RESUMEN
Understanding how flowers develop from undifferentiated stem cells has occupied developmental biologists for decades. Key to unraveling this process is a detailed knowledge of the global regulatory hierarchies that control developmental transitions, cell differentiation and organ growth. These hierarchies may be deduced from gene perturbation experiments, which determine the effects on gene expression after specific disruption of a regulatory gene. Here, we tested experimental strategies for gene perturbation experiments during Arabidopsis thaliana flower development. We used artificial miRNAs (amiRNAs) to disrupt the functions of key floral regulators, and expressed them under the control of various inducible promoter systems that are widely used in the plant research community. To be able to perform genome-wide experiments with stage-specific resolution using the various inducible promoter systems for gene perturbation experiments, we also generated a series of floral induction systems that allow collection of hundreds of synchronized floral buds from a single plant. Based on our results, we propose strategies for performing dynamic gene perturbation experiments in flowers, and outline how they may be combined with versions of the floral induction system to dissect the gene regulatory network underlying flower development.
Asunto(s)
Arabidopsis/crecimiento & desarrollo , Flores/crecimiento & desarrollo , Redes Reguladoras de Genes , Genes de Plantas , Arabidopsis/genética , Técnicas de Silenciamiento del Gen , Datos de Secuencia Molecular , ARN de Planta/genéticaRESUMEN
The floral organ identity factor AGAMOUS (AG) is a key regulator of Arabidopsis thaliana flower development, where it is involved in the formation of the reproductive floral organs as well as in the control of meristem determinacy. To obtain insights into how AG specifies organ fate, we determined the genes and processes acting downstream of this C function regulator during early flower development and distinguished between direct and indirect effects. To this end, we combined genome-wide localization studies, gene perturbation experiments, and computational analyses. Our results demonstrate that AG controls flower development to a large extent by controlling the expression of other genes with regulatory functions, which are involved in mediating a plethora of different developmental processes. One aspect of this function is the suppression of the leaf development program in emerging floral primordia. Using trichome initiation as an example, we demonstrate that AG inhibits an important aspect of leaf development through the direct control of key regulatory genes. A comparison of the gene expression programs controlled by AG and the B function regulators APETALA3 and PISTILLATA, respectively, showed that while they control many developmental processes in conjunction, they also have marked antagonistic, as well as independent activities.
Asunto(s)
Proteína AGAMOUS de Arabidopsis/genética , Proteínas de Arabidopsis/genética , Arabidopsis/genética , Flores/genética , Proteína AGAMOUS de Arabidopsis/metabolismo , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Flores/crecimiento & desarrollo , Flores/ultraestructura , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Immunoblotting , Proteínas de Dominio MADS/genética , Proteínas de Dominio MADS/metabolismo , Meristema/genética , Meristema/crecimiento & desarrollo , Meristema/metabolismo , Microscopía Confocal , Microscopía Electrónica de Rastreo , Microscopía Fluorescente , Mutación , Análisis de Secuencia por Matrices de Oligonucleótidos , Hojas de la Planta/genética , Hojas de la Planta/crecimiento & desarrollo , Hojas de la Planta/metabolismo , Plantas Modificadas Genéticamente , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcriptoma , Tricomas/genética , Tricomas/crecimiento & desarrollo , Tricomas/metabolismoRESUMEN
How different organs are formed from small sets of undifferentiated precursor cells is a key question in developmental biology. To understand the molecular mechanisms underlying organ specification in plants, we studied the function of the homeotic selector genes APETALA3 (AP3) and PISTILLATA (PI), which control the formation of petals and stamens during Arabidopsis flower development. To this end, we characterized the activities of the transcription factors that AP3 and PI encode throughout flower development by using perturbation assays as well as transcript profiling and genomewide localization studies, in combination with a floral induction system that allows a stage-specific analysis of flower development by genomic technologies. We discovered considerable spatial and temporal differences in the requirement for AP3/PI activity during flower formation and show that they control different sets of genes at distinct phases of flower development. The genomewide identification of target genes revealed that AP3/PI act as bifunctional transcription factors: they activate genes involved in the control of numerous developmental processes required for organogenesis and repress key regulators of carpel formation. Our results imply considerable changes in the composition and topology of the gene network controlled by AP3/PI during the course of flower development. We discuss our results in light of a model for the mechanism underlying sex-determination in seed plants, in which AP3/PI orthologues might act as a switch between the activation of male and the repression of female development.