Complex developmental and transcriptional dynamics underlie pollinator-driven evolutionary transitions in nectar spur morphology in Aquilegia (columbine).

PREMISE: Determining the developmental programs underlying morphological variation is key to elucidating the evolutionary processes that generated the stunning biodiversity of the angiosperms. Here, we characterized the developmental and transcriptional dynamics of the elaborate petal nectar spur of Aquilegia (columbine) in species with contrasting pollination syndromes and spur morphologies. METHODS: We collected petal epidermal cell number and length data across four Aquilegia species, two with short, curved nectar spurs of the bee-pollination syndrome and two with long, straight spurs of the hummingbird-pollination syndrome. We also performed RNA-seq on A. brevistyla (bee) and A. canadensis (hummingbird) distal and proximal spur compartments at multiple developmental stages. Finally, we intersected these data sets with a previous QTL mapping study on spur length and shape to identify new candidate loci. RESULTS: The differential growth between the proximal and distal surfaces of curved spurs is primarily driven by differential cell division. However, independent transitions to straight spurs in the hummingbird syndrome have evolved by increasing differential cell elongation between spur surfaces. The RNA-seq data reveal these tissues to be transcriptionally distinct and point to auxin signaling as being involved with the differential cell elongation responsible for the evolution of straight spurs. We identify several promising candidate genes for future study. CONCLUSIONS: Our study, taken together with previous work in Aquilegia, reveals the complexity of the developmental mechanisms underlying trait variation in this system. The framework we established here will lead to exciting future work examining candidate genes and processes involved in the rapid radiation of the genus.

Genetic architecture underlying variation in floral meristem termination in Aquilegia.

Floral organs are produced by floral meristems (FMs), which harbor stem cells in their centers. Since each flower only has a finite number of organs, the stem cell activity of an FM will always terminate at a specific time point, a process termed floral meristem termination (FMT). Variation in the timing of FMT can give rise to floral morphological diversity, but how this process is fine-tuned at a developmental and evolutionary level is poorly understood. Flowers from the genus Aquilegia share identical floral organ arrangement except for stamen whorl number (SWN), making Aquilegia a well-suited system for investigation of this process: differences in SWN between species represent differences in the timing of FMT. By crossing A. canadensis and A. brevistyla, quantitative trait locus (QTL) mapping has revealed a complex genetic architecture with seven QTL. We explored potential candidate genes under each QTL and characterized novel expression patterns of select loci of interest using in situ hybridization. To our knowledge, this is the first attempt to dissect the genetic basis of how natural variation in the timing of FMT is regulated, and our results provide insight into how floral morphological diversity can be generated at the meristematic level.

Genetic architecture of floral traits in bee- and hummingbird-pollinated sister species of Aquilegia (columbine).

Interactions with animal pollinators have helped shape the stunning diversity of flower morphologies across the angiosperms. A common evolutionary consequence of these interactions is that some flowers have converged on suites of traits, or pollination syndromes, that attract and reward specific pollinator groups. Determining the genetic basis of these floral pollination syndromes can help us understand the processes that contributed to the diversification of the angiosperms. Here, we characterize the genetic architecture of a bee-to-hummingbird pollination shift in Aquilegia (columbine) using QTL mapping of 17 floral traits encompassing color, nectar composition, and organ morphology. In this system, we find that the genetic architectures underlying differences in floral color are quite complex, and we identify several likely candidate genes involved in anthocyanin and carotenoid floral pigmentation. Most morphological and nectar traits also have complex genetic underpinnings; however, one of the key floral morphological phenotypes, nectar spur curvature, is shaped by a single locus of large effect.

POPOVICH, encoding a C2H2 zinc-finger transcription factor, plays a central role in the development of a key innovation, floral nectar spurs, in Aquilegia.

