Publications

BACKGROUND AND AIMS: Species in the holoparasitic plant family Rafflesiaceae exhibit one of the most highly modified vegetative bodies in flowering plants. Apart from the flower shoot and associated bracts, the parasite is a mycelium-like endophyte living inside their grapevine hosts. This study provides a comprehensive treatment of the endophytic vegetative body for all three genera of Rafflesiaceae (Rafflesia, Rhizanthes and Sapria), and reports on the cytology and development of the endophyte, including its structural connection to the host, shedding light on the poorly understood nature of this symbiosis. METHODS: Serial sectioning and staining with non-specific dyes, periodic-Schiff's reagent and aniline blue were employed in order to characterize the structure of the endophyte across a phylogenetically diverse sampling. KEY RESULTS: A previously identified difference in the nuclear size between Rafflesiaceae endophytes and their hosts was used to investigate the morphology and development of the endophytic body. The endophytes generally comprise uniseriate filaments oriented radially within the host root. The emergence of the parasite from the host during floral development is arrested in some cases by an apparent host response, but otherwise vegetative growth does not appear to elicit suppression by the host. CONCLUSIONS: Rafflesiaceae produce greatly reduced and modified vegetative bodies even when compared with the other holoparasitic angiosperms once grouped with Rafflesiaceae, which possess some vegetative differentiation. Based on previous studies of seeds together with these findings, it is concluded that the endophyte probably develops directly from a proembryo, and not from an embryo proper. Similarly, the flowering shoot arises directly from the undifferentiated endophyte. These filaments produce a protocorm in which a shoot apex originates endogenously by formation of a secondary morphological surface. This degree of modification to the vegetative body is exceptional within angiosperms and warrants additional investigation. Furthermore, the study highlights a mechanical isolation mechanism by which the host may defend itself from the parasite.

The Medicago truncatula WUSCHEL-related homeobox (WOX) gene, STENOFOLIA (STF), plays a key role in leaf blade outgrowth by promoting cell proliferation at the adaxial-abaxial junction. STF functions primarily as a transcriptional repressor, but the underlying molecular mechanism is unknown. Here, we report the identification of a protein interaction partner and a direct target, shedding light on the mechanism of STF function. Two highly conserved motifs in the C-terminal domain of STF, the WUSCHEL (WUS) box and the STF box, cooperatively recruit TOPLESS (Mt-TPL) family corepressors, and this recruitment is required for STF function, as deletion of these two domains (STFdel) impaired blade outgrowth whereas fusing Mt-TPL to STFdel restored function. The homeodomain motif is required for direct repression of ASYMMETRIC LEAVES2 (Mt-AS2), silencing of which partially rescues the stf mutant phenotype. STF and LAMINALESS1 (LAM1) are functional orthologs. A single amino acid (Asn to Ile) substitution in the homeodomain abolished the repression of Mt-AS2 and STF's ability to complement the lam1 mutant of Nicotiana sylvestris. Our data together support a model in which STF recruits corepressors to transcriptionally repress its targets during leaf blade morphogenesis. We propose that recruitment of TPL/TPL-related proteins may be a common mechanism in the repressive function of modern/WUS clade WOX genes.

Flowers of the lower eudicot Aquilegia (columbine) possess morphological innovations, namely elaborate petal spurs and a fifth distinct organ identity, the staminodium, that are well suited to the investigation of key questions in developmental evolution. The recent evolution of these characteristics combined with a growing set of genetic and genomic resources has provided insight into how the traits arose and diversified. The petal spur appears to represent a key innovation that diversified largely via modification of specific aspects of cell expansion. In the case of the staminodium, gene duplication has played a role in allowing a novel organ identity to be carved out of the traditional ABC program.

The APETALA1/FRUITFULL (AP1/FUL) MADS box transcription factors are best known for the role of AP1 in Arabidopsis sepal and petal identity, the canonical A function of the ABC model of flower development. However, this gene lineage underwent multiple duplication events during angiosperm evolution, providing different taxa with unique gene complements. One such duplication correlates with the origin of the core eudicots, and produced the euAP1 and euFUL clades. Together, euAP1 and euFUL genes function in proper floral meristem identity and repression of axillary meristem growth. Independently, euAP1 genes function in floral meristem and sepal identity, whereas euFUL genes control phase transition, cauline leaf growth and fruit development. To investigate the impact of the core eudicot duplication on the functional diversification of this gene lineage, we studied the role of pre-duplication FUL-like genes in columbine (Aquilegia coerulea). Our results show that AqcFL1 genes are broadly expressed in vegetative and reproductive meristems, leaves and flowers. Virus-induced gene silencing of the loci results in plants with increased branching, shorter inflorescences with fewer flowers, and dramatic changes in leaf shape and complexity. However, aqcfl1 plants have normal flowers and fruits. Our results show that, in contrast to characterized AP1/FUL genes, the AqcFL1 loci are either genetically redundant or have been decoupled from the floral genetic program, and play a major role in leaf morphogenesis. We analyze the results in the context of the core eudicot duplication, and discuss the implications of our findings in terms of the genetic regulation of leaf morphogenesis in Aquilegia and other flowering plants.

