RESUMEN
The regulatory photoreceptor, phytochrome, controls the expression of numerous genes, including its own phyA genes, which are transcriptionally repressed in response to light. Functional analysis of a rice phyA gene promoter, by means of microprojectile-mediated gene transfer, indicates that a GT motif, GCGGTAATT, closely related to elements in the promoters of a number of other light-regulated genes, is critical for expression. Partial complementary DNA clones have been obtained for a rice nuclear protein, designated GT-2, that binds in a highly sequence-specific fashion to this motif. Mutational analysis shows that the paired G's are most crucial to binding. GT-2 has domains related to certain other transcription factors. Northern blot analysis shows that GT-2 messenger RNA levels decline in white light although red and far red light pulses are ineffective.
Asunto(s)
Genes de Plantas , Proteínas Nucleares/metabolismo , Fitocromo/genética , Regiones Promotoras Genéticas , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Desoxirribonucleasa I , Datos de Secuencia Molecular , Mapeo Nucleótido , Sondas de Oligonucleótidos , Oryza/genética , Oryza/metabolismoRESUMEN
The five members of the phytochrome photoreceptor family of Arabidopsis thaliana control morphogenesis differentially in response to light. Genetic analysis has identified a signaling pathway that is specifically activated by phytochrome A. A component in this pathway, SPA1 (for "suppressor of phyA-105"), functions in repression of photomorphogenesis and is required for normal photosensory specificity of phytochrome A. Molecular cloning of the SPA1 gene indicates that SPA1 is a WD (tryptophan-aspartic acid)-repeat protein that also shares sequence similarity with protein kinases. SPA1 can localize to the nucleus, suggesting a possible function in phytochrome A-specific regulation of gene expression.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/fisiología , Luz , Fitocromo/metabolismo , Proteínas de Plantas/química , Transducción de Señal , Secuencia de Aminoácidos , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Núcleo Celular/metabolismo , Clonación Molecular , Oscuridad , Regulación de la Expresión Génica de las Plantas , Datos de Secuencia Molecular , Morfogénesis , Mutación , Señales de Localización Nuclear , Fitocromo A , Proteínas de Plantas/genética , Proteínas de Plantas/fisiología , Proteínas Quinasas/química , ARN Mensajero/genética , ARN Mensajero/metabolismo , Secuencias Repetitivas de Aminoácido , Proteínas Represoras/química , Alineación de SecuenciaRESUMEN
Light signals perceived by the phytochrome family of sensory photoreceptors are transduced to photoresponsive genes by an unknown mechanism. Here, we show that the basic helix-loop-helix transcription factor PIF3 binds specifically to a G-box DNA-sequence motif present in various light-regulated gene promoters, and that phytochrome B binds reversibly to G-box-bound PIF3 specifically upon light-triggered conversion of the photoreceptor to its biologically active conformer. We suggest that the phytochromes may function as integral light-switchable components of transcriptional regulator complexes, permitting continuous and immediate sensing of changes in this environmental signal directly at target gene promoters.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Luz , Células Fotorreceptoras , Fitocromo/metabolismo , Proteínas de Plantas , Regiones Promotoras Genéticas , Factores de Transcripción/metabolismo , Arabidopsis/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Proteínas de Ciclo Celular/genética , ADN de Plantas/genética , ADN de Plantas/metabolismo , Proteínas de Unión al ADN/genética , Perfilación de la Expresión Génica , Genes de Plantas , Secuencias Hélice-Asa-Hélice , Modelos Genéticos , Oligodesoxirribonucleótidos/genética , Oligodesoxirribonucleótidos/metabolismo , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , Fitocromo B , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , Factores de Transcripción/genéticaRESUMEN
The phytochrome family of photoreceptors monitors the light environment and dictates patterns of gene expression that enable the plant to optimize growth and development in accordance with prevailing conditions. The enduring challenge is to define the biochemical mechanism of phytochrome action and to dissect the signaling circuitry by which the photoreceptor molecules relay sensory information to the genes they regulate. Evidence indicates that individual phytochromes have specialized photosensory functions. The amino-terminal domain of the molecule determines this photosensory specificity, whereas a short segment in the carboxyl-terminal domain is critical for signal transfer to downstream components. Heterotrimeric GTP-binding proteins, calcium-calmodulin, cyclic guanosine 5'-phosphate, and the COP-DET-FUS class of master regulators are implicated as signaling intermediates in phototransduction.
