RESUMEN
A large number of genes involved in lateral root (LR) organogenesis have been identified over the last decade using forward and reverse genetic approaches in Arabidopsis thaliana. Nevertheless, how these genes interact to form a LR regulatory network largely remains to be elucidated. In this study, we developed a time-delay correlation algorithm (TDCor) to infer the gene regulatory network (GRN) controlling LR primordium initiation and patterning in Arabidopsis from a time-series transcriptomic data set. The predicted network topology links the very early-activated genes involved in LR initiation to later expressed cell identity markers through a multistep genetic cascade exhibiting both positive and negative feedback loops. The predictions were tested for the key transcriptional regulator AUXIN RESPONSE FACTOR7 node, and over 70% of its targets were validated experimentally. Intriguingly, the predicted GRN revealed a mutual inhibition between the ARF7 and ARF5 modules that would control an early bifurcation between two cell fates. Analyses of the expression pattern of ARF7 and ARF5 targets suggest that this patterning mechanism controls flanking and central zone specification in Arabidopsis LR primordia.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Proteínas de Unión al ADN/genética , Redes Reguladoras de Genes/genética , Raíces de Plantas/genética , Factores de Transcripción/genética , Transcriptoma , Algoritmos , Arabidopsis/citología , Arabidopsis/crecimiento & desarrollo , Diferenciación Celular/genética , Regulación de la Expresión Génica de las Plantas , Mutación , Raíces de Plantas/citología , Raíces de Plantas/crecimiento & desarrollo , Plantas Modificadas Genéticamente , Factores de TiempoRESUMEN
In plants, the AUXIN RESPONSE FACTOR (ARF) transcription factor family regulates gene expression in response to auxin. In the absence of auxin, ARF transcription factors are repressed by interaction with AUXIN/INDOLE 3-ACETIC ACID (Aux/IAA) proteins. Although the C termini of ARF and Aux/IAA proteins facilitate their homo- and heterooligomerization, the molecular basis for this interaction remained undefined. The crystal structure of the C-terminal interaction domain of Arabidopsis ARF7 reveals a Phox and Bem1p (PB1) domain that provides both positive and negative electrostatic interfaces for directional protein interaction. Mutation of interface residues in the ARF7 PB1 domain yields monomeric protein and abolishes interaction with both itself and IAA17. Expression of a stabilized Aux/IAA protein (i.e., IAA16) bearing PB1 mutations in Arabidopsis suggests a multimerization requirement for ARF protein repression, leading to a refined auxin-signaling model.
Asunto(s)
Arabidopsis/metabolismo , Ácidos Indolacéticos/metabolismo , Proteínas de Plantas/metabolismo , Secuencia de Aminoácidos , Datos de Secuencia Molecular , Mutación , Proteínas de Plantas/química , Proteínas de Plantas/genética , Homología de Secuencia de AminoácidoRESUMEN
The plant hormone auxin is a key morphogenetic regulator acting from embryogenesis onwards. Transcriptional events in response to auxin are mediated by the auxin response factor (ARF) transcription factors and the Aux/IAA (IAA) transcriptional repressors. At low auxin concentrations, IAA repressors associate with ARF proteins and recruit corepressors that prevent auxin-induced gene expression. At higher auxin concentrations, IAAs are degraded and ARFs become free to regulate auxin-responsive genes. The interaction between ARFs and IAAs is thus central to auxin signalling and occurs through the highly conserved domain III/IV present in both types of proteins. Here, we report the crystal structure of ARF5 domain III/IV and reveal the molecular determinants of ARF-IAA interactions. We further provide evidence that ARFs have the potential to oligomerize, a property that could be important for gene regulation in response to auxin.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica de las Plantas , Ácidos Indolacéticos/metabolismo , Factores de Transcripción/metabolismo , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Cristalografía por Rayos X , Morfogénesis , Reguladores del Crecimiento de las Plantas , Dominios y Motivos de Interacción de Proteínas , Estructura Terciaria de Proteína , Transducción de SeñalRESUMEN
Auxin transport network, which is important in the integration of plant developmental signals, depends on differential expression of the auxin efflux carrier PIN gene family. We cloned three tomato PIN (referred as SlPIN) cDNAs and examined their expression patterns in fruit and other organs. The expression of SlPIN1 and SlPIN2 was highest in very young fruit immediately after anthesis, whereas the expression of SlPIN3 was low at this same stage of fruit development. SlPIN2::GUS was expressed in ovules at anthesis and in young developing seeds at 4 days after anthesis, while SlPIN1::GUS was expressed in whole fruit. The DR5::GUS auxin-responsive reporter gene was expressed in the fruit and peduncle at anthesis and was higher in the peduncle 4 days after anthesis. These studies suggest that auxin is likely transported from young seeds by SlPIN1 and SlPIN2 and accumulated in peduncles where SlPIN gene expression is low in tomato. The possible role of SlPINs in fruit set was discussed.