The evolution of novel features, such as eyes or wings, that allow organisms to exploit their environment in new ways can lead to increased diversification rates. Therefore, understanding the genetic and developmental mechanisms involved in the origin of these key innovations has long been of interest to evolutionary biologists. In flowering plants, floral nectar spurs are a prime example of a key innovation, with the independent evolution of spurs associated with increased diversification rates in multiple angiosperm lineages due to their ability to promote reproductive isolation via pollinator specialization. As none of the traditional plant model taxa have nectar spurs, little is known about the genetic and developmental basis of this trait. Nectar spurs are a defining feature of the columbine genus Aquilegia (Ranunculaceae), a lineage that has experienced a relatively recent and rapid radiation. We use a combination of genetic mapping, gene expression analyses, and functional assays to identify a gene crucial for nectar spur development, POPOVICH (POP), which encodes a C2H2 zinc-finger transcription factor. POP plays a central role in regulating cell proliferation in the Aquilegia petal during the early phase (phase I) of spur development and also appears to be necessary for the subsequent development of nectaries. The identification of POP opens up numerous avenues for continued scientific exploration, including further elucidating of the genetic pathway of which it is a part, determining its role in the initial evolution of the Aquilegia nectar spur, and examining its potential role in the subsequent evolution of diverse spur morphologies across the genus.

Temporal transcriptional logic of dynamic regulatory networks underlying nitrogen signaling and use in plants.

This study exploits time, the relatively unexplored fourth dimension of gene regulatory networks (GRNs), to learn the temporal transcriptional logic underlying dynamic nitrogen (N) signaling in plants. Our "just-in-time" analysis of time-series transcriptome data uncovered a temporal cascade of cis elements underlying dynamic N signaling. To infer transcription factor (TF)-target edges in a GRN, we applied a time-based machine learning method to 2,174 dynamic N-responsive genes. We experimentally determined a network precision cutoff, using TF-regulated genome-wide targets of three TF hubs (CRF4, SNZ, and CDF1), used to "prune" the network to 155 TFs and 608 targets. This network precision was reconfirmed using genome-wide TF-target regulation data for four additional TFs (TGA1, HHO5/6, and PHL1) not used in network pruning. These higher-confidence edges in the GRN were further filtered by independent TF-target binding data, used to calculate a TF "N-specificity" index. This refined GRN identifies the temporal relationship of known/validated regulators of N signaling (NLP7/8, TGA1/4, NAC4, HRS1, and LBD37/38/39) and 146 additional regulators. Six TFs-CRF4, SNZ, CDF1, HHO5/6, and PHL1-validated herein regulate a significant number of genes in the dynamic N response, targeting 54% of N-uptake/assimilation pathway genes. Phenotypically, inducible overexpression of CRF4 in planta regulates genes resulting in altered biomass, root development, and 15NO3- uptake, specifically under low-N conditions. This dynamic N-signaling GRN now provides the temporal "transcriptional logic" for 155 candidate TFs to improve nitrogen use efficiency with potential agricultural applications. Broadly, these time-based approaches can uncover the temporal transcriptional logic for any biological response system in biology, agriculture, or medicine.

New phylogenetic insights toward developing a natural generic classification of African angraecoid orchids (Vandeae, Orchidaceae).

Despite significant progress made in recent years toward developing an infrafamilial classification of Orchidaceae, our understanding of relationships among and within tribal and subtribal groups of epidendroid orchids remains incomplete. To reassess generic delimitation among one group of these epidendroids, the African angraecoids, phylogenetic relationships were inferred from DNA sequence data from three regions, ITS, matK, and the trnL-trnF intergenic spacer, obtained from a broadly representative sample of taxa. Parsimony and Bayesian analyses yielded highly resolved trees that are in clear agreement and show significant support for many key clades within subtribe Angraecinae s.l. Angraecoid orchids comprise two well-supported clades: an African/American group and an Indian Ocean group. Molecular results also support many previously proposed relationships among genera, but also reveal some unexpected relationships. The genera Aerangis, Ancistrorhynchus, Bolusiella, Campylocentrum, Cyrtorchis, Dendrophylax, Eurychone, Microcoelia, Nephrangis, Podangis and Solenangis are all shown to be monophyletic, but Angraecopsis, Diaphananthe and Margelliantha are polyphyletic. Diaphananthe forms three well-supported clades, one of which might represent a new genus, and Rhipidoglossum is paraphyletic with respect to Cribbia and Rhaesteria, and also includes taxa currently assigned to Margelliantha. Tridactyle too is paraphyletic as Eggelingia is embedded within it. The large genus Angraecum is confirmed to be polyphyletic and several groups will have to be recognized as separate genera, including sections Dolabrifolia and Hadrangis. The recently segregated genus Pectinariella (previously recognized as A. sect. Pectinaria) is polyphyletic and its Continental African species will have to be removed. Similarly, some of the species recently transferred to Angraecoides that were previously placed in Angraecum sects. Afrangraecum and Conchoglossum will have to be moved and described as a new genus.

