RESUMEN
Members of the R2R3-MYB transcription factor subgroup 19 (SG19) have been extensively studied in multiple plant species using different silenced or mutated lines. Some studies have proposed a function in flower opening, others in floral organ development/maturation, or specialized metabolism production. While SG19 members are clearly key players during flower development and maturation, the resulting picture is complex, confusing our understanding in how SG19 genes function. To clarify the function of the SG19 transcription factors, we used a single system, Petunia axillaris, and targeted its two SG19 members (EOB1 and EOB2) by CRISPR-Cas9. Although EOB1 and EOB2 are highly similar, they display radically different mutant phenotypes. EOB1 has a specific role in scent emission while EOB2 has pleiotropic functions during flower development. The eob2 knockout mutants reveal that EOB2 is a repressor of flower bud senescence by inhibiting ethylene production. Moreover, partial loss-of-function mutants (transcriptional activation domain missing) show that EOB2 is also involved in both petal and pistil maturation through regulation of primary and secondary metabolism. Here, we provide new insights into the genetic regulation of flower maturation and senescence. It also emphasizes the function of EOB2 in the adaptation of plants to specific guilds of pollinators.
Asunto(s)
Petunia , Factores de Transcripción , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Regulación de la Expresión Génica de las Plantas , Flores/fisiología , Reproducción , Petunia/metabolismoRESUMEN
BACKGROUND: Theory suggests that the genetic architecture of traits under divergent natural selection influences how easily reproductive barriers evolve and are maintained between species. Divergently selected traits with a simple genetic architecture (few loci with major phenotypic effects) should facilitate the establishment and maintenance of reproductive isolation between species that are still connected by some gene flow. While empirical support for this idea appears to be mixed, most studies test the influence of trait architectures on reproductive isolation only indirectly. Petunia plant species are, in part, reproductively isolated by their different pollinators. To investigate the genetic causes and consequences of this ecological isolation, we deciphered the genetic architecture of three floral pollination syndrome traits in naturally occurring hybrids between the widespread Petunia axillaris and the highly endemic and endangered P. exserta. RESULTS: Using population genetics, Bayesian linear mixed modelling and genome-wide association studies, we found that the three pollination syndrome traits vary in genetic architecture. Few genome regions explain a majority of the variation in flavonol content (defining UV floral colour) and strongly predict the trait value in hybrids irrespective of interspecific admixture in the rest of their genomes. In contrast, variation in pistil exsertion and anthocyanin content (defining visible floral colour) is controlled by many genome-wide loci. Opposite to flavonol content, the genome-wide proportion of admixture between the two species predicts trait values in their hybrids. Finally, the genome regions strongly associated with the traits do not show extreme divergence between individuals representing the two species, suggesting that divergent selection on these genome regions is relatively weak within their contact zones. CONCLUSIONS: Among the traits analysed, those with a more complex genetic architecture are best maintained in association with the species upon their secondary contact. We propose that this maintained genotype-phenotype association is a coincidental consequence of the complex genetic architectures of these traits: some of their many underlying small-effect loci are likely to be coincidentally linked with the actual barrier loci keeping these species partially isolated upon secondary contact. Hence, the genetic architecture of a trait seems to matter for the outcome of hybridization not only then when the trait itself is under selection.
Asunto(s)
Petunia , Petunia/genética , Estudio de Asociación del Genoma Completo , Teorema de Bayes , Hibridación Genética , Reproducción , Polinización/genética , Flores/genéticaRESUMEN
The mechanisms of reproductive isolation that cause phenotypic diversification and eventually speciation are a major topic of evolutionary research. Hybrid necrosis is a post-zygotic isolation mechanism in which cell death develops in the absence of pathogens. It is often due to the incompatibility between proteins from two parents. Here we describe a unique case of hybrid necrosis due to an incompatibility between loci on chromosomes 2 and 7 between two pollinator-isolated Petunia species. Typical immune responses as well as endoplasmic reticulum stress responses are induced in the necrotic line. The locus on chromosome 2 encodes ChiA1, a bifunctional GH18 chitinase/lysozyme. The enzymatic activity of ChiA1 is dispensable for the development of necrosis. We propose that the extremely high expression of ChiA1 involves a positive feedback loop between the loci on chromosomes 2 and 7. ChiA1 is tightly linked to major genes involved in the adaptation to different pollinators, a form of pre-zygotic isolation. This linkage of pre- and post-zygotic barriers strengthens reproductive isolation and probably contributes to rapid diversification and speciation.