Asunto(s)
Células Fotorreceptoras/fisiología , Fitocromo , Fenómenos Fisiológicos de las Plantas , Transducción de Señal/fisiología , Células Fotorreceptoras/química , Fitocromo/química , Fitocromo/genética , Plantas/genéticaRESUMEN
Genetic and molecular studies are beginning to unravel the complexities of the signaling circuitry that plants use to sense and transduce information concerning the prevailing light environment. The past year has witnessed definition of discrete photosensory roles for phytochromes A and B, the cloning of a gene encoding the first apparent blue-light photoreceptor from any organism, the cloning of genes encoding additional members of the COP/DET/FUS class of light-responsive master regulators, and evidence that G proteins, Ca2+/calmodulin, and cGMP may be signaling intermediates in phototransduction.
Asunto(s)
Plantas/efectos de la radiación , Genes de Plantas , Genes de Cambio , Luz , Modelos Biológicos , Mutación , Células Fotorreceptoras/metabolismo , Fitocromo/genética , Fenómenos Fisiológicos de las Plantas , Plantas/genética , Sistemas de Mensajero Secundario , Transducción de Señal , Rayos UltravioletaRESUMEN
The hy1 and hy2 long hypocotyl mutants of Arabidopsis contain normal levels of immunochemically detectable phytochrome A, but the molecule is photochemically nonfunctional. We have investigated the biochemical basis for this lack of function. When the hy1 and hy2 mutants were grown in white light on a medium containing biliverdin IX[alpha], a direct precursor to phytochromobilin, the phytochrome chromophore, the seedlings developed with a morphological phenotype indistinguishable from the light-grown wild-type control. Restoration of a light-grown phenotype in the hy1 mutant was also accomplished by using phycocyanobilin, a tetrapyrrole analog of phytochromobilin. Spectrophotometric and immunochemical analyses of the rescued hy1 and hy2 mutants demonstrated that they possessed wild-type levels of photochemically functional phytochrome that displayed light-induced conformational changes in the holoprotein indistinguishable from the wild type. Moreover, phytochrome A levels declined in vivo in response to white light in rescued hy1 and hy2 seedlings, indicative of biliverdin-dependent formation of photochemically functional phytochrome A that was then subject to normal selective turnover in the far-red-light-absorbing form. Combined, these data suggest that the hy1 and hy2 mutants are inhibited in chromophore biosynthesis at steps prior to the formation of biliverdin IX[alpha], thus potentially causing a global functional deficiency in all members of the phytochrome photoreceptor family.
RESUMEN
To determine the functional homology between phytochromes from evolutionarily divergent species, we used the cauliflower mosaic virus 35S promoter to express a monocot (oat) phytochrome cDNA in a dicot plant (tomato). Immunoblot analysis shows that more than 50% of the transgenic tomato plants synthesize the full-length oat phytochrome polypeptide. Moreover, leaves of light-grown transgenic plants contain appreciably less oat phytochrome than leaves from dark-adapted plants, and etiolated R1 transgenic seedlings have higher levels of spectrally active phytochrome than wild-type tomato seedlings in direct proportion to the level of immunochemically detectable oat polypeptide present. These data suggest that the heterologous oat polypeptide carries a functional chromophore, allowing reversible photoconversion between the two forms of the molecule, and that the far-red absorbing form (Pfr) is recognized and selectively degraded by the Pfr-specific degradative machinery in the dicot cell. The overexpression of oat phytochrome has pleiotropic, phenotypic consequences at all major phases of the life cycle. Adult transgenic tomato plants expressing high levels of the oat protein tend to be dwarfed, with dark green foliage and fruits. R1 transgenic seedlings have short hypocotyls with elevated anthocyanin contents. We conclude that a monocot phytochrome can be synthesized and correctly processed to a biologically active form in a dicot cell, and that the transduction pathway components that interact with the photoreceptor are evolutionarily conserved.