"Hit-and-Run" transcription: de novo transcription initiated by a transient bZIP1 "hit" persists after the "run".

BACKGROUND: Dynamic transcriptional regulation is critical for an organism's response to environmental signals and yet remains elusive to capture. Such transcriptional regulation is mediated by master transcription factors (TF) that control large gene regulatory networks. Recently, we described a dynamic mode of TF regulation named "hit-and-run". This model proposes that master TF can interact transiently with a set of targets, but the transcription of these transient targets continues after the TF dissociation from the target promoter. However, experimental evidence validating active transcription of the transient TF-targets is still lacking. RESULTS: Here, we show that active transcription continues after transient TF-target interactions by tracking de novo synthesis of RNAs made in response to TF nuclear import. To do this, we introduced an affinity-labeled 4-thiouracil (4tU) nucleobase to specifically isolate newly synthesized transcripts following conditional TF nuclear import. Thus, we extended the TARGET system (Transient Assay Reporting Genome-wide Effects of Transcription factors) to include 4tU-labeling and named this new technology TARGET-tU. Our proof-of-principle example is the master TF Basic Leucine Zipper 1 (bZIP1), a central integrator of metabolic signaling in plants. Using TARGET-tU, we captured newly synthesized mRNAs made in response to bZIP1 nuclear import at a time when bZIP1 is no longer detectably bound to its target. Thus, the analysis of de novo transcripomics demonstrates that bZIP1 may act as a catalyst TF to initiate a transcriptional complex ("hit"), after which active transcription by RNA polymerase continues without the TF being bound to the gene promoter ("run"). CONCLUSION: Our findings provide experimental proof for active transcription of transient TF-targets supporting a "hit-and-run" mode of action. This dynamic regulatory model allows a master TF to catalytically propagate rapid and broad transcriptional responses to changes in environment. Thus, the functional read-out of de novo transcripts produced by transient TF-target interactions allowed us to capture new models for genome-wide transcriptional control.

Generic delimitations, biogeography and evolution in the tribe Coleeae (Bignoniaceae), endemic to Madagascar and the smaller islands of the western Indian Ocean.

This study presents the most complete generic phylogenetic framework to date for the tribe Coleeae (Bignoniaceae), which is endemic to Madagascar and the other smaller islands in the western part of the Indian Ocean. The study is based on plastid and nuclear DNA regions and includes 47 species representing the five currently recognized genera (including all the species occurring in the western Indian Ocean region). Bayesian and maximum likelihood analyses supported (i) the monophyly of the tribe, (ii) the monophyly of Phylloctenium, Phyllarthron and Rhodocolea and (iii) the paraphyly of Colea due to the inclusion of species of Ophiocolea. The latter genus was also recovered paraphyletic due to the inclusion of two species of Colea (C. decora and C. labatii). The taxonomic implications of the mutual paraphyly of these two genera are discussed in light of morphological evidence, and it is concluded that the two genera should be merged, and the necessary new nomenclatural combinations are provided. The phylogenetic framework shows Phylloctenium, which is endemic to Madagascar and restricted to dry ecosystems, as basal and sister to the rest of the tribe, suggesting Madagascar to be the centre of origin of this clade. The remaining genera are diversified mostly in humid ecosystems, with evidence of multiple dispersals to the neighboring islands, including at least two to the Comoros, one to Mauritius and one to the Seychelles. Finally, we hypothesize that the ecological success of this tribe might have been triggered by a shift of fruit-dispersal mode from wind to lemur.

The histone methyltransferase SDG8 mediates the epigenetic modification of light and carbon responsive genes in plants.

