molecular-genetic evidence that explains the unique inside-out homeotic floral organ arrangement of the monocotyledonous mycoheterotroph species Lacandonia schismatica (Triuridaceae) from Mexico. Whereas a quarter million flowering plant species bear central carpels surrounded by stamens, L. schismatica stamens occur in the center of the flower and are surrounded by carpels. The simplest explanation for this is that the B-function is displaced toward the flower center. Our analyses of the spatio-temporal pattern of B- and C-function gene expression are consistent with this hypothesis. The hypothesis is further supported by conservation between the B-function genes of L. schismatica and Arabidopsis, as the former are able to rescue stamens in Arabidopsis transgenic complementation lines, and Ls-AP3 and Ls-PI are able to interact with each other and with the corresponding Arabidopsis B-function proteins in yeast. Thus, relatively simple molecular modifications may underlie important morphological shifts in natural populations of extant plant taxa.