RESUMEN
We used a series of in vitro-generated deletion and amino acid substitution derivatives of phytochrome B (phyB) expressed in transgenic Arabidopsis to identify regions of the molecule important for biological activity. Expression of the chromophore-bearing N-terminal domain of phyB alone resulted in a fully photoactive, monomeric molecule lacking normal regulatory activity. Expression of the C-terminal domain alone resulted in a photoinactive, dimeric molecule, also lacking normal activity. Thus, both domains are necessary, but neither is sufficient for phyB activity. Deletion of a small region on each major domain (residues 6 to 57 and 652 to 712, respectively) was shown to compromise phyB activity differentially without interfering with spectral activity or dimerization. Deletion of residues 6 to 57 caused a large increase in the fluence rate of continuous red light (Rc) required for maximal seedling responsiveness, indicating a marked decrease in efficiency of light signal perception or processing per mole of mutant phyB. In contrast, deletion of residues 652 to 712 resulted in a photoreceptor that retained saturation of seedling responsiveness to Rc at low fluence rates but at a response level much below the maximal response elicited by the parent molecule. This deletion apparently reduces the maximal biological activity per mole of phyB without a major decrease in efficiency of signal perception, thus suggesting disruption of a process downstream of signal perception. In addition, certain phyB constructs caused dominant negative interference with endogenous phyA activity in continuous far-red light, suggesting that the two photoreceptors may share reaction partners.
RESUMEN
The photoreceptor phytochrome is encoded by a small multigene family in higher plants. phyA encodes the well-characterized etiolated-tissue phytochrome. The product of the phyB gene, which has properties resembling those of "green tissue" phytochrome, is as yet poorly characterized. We have developed a phytochrome B overexpression system for analysis of the structure and function of this protein. Using newly generated polyclonal and monoclonal antibodies that are selective for phytochrome B, we have demonstrated high levels of expression of full-length rice and Arabidopsis phytochrome B under the control of the cauliflower mosaic virus 35S promoter in transgenic Arabidopsis. The overexpressed phytochrome is spectrally active, undergoes red/far-red-light-dependent conformational changes, is synthesized in its inactive red light-absorbing form, and is stable in the light. Overexpression of phytochrome B is tightly correlated with a short hypocotyl phenotype in transgenic seedlings. This phenotype is strictly light dependent, thus providing direct evidence that phytochrome B is a biologically functional photoreceptor. Based on similarities to phenotypes obtained by overexpression of phytochrome A, it appears that phytochromes A and B can control similar responses in the plant.
RESUMEN
The six long hypocotyl (hy) complementation groups of Arabidopsis (hy1, hy2, hy3, hy4, hy5, and hy6) share the common feature of an elongated hypocotyl when grown in white light. The varied responses of these mutants to irradiations of differing wavelengths have suggested that some of the lines may lack elements of the phytochrome signal transduction pathway. We have performed immunoblot and RNA gel blot analyses of the multiple types of phytochrome present in wild-type and mutant Arabidopsis and provide evidence that mutations at the HY3 locus cause a specific deficiency in phytochrome B. Using an Escherichia coli overexpression system, we have developed and identified monoclonal antibodies that selectively recognize phytochromes A, B, and C from Arabidopsis. In wild-type plants, phytochrome A is highly abundant in etiolated tissue, but rapidly decreases about 200-fold upon illumination. Phytochromes B and C are present at much lower levels in etiolated tissue but are unaffected by up to 24 hr of red light illumination, and together predominate in green seedlings. These data establish that phytochromes B and C are "type 2" or photostable phytochromes. Levels of phytochromes A, B, and C similar to those of the wild type are observed in strains containing mutations at the HY4 and HY5 loci. In contrast, all four hy3 mutant alleles tested here exhibit a modest (twofold to threefold) reduction in phyB transcript and a severe (20- to 50-fold) deficiency in phyB-encoded protein, relative to levels in wild-type plants. The levels of phyA- and phyC-encoded mRNA and protein, however, are indistinguishable from the wild type in these mutants. We conclude that the phenotype conferred by hy3 is due to the reduced levels of the light-stable phytochrome B.