BACKGROUND: Histone methylation modifies the epigenetic state of target genes to regulate gene expression in the context of developmental and environmental changes. Previously, we used a positive genetic screen to identify an Arabidopsis mutant, cli186, which was impaired in carbon and light signaling. Here, we report a deletion of the Arabidopsis histone methyltransferase SDG8 in this mutant (renamed sdg8-5), which provides a unique opportunity to study the global function of a specific histone methyltransferase within a multicellular organism. RESULTS: To assess the specific role of SDG8, we examine how the global histone methylation patterns and transcriptome were altered in the sdg8-5 deletion mutant compared to wild type, within the context of transient light and carbon treatments. Our results reveal that the sdg8 deletion is associated with a significant reduction of H3K36me3, preferentially towards the 3' end of the gene body, accompanied by a reduction in gene expression. We uncover 728 direct targets of SDG8 that have altered methylation in the sdg8-5 mutant and are also bound by SDG8. As a group, this set of SDG8 targets is enriched in specific biological processes including defense, photosynthesis, nutrient metabolism and energy metabolism. Importantly, 64% of these SDG8 targets are responsive to light and/or carbon signals. CONCLUSIONS: The histone methyltransferase SDG8 functions to regulate the H3K36 methylation of histones associated with gene bodies in Arabidopsis. The H3K36me3 mark in turn is associated with high-level expression of a specific set of light and/or carbon responsive genes involved in photosynthesis, metabolism and energy production.

Dissecting the molecular signatures of apical cell-type shoot meristems from two ancient land plant lineages.

Shoot apical meristem (SAM) structure varies markedly within the land plants. The SAMs of many seedless vascular plants contain a conspicuous inverted, pyramidal cell called the apical cell (AC), which is unidentified in angiosperms. In this study, we use transcriptomic sequencing with precise laser microdissections of meristem subdomains to define the molecular signatures of anatomically distinct zones from the AC-type SAMs of a lycophyte (Selaginella moellendorffii) and a monilophyte (Equisetum arvense). The two model species for this study represent vascular plant lineages that diverged > 400 million yr ago. Our data comprise comprehensive molecular signatures for the distinct subdomains within AC-type SAMs, an anatomical anomaly whose functional significance has been debated in the botanical literature for over two centuries. Moreover, our data provide molecular support for distinct gene expression programs between the AC-type SAMs of Selaginella and Equisetum, as compared with the SAM transcriptome of the angiosperm maize. The results are discussed in light of the functional significance and evolutionary success of the AC-type SAM within the embryophytes.

Hit-and-run transcriptional control by bZIP1 mediates rapid nutrient signaling in Arabidopsis.

The dynamic nature of gene regulatory networks allows cells to rapidly respond to environmental change. However, the underlying temporal connections are missed, even in kinetic studies, as transcription factor (TF) binding within at least one time point is required to identify primary targets. The TF-regulated but unbound genes are dismissed as secondary targets. Instead, we report that these genes comprise transient TF-target interactions most relevant to rapid signal transduction. We temporally perturbed a master TF (Basic Leucine Zipper 1, bZIP1) and the nitrogen (N) signal it transduces and integrated TF regulation and binding data from the same cell samples. Our enabling approach could identify primary TF targets based solely on gene regulation, in the absence of TF binding. We uncovered three classes of primary TF targets: (i) poised (TF-bound but not TF-regulated), (ii) stable (TF-bound and TF-regulated), and (iii) transient (TF-regulated but not TF-bound), the largest class. Unexpectedly, the transient bZIP1 targets are uniquely relevant to rapid N signaling in planta, enriched in dynamic N-responsive genes, and regulated by TF and N signal interactions. These transient targets include early N responders nitrate transporter 2.1 and NIN-like protein 3, bound by bZIP1 at 1-5 min, but not at later time points following TF perturbation. Moreover, promoters of these transient targets are uniquely enriched with cis-regulatory motifs coinherited with bZIP1 binding sites, suggesting a recruitment role for bZIP1. This transient mode of TF action supports a classic, but forgotten, "hit-and-run" transcription model, which enables a "catalyst TF" to activate a large set of targets within minutes of signal perturbation.