Nectar spurs (tubular outgrowths of floral organs) have long fascinated biologists. However, given that no model species possess nectar spurs, there is still much to learn about their development. In this study we combined morphological analysis with comparative transcriptomics to gain a global insight into the morphological and molecular basis of spur outgrowth in Linaria. Whole transcriptome sequencing was performed on two related species at three key developmental stages (identified by our morphological analysis), one with a spur (Linaria vulgaris), and one without a spur (Antirrhinum majus). A list of spur-specific genes was selected, on which we performed a gene enrichment analysis. Results from our RNA-seq analysis agreed with our morphological observations. We describe gene activity during spur development and provide a catalogue of spur-specific genes. Our list of spur-specific genes was enriched for genes connected to the plant hormones cytokinin, auxin and gibberellin. We present a global view of the genes involved in spur development in L. vulgaris, and define a suite of genes which are specific to spur development. This work provides candidate genes for spur outgrowth and development in L. vulgaris which can be investigated in future studies.

BACKGROUND: Spur, a structure capable of producing and storing nectar, not only plays a vital role in the pollination process but also promotes the rapid diversification of some plant lineages, which is considered a key innovation in plants. Spur is the focus of many studies, such as evolution and ecological hypothesis, but the current understanding of spur development is limited. High-throughput sequencing of Impatiens uliginosa was carried out to study the molecular mechanism of its spur development, which is believed to provide some insights into the spur development of Impatiens.
RESULTS: Transcriptomic sequencing and analysis were performed on spurs and limbs of I. uliginosa at three developmental stages. A total of 47.83 Gb of clean data were obtained, and 49,716 unigene genes were assembled. After comparison with NR, Swiss-Prot, Pfam, COG, GO and KEGG databases, a total of 27,686 genes were annotated successfully. Through comparative analysis, 19,356 differentially expressed genes were found and enriched into 208 GO terms and 146 KEGG pathways, among which plant hormone signal transduction was the most significantly enriched pathway. One thousand thirty-two transcription factors were identified, which belonged to 33 TF families such as MYB, bHLH and TCP. Twenty candidate genes that may be involved in spur development were screened and verified by qPCR, such as SBP, IAA and ABP.
CONCLUSIONS: Transcriptome data of different developmental stages of spurs were obtained, and a series of candidate genes related to spur development were identified. The importance of genes related to cell cycle, cell division, cell elongation and hormones in spur development was clarified. This study provided valuable information and resources for understanding the molecular mechanism of spur development in Impatiens.

Nectar spur is a hollow extension of certain flower parts and shows strikingly diverse size and shape in Aquilegia. Nectar spur development is involved in cell division and expansion processes. The basic helix-loop-helix (bHLH) transcription factors (TFs) control a diversity of organ morphogenesis, including cell division and cell expansion processes. However, the role of bHLH genes in nectar spur development in Aquilegia is mainly unknown. We conducted a genome-wide identification of the bHLH gene family in Aquilegia to determine structural characteristics and phylogenetic relationships, and to analyze expression profiles of these genes during the development of nectar spur in spurless and spurred species. A total of 120 AqbHLH genes were identified from the Aquilegia coerulea genome. The phylogenetic tree showed that AqbHLH proteins were divided into 15 subfamilies, among which S7 and S8 subfamilies occurred marked expansion. The AqbHLH genes in the same clade had similar motif composition and gene structure characteristics. Conserved residue analysis indicated nineteen residues with conservation of more than 50% were found in the four conserved regions. In the upstream sequence of AqbHLH genes, the light-responsive element was the most abundant cis-acting element. Eighteen AqbHLH genes showed syntenic relationships, and eight genes from four syntenic pairs underwent tandem duplications. According to the expression profiling analysis by public RNA-Seq data and qRT-PCR results, five AqbHLH genes, including AqbHLH027, AqbHLH046, AqbHLH082, AqbHLH083 and AqbHLH092, were differentially expressed between different tissues in A. coerulea at early developmental stages, as well as between spurless and spurred Aquilegia species. Of them, AqbHLH046 was not only highly expressed in spur compared with blade, but also showed higher expression levels in spurred species than spurless specie, suggesting it plays an essential role in the development of spur by regulating cell division. This study lays a foundation to investigate the function of AqbHLH genes family in nectar spur development, and has potential implications for speciation and genetic breeding in the genus Aquilegia.

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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.

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Variations of nectar spur length allow pollinators to utilize resources in novel ways, leading to the different selective pressures on spurs and allowing taxa to diversify. However, the mechanisms underlying spur length variation remain unclear. Interspecific comparisons of spur length suggest that both cell division and anisotropic expansion could explain the changes of spur length, and that hormone-related genes contribute to the process of spur formation. In contrast, little is known about intraspecific spur length variation. In Aquilegia rockii, spur length varies strikingly, ranging from 1 mm to 18 mm. To examine the potential mechanisms underlying spur length variation in A. rockii, we observed cell morphology and analyzed RNA-seq of short- and long-spurred flowers. Scanning electron microscopy revealed that at two positions on spurs there were no differences in either cell density or cell anisotropy between short- and long-spurred flowers, suggesting that in A. rockii changes in cell number may explain variations in spur length. In addition, we screened transcriptomes of short- and long-spurred flowers for differentially expressed genes; this screen identified several genes linked to cell division (e.g., F-box, CDKB2-2, and LST8), a finding which is consistent with our analysis of the cellular morphology of spurs. However, we did not find any highly expressed genes involved in the hormone pathway in long-spurred flowers. In contrast to previous hypotheses that anisotropic cell expansion leads to interspecific spur variation in Aquilegia, our results suggest that cell number changes and related genes are mainly responsible for spur length variations of A. rockii. Furthermore, the underlying mechanisms of similar floral traits in morphology may be quite different, enriching our understanding of the mechanisms of flower diversity in angiosperms.

