RESUMEN
The molecular mechanism underlying phototherapy and light treatment, which utilize various wavelength spectra of light, including near-infrared (NIR), to cure human and plant diseases, is obscure. Here we revealed that NIR light confers antiviral immunity by positively regulating PHYTOCHROME-INTERACTING FACTOR 4 (PIF4)-activated RNA interference (RNAi) in plants. PIF4, a central transcription factor involved in light signaling, accumulates to high levels under NIR light in plants. PIF4 directly induces the transcription of two essential components of RNAi, RNA-DEPENDENT RNA POLYMERASE 6 (RDR6) and ARGONAUTE 1 (AGO1), which play important roles in resistance to both DNA and RNA viruses. Moreover, the pathogenic determinant ßC1 protein, which is evolutionarily conserved and encoded by betasatellites, interacts with PIF4 and inhibits its positive regulation of RNAi by disrupting PIF4 dimerization. These findings shed light on the molecular mechanism of PIF4-mediated plant defense and provide a new perspective for the exploration of NIR antiviral treatment.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Fitocromo , Humanos , Fitocromo/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Interferencia de ARN , Regulación de la Expresión Génica de las PlantasRESUMEN
The effect of red (RL, 660 nm) and blue (BL, 450 nm) light on phy mutant tomato plants was studied. The rates of photosynthesis (Pn) and transpiration, the efficiency of the primary photochemical processes of photosynthesis, the contents of flavonoids and phenolic compounds, the low-molecular-weight antioxidant capacity (Trolox equivalent antioxidant capacity (TEAC)) of leaf extracts, and the expression of light-dependent genes were evaluated. Under RL, BL, and white fluorescent light (WFL), the Pn values decreased in the order: WT > phyb2 > phyaphyb2 > phyaphyb1phyb2, except for the Pn in phyb2 on BL. Phyb2 also had a larger number of stomata under BL and, as a result, it reached maximum transpiration. The noticeable accumulation of flavonoids and phenolic compounds was observed only in the phyb2 and phyaphyb2 mutants upon irradiation with BL, which agrees with the increased TEAC in the leaf extracts. We suggest that the increased antioxidant activity under PHYB2 deficiency and the maintenance of high photosynthesis under BL are based on an increase in the expression of the early signaling transcription factors genes BBX, HY5. The largest decrease in the content of flavonoids and TEAC was manifested with a deficiency in PHYB1, which is probably the key to maintaining the antioxidant status in BL plants.
Asunto(s)
Fitocromo , Solanum lycopersicum , Fitocromo/genética , Fitocromo/metabolismo , Solanum lycopersicum/genética , Solanum lycopersicum/metabolismo , Flavonoides , Antioxidantes/metabolismo , Fotosíntesis/genética , Extractos Vegetales/metabolismoRESUMEN
The transition to tuberization contributes greatly to the adaptability of potato to a wide range of environments. Phytochromes are important light receptors for the growth and development of plants, but the detailed functions of phytochromes remain unclear in potato. In this study, we first confirmed that phytochrome F (StPHYF) played essential roles in photoperiodic tuberization in potato. By suppressing the StPHYF gene, the strict short-day potato genotype exhibited normal tuber formation under long-day (LD) conditions, together with the degradation of the CONSTANTS protein StCOL1 and modulation of two FLOWERING LOCUS T (FT) paralogs, as demonstrated by the repression of StSP5G and by the activation of StSP6A during the light period. The function of StPHYF was further confirmed through grafting the scion of StPHYF-silenced lines, which induced the tuberization of untransformed stock under LDs, suggesting that StPHYF was involved in the production of mobile signals for tuberization in potato. We also identified that StPHYF exhibited substantial interaction with StPHYB both in vitro and in vivo. Therefore, our results indicate that StPHYF plays a role in potato photoperiodic tuberization, possibly by forming a heterodimer with StPHYB.