Asunto(s)
Evolución Biológica , Aislamiento Reproductivo , Adaptación Fisiológica , Ligamiento Genético , NecrosisRESUMEN
Understanding the molecular basis of reproductive isolation and speciation is a key goal of evolutionary genetics. In the South American genus Petunia, the R2R3-MYB transcription factor MYB-FL regulates the biosynthesis of UV-absorbing flavonol pigments, a major determinant of pollinator preference. MYB-FL is highly expressed in the hawkmoth-pollinated P. axillaris, but independent losses of its activity in sister taxa P. secreta and P. exserta led to UV-reflective flowers and associated pollinator shifts in each lineage (bees and hummingbirds, respectively). We created a myb-fl CRISPR mutant in P. axillaris and studied the effect of this single gene on innate pollinator preference. The mutation strongly reduced the expression of the two key flavonol-related biosynthetic genes but only affected the expression of few other genes. The mutant flowers were UV reflective as expected but additionally contained low levels of visible anthocyanin pigments. Hawkmoths strongly preferred the wild-type P. axillaris over the myb-fl mutant, whereas both social and solitary bee preference depended on the level of visible color of the mutants. MYB-FL, with its specific expression pattern, small number of target genes, and key position at the nexus of flavonol and anthocyanin biosynthetic pathways, provides a striking example of evolution by single mutations of large phenotypic effect.
Asunto(s)
Manduca , Factores de Transcripción , Animales , Abejas/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Antocianinas/metabolismo , Manduca/fisiología , Flavonoles , Mutación , Flores/fisiología , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Regulación de la Expresión Génica de las PlantasRESUMEN
Red flower color has arisen multiple times and is generally associated with hummingbird pollination. The majority of evolutionary transitions to red color proceeded from purple lineages and tend to be genetically simple, almost always involving a few loss-of-function mutations of major phenotypic effect. Here we report on the complex evolution of a novel red floral color in the hummingbird-pollinated Petunia exserta (Solanaceae) from a colorless ancestor. The presence of a red color is remarkable because the genus cannot synthesize red anthocyanins and P. exserta retains a nonfunctional copy of the key MYB transcription factor AN2. We show that moderate upregulation and a shift in tissue specificity of an AN2 paralog, DEEP PURPLE, restores anthocyanin biosynthesis in P. exserta. An essential shift in anthocyanin hydroxylation occurred through rebalancing the expression of three hydroxylating genes. Furthermore, the downregulation of an acyltransferase promotes reddish hues in typically purple pigments by preventing acyl group decoration of anthocyanins. This study presents a rare case of a genetically complex evolutionary transition toward the gain of a novel red color.