BACKGROUND: Petal nectar spurs, which facilitate pollination through animal attraction and pollen placement, represent a key innovation promoting diversification in the genus Aquilegia (Ranunculaceae). Identifying the genetic components that contribute to the development of these three-dimensional structures will inform our understanding of the number and types of genetic changes that are involved in the evolution of novel traits. In a prior study, gene expression between two regions of developing petals, the laminar blade and the spur cup, was compared at two developmental stages in the horticultural variety A. coerulea \'Origami\'. Several hundred genes were differentially expressed (DE) between the blade and spur at both developmental stages. In order to narrow in on a set of genes crucial to early spur formation, the current study uses RNA sequencing (RNAseq) to conduct comparative expression analyses of petals from five developmental stages between four Aquilegia species, three with morphologically variable nectar spurs, A. sibirica, A. formosa, and A. chrysantha, and one that lacks nectar spurs, A. ecalcarata.
RESULTS: Petal morphology differed increasingly between taxa across the developmental stages assessed, with petals from all four taxa being indistinguishable pre-spur formation at developmental stage 1 (DS1) and highly differentiated by developmental stage 5 (DS5). In all four taxa, genes involved in mitosis were down-regulated over the course of the assessed developmental stages, however, many genes involved in mitotic processes remained expressed at higher levels later in development in the spurred taxa. A total of 690 genes were identified that were consistently DE between the spurred taxa and A. ecalcarata at all five developmental stages. By comparing these genes with those identified as DE between spur and blade tissue in A. coerulea \'Origami\', a set of only 35 genes was identified that shows consistent DE between petal samples containing spur tissue versus those without spur tissue.
CONCLUSIONS: The results of this study suggest that expression differences in very few loci are associated with the presence and absence of spurs. In general, it appears that the spurless petals of A. ecalcarata cease cell divisions and enter the cell differentiation phase at an earlier developmental time point than those that produce spurs. This much more tractable list of 35 candidates genes will greatly facilitate targeted functional studies to assess the genetic control and evolution of petal spurs in Aquilegia.

Floral nectar spurs are widely considered a key innovation promoting diversification in angiosperms by means of pollinator shifts. We investigated the macroevolutionary dynamics of nectar spurs in the tribe Antirrhineae (Plantaginaceae), which contains 29 genera and 300-400 species (70-80% spurred). The effect of nectar spurs on diversification was tested, with special focus on Linaria, the genus with the highest number of species. We generated the most comprehensive phylogeny of Antirrhineae to date and reconstructed the evolution of nectar spurs. Diversification rate heterogeneity was investigated using trait-dependent and trait-independent methods, and accounting for taxonomic uncertainty. The association between changes in spur length and speciation was examined within Linaria using model testing and ancestral state reconstructions. We inferred four independent acquisitions of nectar spurs. Diversification analyses revealed that nectar spurs are loosely associated with increased diversification rates. Detected rate shifts were delayed by 5-15 Myr with respect to the acquisition of the trait. Active evolution of spur length, fitting a speciational model, was inferred in Linaria, which is consistent with a scenario of pollinator shifts driving diversification. Nectar spurs played a role in diversification of the Antirrhineae, but diversification dynamics can only be fully explained by the complex interaction of multiple biotic and abiotic factors.

OBJECTIVE: Fumarioideae (20 genera, 593 species) is a clade of Papaveraceae (Ranunculales) characterized by flowers that are either disymmetric (i.e. two perpendicular planes of bilateral symmetry) or zygomorphic (i.e. one plane of bilateral symmetry). In contrast, the other subfamily of Papaveraceae, Papaveroideae (23 genera, 230 species), has actinomorphic flowers (i.e. more than two planes of symmetry). Understanding of the evolution of floral symmetry in this clade has so far been limited by the lack of a reliable phylogenetic framework. Pteridophyllum (one species) shares similarities with Fumarioideae but has actinomorphic flowers, and the relationships among Pteridophyllum, Papaveroideae and Fumarioideae have remained unclear. This study reassesses the evolution of floral symmetry in Papaveraceae based on new molecular phylogenetic analyses of the family.
METHODS: Maximum likelihood, Bayesian and maximum parsimony phylogenetic analyses of Papaveraceae were conducted using six plastid markers and one nuclear marker, sampling Pteridophyllum, 18 (90 %) genera and 73 species of Fumarioideae, 11 (48 %) genera and 11 species of Papaveroideae, and a wide selection of outgroup taxa. Floral characters recorded from the literature were then optimized onto phylogenetic trees to reconstruct ancestral states using parsimony, maximum likelihood and reversible-jump Bayesian approaches.
RESULTS: Pteridophyllum is not nested in Fumarioideae. Fumarioideae are monophyletic and Hypecoum (18 species) is the sister group of the remaining genera. Relationships within the core Fumarioideae are well resolved and supported. Dactylicapnos and all zygomorphic genera form a well-supported clade nested among disymmetric taxa.
CONCLUSIONS: Disymmetry of the corolla is a synapomorphy of Fumarioideae and is strongly correlated with changes in the androecium and differentiation of middle and inner tepal shape (basal spurs on middle tepals). Zygomorphy subsequently evolved from disymmetry either once (with a reversal in Dactylicapnos) or twice (Capnoides, other zygomorphic Fumarioideae) and appears to be correlated with the loss of one nectar spur.