RESUMO
Land plants reproduce sexually by developing an embryo from a fertilized egg cell. However, embryos can also be formed from other cell types in many plant species. Thus, a key question is how embryo identity in plants is controlled, and how this process is modified during nonzygotic embryogenesis. The Arabidopsis (Arabidopsis thaliana) zygote divides to produce an embryonic lineage and an extra-embryonic suspensor. Yet, normally quiescent suspensor cells can develop a second embryo when the initial embryo is damaged, or when response to the signaling molecule auxin is locally blocked. Here we used auxin-dependent suspensor embryogenesis as a model to determine transcriptome changes during embryonic reprogramming. We found that reprogramming is complex and accompanied by large transcriptomic changes before anatomical changes. This analysis revealed a strong enrichment for genes encoding components of auxin homeostasis and response among misregulated genes. Strikingly, deregulation among multiple auxin-related gene families converged upon the re-establishment of cellular auxin levels or response. This finding points to a remarkable degree of feedback regulation to create resilience in the auxin response during embryo development. Starting from the transcriptome of auxin-deregulated embryos, we identified an auxin-dependent basic Helix Loop Helix transcription factor network that mediates the activity of this hormone in suppressing embryo development from the suspensor.
Assuntos
Proteínas de Arabidopsis/metabolismo , Ácidos Indolacéticos/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Sementes/metabolismo , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Plantas Geneticamente Modificadas/genética , Sementes/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologiaRESUMO
Plants adapt to different environmental conditions by constantly forming new organs in response to morphogenetic signals. Lateral roots branch from the main root in response to local auxin maxima. How a local auxin maximum translates into a robust pattern of gene activation ensuring the proper growth of the newly formed lateral root is largely unknown. Here, we demonstrate that miR390, TAS3-derived trans-acting short-interfering RNAs (tasiRNAs), and AUXIN RESPONSE FACTORS (ARFs) form an auxin-responsive regulatory network controlling lateral root growth. Spatial expression analysis using reporter gene fusions, tasi/miRNA sensors, and mutant analysis showed that miR390 is specifically expressed at the sites of lateral root initiation where it triggers the biogenesis of tasiRNAs. These tasiRNAs inhibit ARF2, ARF3, and ARF4, thus releasing repression of lateral root growth. In addition, ARF2, ARF3, and ARF4 affect auxin-induced miR390 accumulation. Positive and negative feedback regulation of miR390 by ARF2, ARF3, and ARF4 thus ensures the proper definition of the miR390 expression pattern. This regulatory network maintains ARF expression in a concentration range optimal for specifying the timing of lateral root growth, a function similar to its activity during leaf development. These results also show how small regulatory RNAs integrate with auxin signaling to quantitatively regulate organ growth during development.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , MicroRNAs/genética , Raízes de Plantas/crescimento & desenvolvimento , RNA Interferente Pequeno/genética , Fatores de Transcrição/metabolismo , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Redes Reguladoras de Genes , Genes Reporter , Ácidos Indolacéticos/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , RNA de Plantas/genética , Fatores de Transcrição/genéticaRESUMO
The plant hormone auxin triggers a wide range of developmental and growth responses throughout a plant's life. Most well-known auxin responses involve changes in gene expression that are mediated by a short pathway involving an auxin-receptor/ubiquitin-ligase, DNA-binding auxin response factor (ARF) transcription factors and their interacting auxin/indole-3-acetic acid (Aux/IAA) transcriptional inhibitors. Auxin promotes the degradation of Aux/IAA proteins through the auxin receptor and hence releases the inhibition of ARF transcription factors. Although this generic mechanism is now well understood, it is still unclear how developmental specificity is generated and how individual gene family members of response components contribute to local auxin responses. We have established a collection of transcriptional reporters for the ARF gene family and used these to generate a map of expression during embryogenesis and in the primary root meristem. Our results demonstrate that transcriptional regulation of ARF genes generates a complex pattern of overlapping activities. Genetic analysis shows that functions of co-expressed ARFs converge on the same biological processes, but can act either antagonistically or synergistically. Importantly, the existence of an 'ARF pre-pattern' could explain how cell-type-specific auxin responses are generated. Furthermore, this resource can now be used to probe the functions of ARF in other auxin-dependent processes.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Ácidos Indolacéticos/metabolismo , Família Multigênica , Fatores de Transcrição/metabolismo , Arabidopsis/embriologia , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Regiões Promotoras Genéticas , Fatores de Transcrição/genéticaRESUMO
KEY MESSAGE: We describe a novel set of domain-specific markers that can be used in genetic studies, and we used two examples to show loss of stem cells in a monopteros background. Multicellular organisms can be defined by their ability to establish distinct cell identities, and it is therefore of critical importance to distinguish cell types. One step that leads to cell identity specification is activation of unique sets of transcripts. This property is often exploited in order to infer cell identity; the availability of good domain-specific marker lines is, however, poor in the Arabidopsis embryo. Here we describe a novel set of domain-specific marker lines that can be used in Arabidopsis (embryo) research. Based on transcriptomic data, we selected 12 genes for expression analysis, and according to the observed expression domain during embryogenesis, we divided them into four categories (1-ground tissue; 2-root stem cell; 3-shoot apical meristem; 4-post-embryonic). We additionally show the use of two markers from the "stem cell" category in a genetic study, where we use the absence of the markers to infer developmental defects in the monopteros mutant background. Finally, in order to judge whether the established marker lines also play a role in normal development, we generated loss-of-function resources. None of the analyzed T-DNA insertion, artificial microRNA, or misexpression lines showed any apparent phenotypic difference from wild type, indicating that these genes are not nonredundantly required for development, but also suggesting that marker activation can be considered an output of the patterning process. This set of domain-specific marker lines is therefore a valuable addition to the currently available markers and will help to move toward a generic set of tissue identity markers.