Asunto(s)
Flores/metabolismo , Petunia/metabolismo , Proteínas de Plantas/metabolismo , Solanaceae/metabolismo , Factores de Transcripción/metabolismo , Flores/genética , Petunia/genética , Proteínas de Plantas/genética , Solanaceae/genética , Factores de Transcripción/genéticaRESUMEN
Adaptation to different pollinators is an important driver of speciation in the angiosperms. Genetic approaches such as QTL mapping have been successfully used to identify the underlying speciation genes. However, these methods are often limited by widespread suppression of recombination due to divergence between species. While the mutations that caused the interspecific differences in floral color and scent have been elucidated in a variety of plant genera, the genes that are responsible for morphological differences remain mostly unknown. Differences in floral organ length determine the pollination efficiency of hawkmoths and hummingbirds, and therefore the genes that control these differences are potential speciation genes. Identifying such genes is challenging, especially in non-model species and when studying complex traits for which little prior genetic and biochemical knowledge is available. Here we combine transcriptomics with detailed growth analysis to identify candidate transcription factors underlying interspecific variation in the styles of Petunia flowers. Starting from a set of 2284 genes, stepwise filtering for expression in styles, differential expression between species, correlation with growth-related traits, allele-specific expression in interspecific hybrids, and/or high-impact polymorphisms resulted in a set of 43 candidate speciation genes. Validation by virus-induced gene silencing identified two MYB transcription factors, EOBI and EOBII, that were previously shown to regulate floral scent emission, a trait associated with pollination by hawkmoths.
Asunto(s)
Petunia/fisiología , Proteínas de Plantas/genética , Polinización/fisiología , Factores de Transcripción/genética , Flores/fisiología , Regulación de la Expresión Génica de las Plantas , Silenciador del Gen , Petunia/genética , Petunia/crecimiento & desarrollo , Polinización/genética , Polimorfismo de Nucleótido Simple , Reproducibilidad de los ResultadosRESUMEN
Polar auxin transport lies at the core of many self-organizing phenomena sustaining continuous plant organogenesis. In angiosperms, the shoot apical meristem is a potentially unique system in which the two main modes of auxin-driven patterning-convergence and canalization-co-occur in a coordinated manner and in a fully three-dimensional geometry. In the epidermal layer, convergence points form, from which auxin is canalized towards inner tissue. Each of these two patterning processes has been extensively investigated separately, but the integration of both in the shoot apical meristem remains poorly understood. We present here a first attempt of a three-dimensional model of auxin-driven patterning during phyllotaxis. We base our simulations on a biochemically plausible mechanism of auxin transport proposed by Cieslak et al. (2015) which generates both convergence and canalization patterns. We are able to reproduce most of the dynamics of PIN1 polarization in the meristem, and we explore how the epidermal and inner cell layers act in concert during phyllotaxis. In addition, we discuss the mechanism by which initiating veins connect to the already existing vascular system.
Asunto(s)
Transporte Biológico/fisiología , Ácidos Indolacéticos/metabolismo , Meristema/metabolismo , Modelos Biológicos , Células Vegetales , Arabidopsis/citología , Arabidopsis/metabolismo , Arabidopsis/fisiología , Proteínas de Arabidopsis/metabolismo , Biología Computacional , Simulación por Computador , Células Vegetales/metabolismo , Células Vegetales/fisiología , Hojas de la Planta/citología , Tallos de la Planta/citologíaRESUMEN
Petunia is endemic to South America grasslands; member of this genus exhibit variation in flower colour and shape, attracting bees, hawkmoths or hummingbirds. This group of plants is thus an excellent model system for evolutionary studies of diversification associated with pollinator shifts. Our aims were to identify the legitimate pollinator of Petunia secreta, a rare and endemic species, and to assess the importance of floral traits in pollinator attraction in this Petunia species. To determine the legitimate pollinator, field observations were conducted, and all floral visitors were recorded and evaluated. We also measured the nectar volume and sugar concentration. To characterize morphological cues for pollinators, we assessed the ultraviolet (UV)-light response in detached flowers, and characterized the floral pigments and pollen volatile scents for four different Petunia species that present different pollination syndromes. Petunia secreta shares the most recent ancestor with a white hawkmoth-pollinated species, P. axillaris, but presents flavonols and anthocyanin pigments responsible for the pink corolla colour and UV-light responses that are common to bee-pollinated Petunia species. Our study showed that a solitary bee in the genus Pseudagapostemon was the most frequent pollinator of P. secreta, and these bees collect only pollen as a reward. Despite being mainly bee-pollinated, different functional groups of pollinators visit P. secreta. Nectar volume, sugar concentration per flower, morphology and components of pollen scent would appear to be attractive to several different pollinator groups. Notably, the corolla includes a narrow tube with nectar at its base that cannot be reached by Pseudagapostemon, and flowers of P. secreta appear to follow an evolutionary transition, with traits attractive to several functional groups of pollinators. Additionally, the present study shows that differences in the volatiles of pollen scent are relevant for plant mutualistic and antagonist interactions in Petunia species and that pollen scent profile plays a key role in characterizing pollination syndromes.