Asunto(s)
Proteínas Portadoras/genética , Ácidos Indolacéticos/metabolismo , Solanum lycopersicum/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Clonación Molecular , Cartilla de ADN , ADN Complementario , Genes de Plantas , Genes Reporteros , Solanum lycopersicum/genética , Solanum lycopersicum/crecimiento & desarrollo , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Homología de Secuencia de AminoácidoRESUMEN
In addition to RNA polymerases I, II, and III, the essential RNA polymerases present in all eukaryotes, plants have two additional nuclear RNA polymerases, abbreviated as Pol IV and Pol V, that play nonredundant roles in siRNA-directed DNA methylation and gene silencing. We show that Arabidopsis Pol IV and Pol V are composed of subunits that are paralogous or identical to the 12 subunits of Pol II. Four subunits of Pol IV are distinct from their Pol II paralogs, six subunits of Pol V are distinct from their Pol II paralogs, and four subunits differ between Pol IV and Pol V. Importantly, the subunit differences occur in key positions relative to the template entry and RNA exit paths. Our findings support the hypothesis that Pol IV and Pol V are Pol II-like enzymes that evolved specialized roles in the production of noncoding transcripts for RNA silencing and genome defense.
Asunto(s)
Proteínas de Arabidopsis/química , ARN Polimerasas Dirigidas por ADN/química , Subunidades de Proteína/química , Interferencia de ARN , Secuencia de Aminoácidos , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Metilación de ADN , ADN de Plantas/metabolismo , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Silenciador del Gen , Modelos Biológicos , Datos de Secuencia Molecular , Filogenia , Plantas Modificadas Genéticamente , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , ARN Interferente Pequeño/metabolismo , ARN no Traducido/metabolismo , Alineación de SecuenciaRESUMEN
In plants, both endogenous mechanisms and environmental signals regulate developmental transitions such as seed germination, induction of flowering, leaf senescence and shedding of senescent organs. Auxin response factors (ARFs) are transcription factors that mediate responses to the plant hormone auxin. We have examined Arabidopsis lines carrying T-DNA insertions in AUXIN RESPONSE FACTOR1 (ARF1) and ARF2 genes. We found that ARF2 promotes transitions between multiple stages of Arabidopsis development. arf2 mutant plants exhibited delays in several processes related to plant aging, including initiation of flowering, rosette leaf senescence, floral organ abscission and silique ripening. ARF2 expression was induced in senescing leaves. ARF2 regulated leaf senescence and floral organ abscission independently of the ethylene and cytokinin response pathways. arf1 mutations enhanced many arf2 phenotypes, indicating that ARF1 acts in a partially redundant manner with ARF2. However, unlike arf2 mutations, an arf1 mutation increased transcription of Aux/IAA genes in Arabidopsis flowers, supporting previous biochemical studies that indicated that ARF1 is a transcriptional repressor. Two other ARF genes, NPH4/ARF7 and ARF19, were also induced by senescence, and mutations in these genes enhanced arf2 phenotypes. NPH4/ARF7 and ARF19 function as transcriptional activators, suggesting that auxin may control senescence in part by activating gene expression.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Proteínas de Unión al ADN/metabolismo , Flores/crecimiento & desarrollo , Flores/metabolismo , Regulación de la Expresión Génica de las Plantas , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Citocininas/farmacología , Proteínas de Unión al ADN/genética , Etilenos/farmacología , Flores/genética , Frutas/genética , Frutas/crecimiento & desarrollo , Frutas/metabolismo , Mutación/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fenotipo , Hojas de la Planta/genética , Hojas de la Planta/crecimiento & desarrollo , Hojas de la Planta/metabolismo , Proteínas Represoras/genética , Transducción de Señal/efectos de los fármacos , Factores de Transcripción/genéticaRESUMEN