Assuntos
Antígenos de Diferenciação/genética , Arabidopsis/embriologia , Arabidopsis/genética , Arabidopsis/citologia , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Meristema , Raízes de Plantas/citologia , Brotos de Planta/citologia , Sementes/citologia , Sementes/crescimento & desenvolvimento , Células-Tronco/citologia , Células-Tronco/metabolismoRESUMO
Establishment of the embryonic axis foreshadows the main body axis of adults both in plants and in animals, but underlying mechanisms are considered distinct. Plants utilize directional, cell-to-cell transport of the growth hormone auxin to generate an asymmetric auxin response that specifies the embryonic apical-basal axis. The auxin flow directionality depends on the polarized subcellular localization of PIN-FORMED (PIN) auxin transporters. It remains unknown which mechanisms and spatial cues guide cell polarization and axis orientation in early embryos. Herein, we provide conceptually novel insights into the formation of embryonic axis in Arabidopsis by identifying a crucial role of localized tryptophan-dependent auxin biosynthesis. Local auxin production at the base of young embryos and the accompanying PIN7-mediated auxin flow toward the proembryo are required for the apical auxin response maximum and the specification of apical embryonic structures. Later in embryogenesis, the precisely timed onset of localized apical auxin biosynthesis mediates PIN1 polarization, basal auxin response maximum, and specification of the root pole. Thus, the tight spatiotemporal control of distinct local auxin sources provides a necessary, non-cell-autonomous trigger for the coordinated cell polarization and subsequent apical-basal axis orientation during embryogenesis and, presumably, also for other polarization events during postembryonic plant life.
Assuntos
Arabidopsis/embriologia , Ácidos Indolacéticos/metabolismo , Reguladores de Crescimento de Plantas/fisiologia , Sementes/crescimento & desenvolvimento , Proteínas de Arabidopsis/metabolismo , Padronização Corporal/efeitos dos fármacos , Ácidos Indolacéticos/farmacologia , Proteínas de Membrana Transportadoras/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Reguladores de Crescimento de Plantas/farmacologia , Transporte Proteico , Sementes/efeitos dos fármacosRESUMO
The cell types of the plant root are first specified early during embryogenesis and are maintained throughout plant life. Auxin plays an essential role in embryonic root initiation, in part through the action of the ARF5/MP transcription factor and its auxin-labile inhibitor IAA12/BDL. MP and BDL function in embryonic cells but promote auxin transport to adjacent extraembryonic suspensor cells, including the quiescent center precursor (hypophysis). Here we show that a cell-autonomous auxin response within this cell is required for root meristem initiation. ARF9 and redundant ARFs, and their inhibitor IAA10, act in suspensor cells to mediate hypophysis specification and, surprisingly, also to prevent transformation to embryo identity. ARF misexpression, and analysis of the short suspensor mutant, demonstrates that lineage-specific expression of these ARFs is required for normal embryo development. These results imply the existence of a prepattern for a cell-type-specific auxin response that underlies the auxin-dependent specification of embryonic cell types.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/embriologia , Arabidopsis/metabolismo , Linhagem da Célula , Ácidos Indolacéticos/farmacologia , Raízes de Plantas/embriologia , Sementes/crescimento & desenvolvimento , Fator 1 de Ribosilação do ADP/metabolismo , Arabidopsis/efeitos dos fármacos , Transferência Ressonante de Energia de Fluorescência , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Hibridização In Situ , Reguladores de Crescimento de Plantas/farmacologia , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas/efeitos dos fármacos , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Sementes/efeitos dos fármacos , Sementes/metabolismo , Transdução de SinaisRESUMO
The basic mechanism of auxin as a modulator of gene expression is now well understood. Interactions among three components are required for this process. Auxin is first perceived by its receptor, which then promotes degradation of inhibitors of auxin response transcription factors. These in turn are released from inhibition and modify expression of target genes. How this simple signaling pathway is able to regulate a diverse range of auxin responses is not as well understood, however a clue lies in the existence of large gene families for all components. Recent data indicates that diversification of gene expression patterns, protein activity, and protein-protein interactions among components establishes a matrix of response machineries that generates specific outputs from the generic auxin signal.