RESUMEN
A persistent question in evolutionary biology is how complex phenotypes evolve and whether phenotypic transitions are reversible. Multiple losses of floral pigmentation have been documented in the angiosperms, but color re-gain has not yet been described, supporting that re-gain is unlikely. Pollinator-mediated selection in Petunia has resulted in several color shifts comprised of both losses and gains of color. The R2R3-MYB transcription factor AN2 has been identified as a major locus responsible for shifts in pollinator preference. Whereas the loss of visible color has previously been attributed to repeated pseudogenization of AN2, here, we describe the mechanism of an independent re-gain of floral color via AN2 evolution. In P. secreta, purple color is restored through the improbable resurrection of AN2 gene function from a non-functional AN2-ancestor by a single reading-frame-restoring mutation. Thus, floral color evolution in Petunia is mechanistically dependent on AN2 functionality, highlighting its role as a hotspot in color transitions and a speciation gene for the genus.
Asunto(s)
Evolución Biológica , Petunia/genética , Pigmentos Biológicos/genética , Polinización , Flores/fisiología , Especiación Genética , Petunia/fisiología , Pigmentación/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Plants are able to sense external mechanical stress, such as those due to gravity or obstacles, and alter their growth accordingly [1-8]. Like animals [9, 10], plants can also sense internal mechanical stress that plays a role in regulating their development [11-19]. The internal mechanical stresses also known as tissue stress can result from geometry, cell type, or differential growth [19-21]. In a number of tissues, microtubules have been observed to align with mechanical stress predicted from their geometry. In the unidirectionally growing hypocotyl, the predicted tissue stresses do not reflect its cylindrical geometry. The epidermal layer experiences and resists the tensile stress coming from the expansion of the inner layers [22, 23]; this is known as the epidermal-growth-control hypothesis. Here, we use our recently developed automated confocal micro-extensometer (ACME) [24] to apply relative compressive or tensile stresses to the intact Arabidopsis hypocotyls while monitoring growth and microtubule orientation in the different layers. A finite element model revealed that under relative tension, the pattern of tissue stresses was similar to that in the intact growing hypocotyl, while when relative compression was applied, the pattern of tissue stresses was overcome and the maximum stress direction in the epidermis changed to reflect what one would predict based on the geometry of the hypocotyl. Consistent with this, the microtubules in the epidermis changed orientation under relative compression. Once the direction of stress in the epidermis was altered, the growth of the organ increased.
Asunto(s)
Arabidopsis/fisiología , Hipocótilo/citología , Microtúbulos/fisiología , Epidermis de la Planta/citología , Arabidopsis/crecimiento & desarrollo , Fenómenos Biomecánicos , Estrés MecánicoRESUMEN
How complex developmental-genetic networks are translated into organs with specific 3D shapes remains an open question. This question is particularly challenging because the elaboration of specific shapes is in essence a question of mechanics. In plants, this means how the genetic circuitry affects the cell wall. The mechanical properties of the wall and their spatial variation are the key factors controlling morphogenesis in plants. However, these properties are difficult to measure and investigating their relation to genetic regulation is particularly challenging. To measure spatial variation of mechanical properties, one must determine the deformation of a tissue in response to a known force with cellular resolution. Here, we present an automated confocal micro-extensometer (ACME), which greatly expands the scope of existing methods for measuring mechanical properties. Unlike classical extensometers, ACME is mounted on a confocal microscope and uses confocal images to compute the deformation of the tissue directly from biological markers, thus providing 3D cellular scale information and improved accuracy. Additionally, ACME is suitable for measuring the mechanical responses in live tissue. As a proof of concept, we demonstrate that the plant hormone gibberellic acid induces a spatial gradient in mechanical properties along the length of the Arabidopsis thaliana hypocotyl.