BACKGROUND: Epigenetic regulation is necessary for maintaining gene expression patterns in multicellular organisms. The Polycomb Group (PcG) proteins form several complexes with important and deeply conserved epigenetic functions in both the plant and animal kingdoms. One such complex, the Polycomb Repressive Complex 2 (PRC2), is critical to many developmental processes in plants including the regulation of major developmental transitions. In addition, PRC2 restricts the expression domain of various transcription factor families in Arabidopsis, including the class I KNOX genes and several of the ABCE class MADS box genes. While the functions of these transcription factors are known to be deeply conserved, whether or not their regulation by PRC2 is similarly conserved remains an open question. RESULTS: Here we use virus-induced gene silencing (VIGS) to characterize the function of the PRC2 complex in lateral organ development of Aquilegia x coerulea 'Origami', a member of the lower eudicot order Ranunculales. Leaves with PRC2 down-regulation displayed a range of phenotypes including ruffled or curled laminae, additional lobing, and an increased frequency of higher order branching. Sepals and petals were also affected, being narrowed, distorted, or, in the case of the sepals, exhibiting partial homeotic transformation. Many of the petal limbs also had a particularly intense yellow coloration due to an accumulation of carotenoid pigments. We show that the A. x coerulea floral MADS box genes AGAMOUS1 (AqAG1), APETALA3-3 (AqAP3-3) and SEPALLATA3 (AqSEP3) are up-regulated in many tissues, while expression of the class I KNOX genes and several candidate genes involved in carotenoid production or degradation are largely unaffected. CONCLUSIONS: PRC2 targeting of several floral MADS box genes may be conserved in dicots, but other known targets do not appear to be. In the case of the type I KNOX genes, this may reflect a regulatory shift associated with the evolution of compound leaves.

The structural homology of the daffodil corona has remained a source of debate throughout the history of botany. Over the years it has been separately referred to as a modified petal stipule, stamen and tepal. Here we provide insights from anatomy and molecular studies to clarify the early developmental stages and position of corona initiation in Narcissus bulbocodium. We demonstrate that the corona initiates as six separate anlagen from hypanthial tissue between the stamens and perianth. Scanning electron microscope images and serial sections demonstrate that corona initiation occurs late in development, after the other floral whorls are fully developed. To define more precisely the identity of the floral structures, daffodil orthologues of the ABC floral organ identity genes were isolated and expression patterns were examined in perianth, stamens, carpel, hypanthial tube and corona tissue. Coupled with in situ hybridisation experiments, these analyses showed that the expression pattern of the C-class gene NbAGAMOUS in the corona is more similar to that of the stamens than that of the tepals. In combination, our results demonstrate that the corona of the daffodil N. bulbocodium exhibits stamen-like identity, develops independently from the orthodox floral whorls and is best interpreted as a late elaboration of the region between the petals and stamens associated with epigyny and the hypanthium.

Rafflesiaceae, which produce the world's largest flowers, have captivated the attention of biologists for nearly two centuries. Despite their fame, however, the developmental nature of the floral organs in these giants has remained a mystery. Most members of the family have a large floral chamber defined by a diaphragm. The diaphragm encloses the reproductive organs where pollination by carrion flies occurs. In lieu of a functional genetic system to investigate floral development in these highly specialized holoparasites, we used comparative studies of structure, development, and gene-expression patterns to investigate the homology of their floral organs. Our results surprisingly demonstrate that the otherwise similar floral chambers in two Rafflesiaceae subclades, Rafflesia and Sapria, are constructed very differently. In Rafflesia, the diaphragm is derived from the petal whorl. In contrast, in Sapria it is derived from elaboration of a unique ring structure located between the perianth and the stamen whorl, which, although developed to varying degrees among the genera, appears to be a synapomorphy of the Rafflesiaceae. Thus, the characteristic features that define the floral chamber in these closely related genera are not homologous. These differences refute the prevailing hypothesis that similarities between Sapria and Rafflesia are ancestral in the family. Instead, our data indicate that Rafflesia-like and Sapria-like floral chambers represent two distinct derivations of this morphology. The developmental repatterning we identified in Rafflesia, in particular, may have provided architectural reinforcement, which permitted the explosive growth in floral diameter that has arisen secondarily within this subclade.