Pollination in flowering plants requires that anthers release pollen when the gynoecium is competent to support fertilization. We show that in Arabidopsis thaliana, two paralogous auxin response transcription factors, ARF6 and ARF8, regulate both stamen and gynoecium maturation. arf6 arf8 double-null mutant flowers arrested as infertile closed buds with short petals, short stamen filaments, undehisced anthers that did not release pollen and immature gynoecia. Numerous developmentally regulated genes failed to be induced. ARF6 and ARF8 thus coordinate the transition from immature to mature fertile flowers. Jasmonic acid (JA) measurements and JA feeding experiments showed that decreased jasmonate production caused the block in pollen release, but not the gynoecium arrest. The double mutant had altered auxin responsive gene expression. However, whole flower auxin levels did not change during flower maturation, suggesting that auxin might regulate flower maturation only under specific environmental conditions, or in localized organs or tissues of flowers. arf6 and arf8 single mutants and sesquimutants (homozygous for one mutation and heterozygous for the other) had delayed stamen development and decreased fecundity, indicating that ARF6 and ARF8 gene dosage affects timing of flower maturation quantitatively.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Ciclopentanos/metabolismo , Proteínas de Unión al ADN/metabolismo , Flores/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Reguladores del Crecimiento de las Plantas/metabolismo , Arabidopsis/fisiología , Proteínas de Arabidopsis/genética , Cartilla de ADN , Proteínas de Unión al ADN/genética , Flores/metabolismo , Flores/ultraestructura , Dosificación de Gen/fisiología , Ácidos Indolacéticos/metabolismo , Microscopía Electrónica de Rastreo , Mutación/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Oxilipinas , Fenotipo , Plantas Modificadas Genéticamente , Polen/fisiologíaRESUMEN
Auxin response factors (ARFs) bind auxin response promoter elements and mediate transcriptional responses to auxin. Five of the 22 ARF genes in Arabidopsis thaliana encode ARFs with glutamine-rich middle domains. Four of these can activate transcription and have been ascribed developmental functions. We show that ARF19, the fifth Q-rich ARF, also activates transcription. Mutations in ARF19 have little effect on their own, but in combination with mutations in NPH4/ARF7, encoding the most closely related ARF, they cause several phenotypes including a drastic decrease in lateral and adventitious root formation and a decrease in leaf cell expansion. These results indicate that auxin induces lateral roots and leaf expansion by activating NPH4/ARF7 and ARF19. Auxin induces the ARF19 gene, and NPH4/ARF7 and ARF19 together are required for expression of one of the arf19 mutant alleles, suggesting that a positive feedback loop regulates leaf expansion and/or lateral root induction.
Asunto(s)
Proteínas de Arabidopsis/fisiología , Arabidopsis/crecimiento & desarrollo , Arabidopsis/genética , Hojas de la Planta/crecimiento & desarrollo , Raíces de Plantas/crecimiento & desarrollo , Transactivadores/fisiología , Factores de Transcripción/fisiología , Relación Dosis-Respuesta a Droga , Regulación de la Expresión Génica de las Plantas , Ácidos Indolacéticos/farmacología , Ácidos Indolacéticos/fisiología , Fenotipo , Transcripción GenéticaRESUMEN
The complete genomic sequence for Arabidopsis provides the opportunity to combine phylogenetic and genomic approaches to study the evolution of gene families in plants. The Aux/IAA and ARF gene families, consisting of 29 and 23 loci in Arabidopsis, respectively, encode proteins that interact to mediate auxin responses and regulate various aspects of plant morphological development. We developed scenarios for the genomic proliferation of the Aux/IAA and ARF families by combining phylogenetic analysis with information on the relationship between each locus and the previously identified duplicated genomic segments in Arabidopsis. This analysis shows that both gene families date back at least to the origin of land plants and that the major Aux/IAA and ARF lineages originated before the monocot-eudicot divergence. We found that the extant Aux/IAA loci arose primarily through segmental duplication events, in sharp contrast to the ARF family and to the general pattern of gene family proliferation in Arabidopsis. Possible explanations for the unusual mode of Aux/IAA duplication include evolutionary constraints imposed by complex interactions among proteins and pathways, or the presence of long-distance cis-regulatory sequences. The antiquity of the two gene families and the unusual mode of Aux/IAA diversification have a number of potential implications for understanding both the functional and evolutionary roles of these genes.