Asunto(s)
Arabidopsis/citología , Microscopía Confocal/instrumentación , Células Vegetales/química , Automatización , Fenómenos Biomecánicos , Pared Celular/efectos de los fármacos , Pared Celular/fisiología , Elasticidad , Giberelinas/farmacología , Hipocótilo/citología , Hipocótilo/efectos de los fármacos , Hipocótilo/crecimiento & desarrollo , Hipocótilo/efectos de la radiación , Luz , Modelos Biológicos , Células Vegetales/efectos de los fármacos , Estrés Fisiológico/efectos de los fármacosRESUMEN
Leaves and flowers are arranged in regular patterns around the stem of a plant, a phenomenon known as phyllotaxis. Different arrangements occur, such as distichous, decussate or spiral (Figure 1). Most prevalent in nature are spirals in which the average divergence angles between successive organs are close to 137.5°, the so-called 'golden angle'. It is this exact number that has given phyllotaxis its special flavor as a quantitative developmental problem, and over the centuries, it has enjoyed the attention of scientists far beyond botany. In the 1830s mathematicians described the spirals as they related to the Fibonacci numbers, and in the 1860s improved microscopes made it possible for botanists to observe the initiation of leaf and flower primordia in a diversity of plants. This descriptive work led to the conclusion that new organ primordia form in the first available space between existing primordia, a conclusion still valid today. But how does it work? Ideas from the early 20th century suggested that an inhibitor produced by existing primordia diffuses towards the shoot apical meristem: where the concentration of the inhibitor falls below a threshold value, an organ is initiated. Other models dating back to the 1870s have tried to explain phyllotactic patterning by applying the laws of mechanics. Such models went through a long period of marginal interest, but have experienced a remarkable renaissance over the past 20 years. In this Primer I will give a broad overview of phyllotaxis, its emergence from the shoot apical meristem, how auxin and its transporter function as a 'pattern generator', and the role of tissue mechanics and computational modeling.
Asunto(s)
Flores/crecimiento & desarrollo , Modelos Biológicos , Hojas de la Planta/crecimiento & desarrollo , Tallos de la Planta/crecimiento & desarrollo , Tipificación del Cuerpo , Flores/anatomía & histología , Flores/metabolismo , Ácidos Indolacéticos/metabolismo , Reguladores del Crecimiento de las Plantas/metabolismo , Hojas de la Planta/anatomía & histología , Hojas de la Planta/metabolismo , Tallos de la Planta/anatomía & histología , Tallos de la Planta/efectos de los fármacosRESUMEN
The interactions of plants with their pollinators are thought to be a driving force in the evolution of angiosperms. Adaptation to a new pollinator involves coordinated changes in multiple floral traits controlled by multiple genes. Surprisingly, such complex genetic shifts have happened numerous times during evolution. Here we report on the genetic basis of the changes in one such trait, floral scent emission, in the genus Petunia (Solanaceae). The increase in the quantity and complexity of the volatiles during the shift from bee to hawkmoth pollination was due to de novo expression of the genes encoding benzoic acid/salicylic acid carboxyl methyltransferase (BSMT) and benzoyl-CoA:benzylalcohol/2-phenylethanol benzoyltransferase (BPBT) together with moderately increased transcript levels for most enzymes of the phenylpropanoid/benzenoid pathway. Loss of cinnamate-CoA ligase (CNL) function as well as a reduction in the expression of the MYB transcription factor ODO1 explain the loss of scent during the transition from moth to hummingbird pollination. The CNL gene in the hummingbird-adapted species is inactive due to a stop codon, but also appears to have undergone further degradation over time. Therefore, we propose that loss of scent happened relatively early in the transition toward hummingbird pollination, and probably preceded the loss of UV-absorbing flavonols. The discovery that CNL is also involved in the loss of scent during the transition from outcrossing to selfing in Capsella (Brassicaceae) (see the accompanying paper) raises interesting questions about the possible causes of deep evolutionary conservation of the targets of evolutionary change.