Absence of petals, or being apetalous, is usually one of the most important features that characterizes a group of flowering plants at high taxonomic ranks (i.e., family and above). The apetalous condition, however, appears to be the result of parallel or convergent evolution with unknown genetic causes. Here we show that within the buttercup family (Ranunculaceae), apetalous genera in at least seven different lineages were all derived from petalous ancestors, indicative of parallel petal losses. We also show that independent petal losses within this family were strongly associated with decreased or eliminated expression of a single floral organ identity gene, APETALA3-3 (AP3-3), apparently owing to species-specific molecular lesions. In an apetalous mutant of Nigella, insertion of a transposable element into the second intron has led to silencing of the gene and transformation of petals into sepals. In several naturally occurring apetalous genera, such as Thalictrum, Beesia, and Enemion, the gene has either been lost altogether or disrupted by deletions in coding or regulatory regions. In Clematis, a large genus in which petalous species evolved secondarily from apetalous ones, the gene exhibits hallmarks of a pseudogene. These results suggest that, as a petal identity gene, AP3-3 has been silenced or down-regulated by different mechanisms in different evolutionary lineages. This also suggests that petal identity did not evolve many times independently across the Ranunculaceae but was lost in numerous instances. The genetic mechanisms underlying the independent petal losses, however, may be complex, with disruption of AP3-3 being either cause or effect.

Malpighiaceae possess flowers with a unique bilateral symmetry (zygomorphy), which is a hypothesized adaptation associated with specialization on neotropical oil bee pollinators. Gene expression of two representatives of the CYC2 lineage of floral symmetry TCP genes, CYC2A and CYC2B, demarcate the adaxial (dorsal) region of the flower in the characteristic zygomorphic flowers of most Malpighiaceae. Several clades within the family, however, have independently lost their specialized oil bee pollinators and reverted to radial flowers (actinomorphy) like their ancestors. Here, we investigate CYC2 expression associated with four independent reversals to actinomorphy. We demonstrate that these reversals are always associated with alteration of the highly conserved CYC2 expression pattern observed in most New World (NW) Malpighiaceae. In NW Lasiocarpus and Old World (OW) Microsteria, the expression of CYC2-like genes has expanded to include the ventral region of the corolla. Thus, the pattern of gene expression in these species has become radialized, which is comparable to what has been reported in the radial flowered legume clade Cadia. In striking contrast, in NW Psychopterys and OW Sphedamnocarpus, CYC2-like expression is entirely absent or at barely detectable levels. This is more similar to the pattern of CYC2 expression observed in radial flowered Arabidopsis. These results collectively indicate that, regardless of geographic distribution, reversals to similar floral phenotypes in this large tropical angiosperm clade have evolved via different genetic changes from an otherwise highly conserved developmental program.

In the wild, organismal life cycles occur within seasonal cycles, so shifts in the timing of developmental transitions can alter the seasonal environment experienced subsequently. Effects of genes that control the timing of prior developmental events can therefore be magnified in the wild because they determine seasonal conditions experienced by subsequent life stages, which can influence subsequent phenotypic expression. We examined such environmentally induced pleiotropy of developmental-timing genes in a field experiment with Arabidopsis thaliana. When studied in the field under natural seasonal variation, an A. thaliana seed-dormancy gene, Delay Of Germination 1 (DOG1), was found to influence not only germination, but also flowering time, overall life history, and fitness. Flowering time of the previous generation, in turn, imposed maternal effects that altered germination, the effects of DOG1 alleles, and the direction of natural selection on these alleles. Thus under natural conditions, germination genes act as flowering genes and potentially vice versa. These results illustrate how seasonal environmental variation can alter pleiotropic effects of developmental-timing genes, such that effects of genes that regulate prior life stages ramify to influence subsequent life stages. In this case, one gene acting at the seed stage impacted the entire life cycle.

Aquilegia Origami is an emerging model system for ecology and evolution, which has numerous genetic and genomic tools. Virus-induced gene silencing (VIGS) has been established as an effective approach to study gene function in Aquilegia. In the current protocol, we demonstrate VIGS using Agrobacterium strain GV3101 carrying tobacco rattle virus (TRV)-based constructs to infect Aquilegia coerulea "Origami" plants via vacuum infiltration.