RESUMEN
We have examined phytochrome-regulated transcription of phytochrome (phy) and chlorophyll a/b binding protein (cab) genes in dark-grown Avena seedlings by using run-on transcription in isolated nuclei. Kinetic analysis of phy transcription following pulse-light treatments to produce various amounts of Pfr, the active form of phytochrome, leads to these conclusions. (i) Transcription decreases rapidly (discernible within 5 min) after Pfr formation, reaching an essentially undetectable level by 1 h. (ii) The response is very sensitive; less than 1% Pfr is sufficient to produce maximum feedback repression over the first 30 min. (iii) The duration of transcriptional repression is proportional to the Pfr concentration; derepression begins once the concentration falls below some saturation level because of degradation of Pfr. Concurrent analysis of cab transcription leads to these conclusions. (i) After Pfr formation, transcription increases approximately 10-fold by 3 h, but this response is not detectable until after a 30-min lag. (ii) Detectable induction of cab requires a greater than 30-fold-higher Pfr level than is needed to repress phy expression. (iii) Transcription returns to the preirradiation level considerably sooner than does phy transcription (less than 12 h versus greater than 24 h respectively), indicating that a high level of Pfr is needed to sustain the increased transcription of cab. Taken together, these results suggest that differences in the phytochrome signal transduction pathway are responsible for the distinct patterns of regulation of these genes. Full repression of phy occurs even when protein synthesis is inhibited greater than 90% by cycloheximide and chloramphenicol. In conjunction with the rapidity of the response to Pfr, this result provides evidence that feedback repression of phy gene transcription does not require expression of an intervening regulatory gene(s). Thus, phy is the first gene for which there is evidence for direct control of transcription by the phytochrome signal transduction chain.
Asunto(s)
Fitocromo/farmacología , Proteínas de Plantas/farmacología , Plantas/genética , Transcripción Genética/efectos de los fármacos , Cloranfenicol/farmacología , Clorofila/genética , Cicloheximida/farmacología , Sondas de ADN , Regulación de la Expresión Génica/efectos de los fármacos , Luz , Complejos de Proteína Captadores de Luz , Proteínas del Complejo del Centro de Reacción Fotosintética , Fitocromo/genética , Proteínas de Plantas/genética , Plantas/efectos de los fármacos , Plantas/efectos de la radiación , ARN/biosíntesis , Transcripción Genética/efectos de la radiaciónRESUMEN
Accumulating evidence indicates that individual members of the phytochrome family of photoreceptors have differential but interactive roles in controlling plant responses to light. To investigate possible cross-regulation of these receptors, we have identified monoclonal antibodies that specifically detect each of the five Arabidopsis phytochromes, phyA to phyE (phytochrome A holoprotein; PHYA, phytochrome A apoprotein; PHYA, phytochrome A gene; phyA, mutant allele of phytochrome A gene), on immunoblots and have used them to analyze the effects of phyA and phyB null mutations on the levels of all five family members. In phyB mutants, but not in phyA mutants, a four- to six-fold reduction in the level of phyC is observed in tissues grown either in the dark or in the light. Coordinate expression of phyB and phyC is induced in the phyB mutant background by the presence of a complementing PHYB transgene. However, in transgenic lines that overexpress phyB 15- to 20-fold, phyC is not similarly overexpressed. In these overexpressor lines, the levels of phyA, phyC, and phyD are increased two- to four-fold over normal in light-grown but not dark-grown seedlings. These observations indicate that molecular mechanisms for coordination or cross-regulation of phytochrome levels are active in Arabidopsis and have implications for the interpretation of phytochrome mutants and overexpressor lines.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Apoproteínas/inmunología , Proteínas de Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Mutación/genética , Células Fotorreceptoras , Fitocromo/genética , Factores de Transcripción , Anticuerpos Monoclonales/aislamiento & purificación , Especificidad de Anticuerpos , Apoproteínas/análisis , Apoproteínas/metabolismo , Regulación de la Expresión Génica de las Plantas , Immunoblotting , Peso Molecular , Fitocromo/inmunología , Fitocromo/metabolismo , Fitocromo A , Fitocromo B , Proteínas de Plantas/análisis , Procesamiento Postranscripcional del ARNRESUMEN
