RESUMEN
Nicotine biosynthesis in Nicotiana tabacum is under genetic control by the A and B loci. Plants containing semi-dominant mutations at both the A and B loci (i.e. aabb genotype) have lower nicotine levels, reduced nicotine biosynthetic enzyme activities, and reduced mRNA levels of the corresponding biosynthetic genes. The A and B loci therefore appear to be coordinate regulators of several nicotine biosynthetic genes and define a group of co-regulated genes called the A-B regulon. To investigate the composition of genes in the A-B regulon, a fluorescent differential display (FDD) screen was used to randomly sample the transcriptomes of wild type and mutant aabb roots. This resulted in the isolation of 64 FDD clones, representing 49 unique genes or gene families. Four genes associated with nicotine biosynthesis were identified, whereas most of the other FDD clones were homologous with an assortment of stress response genes. Thirty-three genes or gene families showed reproducible aabb genotype effects, representing seven distinct mRNA expression patterns in response media treatments that increase the mRNA levels of known alkaloid biosynthetic genes. Thus, the A and B loci regulate the mRNA levels of some target genes differently than others. Eleven genes or gene families showed only treatment-specific effects, representing four mRNA accumulation patterns. These results indicate the A-B regulon is complex network of differentially expressed stress response genes, only a small subset of which are involved in nicotine biosynthesis.
Asunto(s)
Regulación de la Expresión Génica de las Plantas/genética , Genes Reguladores/genética , Nicotiana/genética , Nicotina/biosíntesis , Acetatos/farmacología , Alcaloides/biosíntesis , Secuencia de Bases , Ciclopentanos/farmacología , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Genes de Plantas/genética , Genotipo , Datos de Secuencia Molecular , Mutación , Oxilipinas , Reguladores del Crecimiento de las Plantas/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Homología de Secuencia de Ácido Nucleico , Nicotiana/efectos de los fármacos , Nicotiana/metabolismoRESUMEN
Using fluorescent differential display, we identified, from approximately 8000 displayed bands, a DNA fragment showing rapid induction in response to red light irradiation. This EARLY-PHYTOCHROME-RESPONSIVE1 gene (EPR1) encodes a novel nucleus-localized MYB protein harboring a single MYB domain that is highly similar to the circadian oscillator proteins CCA1 and LHY. EPR1 is regulated by both phytochrome A and phytochrome B, and the red-light induction of EPR1 is not inhibited by cycloheximide, demonstrating that EPR1 represents a primary phytochrome-responsive gene. Our results show that EPR1 overexpression results in enhanced far-red light-induced cotyledon opening and delayed flowering. In wild-type Arabidopsis plants grown in continuous light, the EPR1 transcript exhibits circadian rhythmicity similar to that of CCA1 and LHY. Moreover, EPR1 suppresses its own expression, suggesting that this protein is part of a regulatory feedback loop. Constitutive expression of CCA1 and LHY results in the loss of EPR1 rhythmicity, whereas increased levels of EPR1 have no effect on the central oscillator. We propose that EPR1 is a component of a slave oscillator that contributes to the refinement of output pathways, ultimately mediating the correct oscillatory behavior of target genes.
Asunto(s)
Proteínas de Arabidopsis/fisiología , Ritmo Circadiano/fisiología , Proteínas Nucleares/fisiología , Proteínas Proto-Oncogénicas c-myb/fisiología , Secuencia de Aminoácidos , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Secuencia de Bases , Núcleo Celular/genética , Núcleo Celular/fisiología , Cartilla de ADN , ADN Complementario/genética , Datos de Secuencia Molecular , Proteínas Nucleares/química , Proteínas Nucleares/genética , Plantas Modificadas Genéticamente , Reacción en Cadena de la Polimerasa , Proteínas Proto-Oncogénicas c-myb/química , Proteínas Proto-Oncogénicas c-myb/genética , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
We screened for auxin-induced genes with an expression correlated to the auxin-induced growth response from rice coleoptiles by fluorescent differential display. A rice homologue of the auxin response factor (ARF) family of transcriptional regulators, OsARF1, was identified. An OsARF1:GFP fusion protein was localized to the nucleus. Steady-state levels of OsARF1 mRNA correlated positively with auxin-dependent differential growth: gravitropic stimulation enhanced the amount of OsARF1 transcript in the lower, faster-growing flank accompanied by a decrease in the upper flank of gravitropically stimulated rice coleoptiles. Exogenous auxin up-regulated the steady-state level of OsARF1 mRNA within 15-30 min. This up-regulation is independent of de novo protein synthesis. Thus, OsARF1 is the first ARF that classifies as an early auxin-responsive gene. The observed auxin-dependent regulation comprises a new level of regulation in auxin-induced gene expression and is discussed as a possible feedback mechanism in plant growth control.