Epigenetic regulation is important for maintaining gene expression patterns in multicellular organisms. The Polycomb Group (PcG) proteins form several complexes with important and deeply conserved epigenetic functions in both the plant and animal kingdoms. The plant Polycomb Repressive Complex 2 (PRC2) contains four core proteins, Enhancer of Zeste (E(z)), Suppressor of Zeste 12 (Su(z)12), Extra Sex Combs (ESC), and Multicopy Suppressor of IRA 1 (MSI1), and functions in many developmental transitions. In some plant species, including rice and Arabidopsis, duplications in the core PRC2 proteins allow the formation of PRC2s with distinct developmental functions. In addition, members of the plant specific VEL PHD family have been shown to associate with the PRC2 complex in Arabidopsis and may play a role in targeting the PRC2 to specific loci. Here we examine the evolution and expression of the PRC2 and VEL PHD families in Aquilegia, a member of the lower eudicot order Ranunculales and an emerging model for the investigation of plant ecology, evolution and developmental genetics. We find that Aquilegia has a relatively simple PRC2 with only one homolog of Su(z)12, ESC and MSI1 and two ancient copies of E(z), AqSWN and AqCLF. Aquilegia has four members of the VEL PHD family, three of which appear to be closely related to Arabidopsis proteins known to associate with the PRC2. The PRC2 and VEL PHD family proteins are expressed at a relatively constant level throughout Aquilegia vulgaris development, with the VEL PHD family and MSI1 expressed at higher levels during and after vernalization and in the inflorescence. Both AqSWN and AqCLF are expressed in Aquilegia endosperm but neither copy is imprinted.

The study of developmental genetics is providing insights into how plant morphology can and does evolve, and into the fundamental nature of specific organs. This new understanding has the potential to revise significantly the way we think about seed plant evolution, especially with regard to reproductive structures. Here, we have sought to take a step in bridging the divide between genetic data and critical fields such as paleobotany and systematics. We discuss the evidence for several evolutionarily important interpretations, including the possibility that ovules represent meristematic axes with their own type of lateral determinate organs (integuments) and a model that considers carpels as analogs of complex leaves. In addition, we highlight the aspects of reproductive development that are likely to be highly labile and homoplastic, factors that have major implications for the understanding of seed plant relationships. Although these hypotheses may suggest that some long-standing interpretations are misleading, they also open up whole new avenues for comparative study and suggest concrete best practices for evolutionary analyses of development.

The role of petal spurs and specialized pollinator interactions has been studied since Darwin. Aquilegia petal spurs exhibit striking size and shape diversity, correlated with specialized pollinators ranging from bees to hawkmoths in a textbook example of adaptive radiation. Despite the evolutionary significance of spur length, remarkably little is known about Aquilegia spur morphogenesis and its evolution. Using experimental measurements, both at tissue and cellular levels, combined with numerical modelling, we have investigated the relative roles of cell divisions and cell shape in determining the morphology of the Aquilegia petal spur. Contrary to decades-old hypotheses implicating a discrete meristematic zone as the driver of spur growth, we find that Aquilegia petal spurs develop via anisotropic cell expansion. Furthermore, changes in cell anisotropy account for 99 per cent of the spur-length variation in the genus, suggesting that the true evolutionary innovation underlying the rapid radiation of Aquilegia was the mechanism of tuning cell shape.

BACKGROUND: Variation in fruit morphology is important for plant fitness because it influences dispersal capabilities. Approximately half the members of tribe Brassiceae (Brassicaceae) exhibit fruits with segmentation and variable dehiscence, called heteroarthrocarpy. The knowledge of the genetics of fruit patterning in Arabidopsis offers the opportunity to ask: (1) whether this genetic pathway is conserved in taxa with different fruit morphologies; (2) how the pathway may be modified to produce indehiscence; and (3) whether the pathway has been recruited for a novel abscission zone. METHODS: We identified homologs of ALCATRAZ, FRUITFULL, INDEHISCENT, SHATTERPROOF, and REPLUMLESS from two taxa, representing different types of heteroarthrocarpy. Comparative gene expression of twelve loci was assessed to address how their expression may have been modified to produce heteroarthrocarpy. RESULTS: Studies demonstrated overall conservation in gene expression patterns between dehiscent segments of Erucaria erucarioides and Arabidopsis, with some difference in expression of genes that position the valve margin. In contrast, indehiscence in heteroarthrocarpic fruit segments was correlated with the elimination of the entire valve margin pathway in Erucaria and Cakile lanceolata as well as its absence from a novel lateral abscission zone. CONCLUSIONS: These findings suggest that modifications in the valve margin positioning genes are responsible for differences between heteroarthrocarpic and Arabidopsis-like fruits and support the hypothesis that heteroarthrocarpy evolved via repositioning the valve margin. They also highlight conservation in the dehiscence pathway across Brassicaceae.