Phytochrome wild-type gene-[beta]-glucuronidase (PHY-GUS) gene fusions were used in transgenic Arabidopsis to compare the activity levels and light regulation of the PHYA and PHYB promoters and to identify the photoreceptors mediating this regulation. In dark-grown seedlings, both promoters are 4-fold more active in shoots than in roots,but the PHYA promoter is nearly 20-fold more active than that of PHYB in both organs. In shoots, white light represses the activities of the PHYA and PHYB promoters 10- and 2-fold, respectively, whereas in roots light has no effect on the PHYA promoter but increases PHYB promoter activity 2-fold. Consequently, PHYA promoter activity remains higher than that of PHYB in light in both shoots (5-fold) and roots (11-fold). Experiments with narrow-waveband light and photomorphogenic mutants suggest that no single photoreceptor is necessary for full white-light-directed PHYA repression in shoots, but that multiple, independent photoreceptor pathways are sufficient alone or in combination. In contrast, phytochrome B appears both necessary and sufficient for a light-mediated decrease in PHYB activity in shoots, and phytochrome A mediates a far-red-light-stimulated increase in PHYB promoter activity. Together, the data indicate that the PHYA and PHYB genes are regulated in divergent fashion at the transcriptional level, both developmentally and by the spectral distribution of the prevailing light, and that this regulation may be important to the photosensory function of the two photoreceptors.
RESUMEN
We have isolated genomic clones for three loci encoding the phytochrome polypeptide of Zea mays, and have determined the entire sequence of one of them (phyA1) together with approximately 1 kb of 5' flanking DNA. The structure of this gene is highly conserved in comparison with other phytochrome-encoding genes (phy). The deduced amino acid (aa) sequence indicates that the maize phytochrome protein is 1130 aa long (125 kDa). Overall aa sequence identity is 88% with Avena and rice type A phytochromes and 65% with the type A phytochromes of the dicots, pea, zucchini and Arabidopsis. Northern analysis indicates that maize phy transcripts are down-regulated only two- to threefold in etiolated seedlings 3 h after a red light pulse, in contrast to Avena where a ten- to 20-fold decrease is observed. On the other hand, a more than tenfold reduction in maize phy mRNA abundance occurs in seedlings transferred to white light for 24 h. Several conserved sequence elements have been identified by comparison of the maize phyA1 and other monocot phy promoters, suggesting that these common regions may be regulatory elements involved in phy expression.
Asunto(s)
Genes de Plantas , Fitocromo/genética , Proteínas de Plantas/genética , Zea mays/genética , Secuencia de Aminoácidos , Secuencia de Bases , Northern Blotting , Southern Blotting , Clonación Molecular , Regulación de la Expresión Génica/efectos de la radiación , Luz , Datos de Secuencia Molecular , Conformación Proteica , Mapeo Restrictivo , Transcripción GenéticaRESUMEN
The amino acid (aa) sequence of Cucurbita phytochrome has been deduced from the nucleotide (nt) sequence of a cDNA clone which was initially identified by hybridization to an Avena phytochrome cDNA clone. Cucurbita, a dicot, and Avena, a monocot, represent evolutionarily divergent groups of plants. The Cucurbita phytochrome polypeptide is 1123 aa in length, corresponding to 125 kDa. Overall, the Cucurbita and Avena phytochrome sequences are 65% homologous at both the nt and aa levels but this sequence conservation is not evenly distributed. Most of the N-terminal two-thirds of the aligned polypeptide chains exhibits localized regions of high conservation, while the extreme N terminus and the C-terminal one-third are less homologous. Comparison of the predicted hydropathic properties of these polypeptides also indicates conservation of domains of phytochrome structure. The possible correlation of these conserved structural features with previously identified functional domains of phytochrome is discussed.