Asunto(s)
Evolución Biológica , Flores/genética , Flores/fisiología , Regulación de la Expresión Génica de las Plantas/fisiología , Odorantes , Polinización/fisiología , Animales , Mapeo Cromosómico , Cromosomas de las Plantas/genética , Especiación Genética , Genotipo , Mariposas Nocturnas/fisiología , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Polimorfismo de Nucleótido Simple , Sitios de Carácter CuantitativoRESUMEN
The differentiation of a leaf - from its inception as a semicircular bulge on the surface of the shoot apical meristem into a flattened structure with specialized upper and lower surfaces - is one of the most intensely studied processes in plant developmental biology. The large body of contemporary data on leaf dorsiventrality has its origin in the pioneering experiments of Ian Sussex, who carried out these studies as a PhD student in the early 1950s. Here, we review his original experiments in their historical context and describe our current understanding of this surprisingly complex process. Finally, we postulate possible candidates for the 'Sussex signal' - the elusive meristem-derived factor that first ignited interest in this important developmental problem.
Asunto(s)
Ácidos Indolacéticos/metabolismo , Hojas de la Planta/metabolismo , Proteínas de Plantas/metabolismo , Cromatina/metabolismo , Regulación de la Expresión Génica de las PlantasRESUMEN
The lifespan of plants ranges from a few weeks in annuals to thousands of years in trees. It is hard to explain such extreme longevity considering that DNA replication errors inevitably cause mutations. Without purging through meiotic recombination, the accumulation of somatic mutations will eventually result in mutational meltdown, a phenomenon known as Muller's ratchet. Nevertheless, the lifespan of trees is limited more often by incidental disease or structural damage than by genetic aging. The key determinants of tree architecture are the axillary meristems, which form in the axils of leaves and grow out to form branches. The number of branches is low in annual plants, but in perennial plants iterative branching can result in thousands of terminal branches. Here, we use stem cell ablation and quantitative cell-lineage analysis to show that axillary meristems are set aside early, analogous to the metazoan germline. While neighboring cells divide vigorously, axillary meristem precursors maintain a quiescent state, with only 7-9 cell divisions occurring between the apical and axillary meristem. During iterative branching, the number of branches increases exponentially, while the number of cell divisions increases linearly. Moreover, computational modeling shows that stem cell arrangement and positioning of axillary meristems distribute somatic mutations around the main shoot, preventing their fixation and maximizing genetic heterogeneity. These features slow down Muller's ratchet and thereby extend lifespan.
Asunto(s)
Arabidopsis/fisiología , Meristema/crecimiento & desarrollo , Solanum lycopersicum/citología , Solanum lycopersicum/fisiología , Arabidopsis/citología , Arabidopsis/crecimiento & desarrollo , División Celular , Linaje de la Célula , Longevidad , Solanum lycopersicum/crecimiento & desarrollo , Meristema/citologíaRESUMEN
Adaptations to new pollinators involve multiple floral traits, each requiring coordinated changes in multiple genes. Despite this genetic complexity, shifts in pollination syndromes have happened frequently during angiosperm evolution. Here we study the genetic basis of floral UV absorbance, a key trait for attracting nocturnal pollinators. In Petunia, mutations in a single gene, MYB-FL, explain two transitions in UV absorbance. A gain of UV absorbance in the transition from bee to moth pollination was determined by a cis-regulatory mutation, whereas a frameshift mutation caused subsequent loss of UV absorbance during the transition from moth to hummingbird pollination. The functional differences in MYB-FL provide insight into the process of speciation and clarify phylogenetic relationships between nascent species.