PREMISE OF STUDY: Many angiosperms have fruit morphologies that result in seeds from the same plant having different dispersal capabilities. A prime example is found in the Brassiceae (Brassicaceae), which has many members with segmented or heteroarthrocarpic fruits. Since only 40% of the genera are heteroarthrocarpic, this tribe provides an opportunity to study the evolution of an ecologically significant novelty and its variants. METHODS: We analyzed nuclear (PHYA) and plastid (matK) sequences from 66 accessions using maximum parsimony, maximum likelihood, and Bayesian inference approaches. The evolution of heteroarthrocarpy and its variants was evaluated using maximum parsimony and maximum likelihood ancestral state reconstructions. KEY RESULTS: Although nuclear and plastid phylogenies are incongruent with each other, the following findings are consistent: (1) Cakile, Crambe, Vella, and Zilla lineages are monophyletic; (2) the Nigra lineage is not monophyletic; and (3) within the Cakile clade, Cakile, Didesmus, and Erucaria are paraphyletic. Despite differences in the matK and PHYA topologies at both deep and shallow nodes, similar patterns of morphological evolution emerge. Heteroarthrocarpy, a complex morphological trait, has evolved multiple times across the tribe. Moreover, there are convergent transitions in dehiscence capabilities and fruit disarticulation across the tribe. CONCLUSIONS: We present the first explicit analysis of fruit evolution within the Brassiceae, which exemplifies evolutionary lability. The repeated loss and gain of segment dehiscence and disarticulation suggests conservation in the genetic pathway controlling abscission with differential expression across taxa. This study provides a strong foundation for future studies of mechanisms underlying variation in dispersal capabilities of Brassiceae.

BACKGROUND: Flowering is a critical transition in plant development, the timing of which can have considerable fitness consequences. Until recently, research into the genetic control of flowering time and its associated developmental changes was focused on core eudicots (for example, Arabidopsis) or monocots (for example, Oryza). Here we examine the flowering response of Aquilegia formosa, a member of the eudicot order Ranunculales that is emerging as an important model for the investigation of plant ecology and evolution. RESULTS: We have determined that A. formosa has a strong vernalization requirement but little or no photoperiod response, making it a day neutral (DN) plant. Consistent with this, the Aquilegia homolog of FLOWERING LOCUS T (AqFT) is expressed in both long and short days but surprisingly, the locus is expressed before the transition to flowering. In situ hybridizations with homologs of several Arabidopsis Floral Pathway Integrators (FPIs) do not suggest conserved functions relative to Arabidopsis, the potential exceptions being AqLFY and AqAGL24.2. CONCLUSIONS: In Aquilegia, vernalization is critical to flowering but this signal is not strictly required for the transcriptional activation of AqFT. The expression patterns of AqLFY and AqAGL24.2 suggest a hypothesis for the development of Aquilegia's determinate inflorescence whereby their differential expression controls the progression of each meristem from inflorescence to floral identity. Interestingly, none of the Aquilegia expression patterns are consistent with a function in floral repression which, combined with the lack of a FLC homolog, means that new candidate genes must be identified for the control of vernalization response in Aquilegia.

In order to maximize reproductive success, plants have evolved different strategies to control the critical developmental shift marked by the transition to flowering. As plants have adapted to diverse environments across the globe, these strategies have evolved to recognize and respond to local seasonal cues through the induction of specific downstream genetic pathways, thereby ensuring that the floral transition occurs in favorable conditions. Determining the genetic factors involved in controlling the floral transition in many species is key to understanding how this trait has evolved. Striking genetic discoveries in Arabidopsis thaliana (Arabidopsis) and Oryza sativa (rice) revealed that similar genes in both species control flowering in response to photoperiod, suggesting that this genetic module could be conserved between distantly related angiosperms. However, as we have gained a better understanding of the complex evolution of these genes and their functions in other species, another possibility must be considered: that the genetic module controlling flowering in response to photoperiod is the result of convergence rather than conservation. In this review, we show that while data clearly support a central role of FLOWERING LOCUS T (FT) homologs in floral promotion across a diverse group of angiosperms, there is little evidence for a conserved role of CONSTANS (CO) homologs in the regulation of these loci. In addition, although there is an element of conserved function for FT homologs, even this component has surprising complexity in its regulation and evolution.