Asunto(s)
Fitocromo/genética , Proteínas de Plantas/genética , Plantas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Evolución Biológica , ADN/genética , Fitocromo/metabolismo , Plantas/metabolismo , Especificidad de la EspecieRESUMEN
We have isolated and characterized a gene encoding the phytochrome polypeptide of Avena. Based on nucleotide sequence identity with previously sequenced cDNA clones this gene is designated as type 3 (phy3). The gene is about 5.9 kb long with six exons and five introns, one each of the latter in the 5' and 3'-untranslated regions. The largest exon encodes the entire 74-kDa, chromophore-bearing, N-terminal domain of the photo-receptor postulated to be directly involved in its mechanism of action. The transcription start point, identified by mung-bean nuclease digestion, is located 24 to 35 bp downstream from a tandem TATA box. Sequence elements homologous to a number of motifs implicated as upstream regulatory elements in other genes are present in the 5'-flanking DNA of phy3. Particularly intriguing are three elements at positions -140, -470 and -650. These elements share homology with the 'GT' motif postulated to be a component of the light-regulatory element of genes encoding the small subunit of ribulose bisphosphate carboxylase.
Asunto(s)
Fitocromo/genética , Proteínas de Plantas/genética , Poaceae/genética , Bacteriófago lambda/genética , Secuencia de Bases , Mapeo Cromosómico , Clonación Molecular , ADN/análisis , Regulación de la Expresión Génica , Datos de Secuencia Molecular , Transcripción GenéticaRESUMEN
In monocotyledons, the expression of the oat phytochrome A gene (PHYA) is down-regulated by phytochrome itself. This autoregulatory repression is the most rapid light-induced effect on gene expression reported in plants to date. A functional analysis of the oat PHYA3 gene minimal promoter in a rice transient expression assay has identified two promoter elements, PE1 and PE3, that interact synergistically in positive regulation. We have isolated an oat cDNA clone (pO2) that encodes a DNA-binding protein that binds to the PE1 region of the oat PHYA3 gene promoter. The in vitro binding properties of the pO2-encoded protein, towards DNA probes containing either the PE1 sequence or linker-substitution mutations in PE1, correlate with the activity of these DNA elements in the rice transient expression assay. These mutations are known to abolish expression of a reporter gene in vivo. Binding of these linker-substitution mutants to the pO2-encoded protein in vitro was lower by one to two orders of magnitude than the binding of the native PE1 region. We suggest, therefore, that the pO2 clone may encode the putative nuclear factor, oat PF1, that is involved in positive regulation of PHYA3 by binding to PE1 in vivo. pO2 encodes a 170-amino-acid-long protein that contains three repeats of the 'AT-hook' DNA-binding motif found in high mobility group I-Y (HMGI-Y) proteins. Oat PF1 is highly similar to rice PF1 and to the protein encoded by soybean cDNA SB16. They all have a strong similarity in their N-terminus to the pea H1 histone, and the presence of several AT-hook DNA-binding motifs in their C-terminal halves.
Asunto(s)
Avena/genética , Proteínas de Unión al ADN/genética , Fitocromo/genética , Proteínas de Plantas/genética , Regiones Promotoras Genéticas , Clonación Molecular , ADN Complementario , Proteínas de Unión al ADN/metabolismo , Genes de Plantas , Oryza/genética , Fitocromo/metabolismo , Fitocromo A , Proteínas de Plantas/metabolismoRESUMEN
We have determined the sequence of the phytochrome A gene (PHYA) and its flanking DNA from Arabidopsis thaliana and have identified transcription start sites for three nested transcripts of increasing length. The overall structure of the gene is similar as regards exon/intron organization to other angiosperm PHY genes characterized. The triple transcription start site arrangement is similar to that of pea PHYA but different from the single start site of oat, rice and maize PHYA genes, indicating a possible monocot-dicot difference. Comparison of the Arabidopsis PHYA promoter sequence with others available indicates that both pea and Arabidopsis promoters contain a DNA element with a core sequence motif identical to one conserved in all existing monocot PHYA sequences and defined by functional assay in the oat PHYA gene as repressor element, RE1, responsible for negative light regulation.