Asunto(s)
Flores/efectos de la radiación , Manduca/fisiología , Petunia/efectos de la radiación , Polinización , Rayos Ultravioleta , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN de Plantas , Datos de Secuencia Molecular , Petunia/genética , Petunia/fisiología , ReproducciónRESUMEN
Morphogenesis emerges from complex multiscale interactions between genetic and mechanical processes. To understand these processes, the evolution of cell shape, proliferation and gene expression must be quantified. This quantification is usually performed either in full 3D, which is computationally expensive and technically challenging, or on 2D planar projections, which introduces geometrical artifacts on highly curved organs. Here we present MorphoGraphX ( www.MorphoGraphX.org), a software that bridges this gap by working directly with curved surface images extracted from 3D data. In addition to traditional 3D image analysis, we have developed algorithms to operate on curved surfaces, such as cell segmentation, lineage tracking and fluorescence signal quantification. The software's modular design makes it easy to include existing libraries, or to implement new algorithms. Cell geometries extracted with MorphoGraphX can be exported and used as templates for simulation models, providing a powerful platform to investigate the interactions between shape, genes and growth.
Asunto(s)
Algoritmos , Arabidopsis/ultraestructura , Procesamiento de Imagen Asistido por Computador/métodos , Imagenología Tridimensional/métodos , Programas Informáticos , Animales , Anisotropía , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Cassia/genética , Cassia/crecimiento & desarrollo , Cassia/ultraestructura , Proliferación Celular , Forma de la Célula , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/ultraestructura , Flores/genética , Flores/crecimiento & desarrollo , Flores/ultraestructura , Frutas/genética , Frutas/crecimiento & desarrollo , Frutas/ultraestructura , Expresión Génica , Procesamiento de Imagen Asistido por Computador/estadística & datos numéricos , Imagenología Tridimensional/instrumentación , Imagenología Tridimensional/estadística & datos numéricos , Solanum lycopersicum/genética , Solanum lycopersicum/crecimiento & desarrollo , Solanum lycopersicum/ultraestructura , Microscopía Confocal , Microtúbulos/genética , Microtúbulos/ultraestructura , Morfogénesis/genética , Desarrollo de la Planta/genética , Imagen de Lapso de Tiempo/instrumentación , Imagen de Lapso de Tiempo/métodos , Imagen de Lapso de Tiempo/estadística & datos numéricosRESUMEN
The spatial arrangement of leaves and flowers around the stem, known as phyllotaxis, is controlled by an auxin-dependent reiterative mechanism that leads to regular spacing of the organs and thereby to remarkably precise phyllotactic patterns. The mechanism is based on the active cellular transport of the phytohormone auxin by cellular influx and efflux carriers, such as AUX1 and PIN1. Their important role in phyllotaxis is evident from mutant phenotypes, but their exact roles in space and time are difficult to address due to the strong pleiotropic phenotypes of most mutants in phyllotaxis. Models of phyllotaxis invoke the accumulation of auxin at leaf initials and removal of auxin through their developing vascular strand, the midvein. We have developed a precise microsurgical tool to ablate the midvein at high spatial and temporal resolution in order to test its function in leaf formation and phyllotaxis. Using amplified femtosecond laser pulses, we ablated the internal tissues in young leaf primordia of tomato (Solanum lycopersicum) without damaging the overlying L1 and L2 layers. Our results show that ablation of the future midvein leads to a transient accumulation of auxin in the primordia and to an increase in their width. Phyllotaxis was transiently affected after midvein ablations, but readjusted after two plastochrons. These results indicate that the developing midvein is involved in the basipetal transport of auxin through young primordia, which contributes to phyllotactic spacing and stability.