Asunto(s)
Fitocromo/metabolismo , Solanum tuberosum/fisiología , Genotipo , Fotoperiodo , Fitocromo/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Tubérculos de la Planta/genética , Tubérculos de la Planta/fisiología , Tubérculos de la Planta/efectos de la radiación , Solanum tuberosum/genética , Solanum tuberosum/efectos de la radiaciónRESUMEN
The WRKY proteins belong to a superfamily of TFs that play pivotal roles in responses to a wide range of biotic, abiotic, developmental and physiologic cues. Here, we assayed the accumulation of basal WRKY27 transcripts in diverse tissue including root, shoot, leaf and flowers. We demonstrated that plants over-expressing WRKY27 transcript levels exhibit growth aberrations and fertility defects. Scanning electron microscopic data suggest that WRKY27 overexpressor plants exhibit pollen dehiscence defects. Our fluorescein diacetate hydrolysis assay showed that flowers of plants overexpressing WRKY27 display significantly decreased pollen viability. These sterility-related phenotypes were not rescued by the exogenous applications of different phytohormones. Our results indicate the involvement of WRKY27 in particular for proper plant biomass accumulation and male fertility.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Arabidopsis/fisiología , Infertilidad Vegetal/fisiología , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Fitocromo/genética , Fitocromo/metabolismo , Reguladores del Crecimiento de las Plantas/genética , Reguladores del Crecimiento de las Plantas/metabolismo , Infertilidad Vegetal/genética , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Plantas Modificadas Genéticamente/fisiología , Polen/genética , Polen/metabolismoRESUMEN
BPG2 (Brz-insensitive pale green 2) is a dark-repressible and light-inducible gene that is required for the greening process in Arabidopsis. Light pulse experiments suggested that light-regulated gene expression of BPG2 is mediated by phytochrome. The T-DNA insertion mutant bpg2-2 exhibited a reduced level of chlorophyll and carotenoid pigmentation in the plastids. Measurements of time resolved chlorophyll fluorescence and of fluorescence emission at 77 K indicated defective photosystem II and altered photosystem I functions in bpg2 mutants. Kinetic analysis of chlorophyll fluorescence induction suggested that the reduction of the primary acceptor (QA) is impaired in bpg2. The observed alterations resulted in reduced photosynthetic efficiency as measured by the electron transfer rate. BPG2 protein is localized in the plastid stroma fraction. Co-immunoprecipitation of a formaldehyde cross-linked RNA-protein complex indicated that BPG2 protein binds with specificity to chloroplast 16S and 23S ribosomal RNAs. The direct physical interaction with the plastid rRNAs supports an emerging model whereby BPG2 provides light-regulated ribosomal RNA processing functions, which are rate limiting for development of the plastid and its photosynthetic apparatus.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Cloroplastos/genética , Proteínas de Unión al GTP/genética , Regulación de la Expresión Génica de las Plantas/efectos de la radiación , ARN Ribosómico 16S/genética , ARN Ribosómico 23S/genética , Arabidopsis/fisiología , Arabidopsis/efectos de la radiación , Arabidopsis/ultraestructura , Proteínas de Arabidopsis/metabolismo , Carotenoides/metabolismo , Clorofila/metabolismo , Cloroplastos/metabolismo , Proteínas de Unión al GTP/metabolismo , Inmunoprecipitación , Luz , Mutagénesis Insercional , Cebollas/genética , Cebollas/metabolismo , Fotosíntesis , Complejo de Proteína del Fotosistema I/metabolismo , Complejo de Proteína del Fotosistema II/metabolismo , Fitocromo/genética , Fitocromo/metabolismo , Estomas de Plantas/metabolismo , ARN del Cloroplasto/genética , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
Tuberization is one of the multiple outputs of a single-input phytochrome B sensory system, involving several regulatory genes. Phytochrome B- and GA-mediated photoperiodic perception occurs in the leaf, and then the RNA acts as a systemic signal in the long-distance signaling pathway to initiate tuberization in the subapical region of an underground stolon. There is good evidence that flowering and tuberizing signals might be similar. Is there a cross-talk with an oxidative burst-mediated redox signaling pathway during tuberization? Is the lipoxygenase cascade involved in the formation of the perimedullary tissue in a growing tuber? Do aquaporins regulate cell division, expansion and elongation during stolon growth and tuber induction in potato? Is the adaptive diversity for tuberization under varying photoperiods a micro-evolutionary indicator of differential transduction of cell-to-cell signal molecules under spatial and temporal expression of regulatory genes encoding transcriptional activators? Taking these views into consideration, the review presents an interim synthesis of a signaling network regulating in planta tuberization in potato.
Asunto(s)
Transducción de Señal/fisiología , Solanum tuberosum/metabolismo , Modelos Biológicos , Fitocromo/genética , Fitocromo/metabolismo , Tubérculos de la Planta/crecimiento & desarrollo , Tubérculos de la Planta/metabolismo , ARN de Planta/genética , ARN de Planta/metabolismo , Transducción de Señal/genética , Solanum tuberosum/genética , Solanum tuberosum/crecimiento & desarrolloRESUMEN
Coimmunoprecipitation of members of the phytochrome red/farred photoreceptor family from plant extracts has been used to analyze their heteromeric binding interactions. Phytochrome (phy)B or phyD apoproteins with six myc epitopes fused to their N termini are biologically active when expressed in Arabidopsis. Immunoprecipitation of either of these tagged proteins from seedling extracts coprecipitates additional type II phytochromes: six myc (myc6)-phyB coprecipitates phyC-phyE; and myc6-phyD coprecipitates phyB and phyE. No interaction of the epitope-tagged proteins with type I phyA was detected. Gel filtration chromatography shows that all five of the Arabidopsis phytochromes are present in seedlings as dimers, and that the heteromeric type II phytochrome complexes migrate at molecular masses characteristic of heterodimers. Similar levels of heterodimer formation are observed in extracts of dark-grown seedlings, where the phytochromes are cytosolic, and light-grown seedlings, where they are predominantly nuclear. These findings indicate that Arabidopsis, which until now has been thought to contain five homodimeric forms of phytochrome, in fact contains multiple species of both homodimeric and heterodimeric phytochromes. The conservation of the phytochrome family throughout angiosperms suggests that heterodimeric red/far-red receptors may be present in many flowering plants.
Asunto(s)
Arabidopsis/química , Arabidopsis/genética , Células Fotorreceptoras , Fitocromo/química , Fitocromo/genética , Factores de Transcripción , Proteínas de Arabidopsis , Oscuridad , Dimerización , Epítopos , Iluminación , Fitocromo/metabolismo , Fitocromo B , Plantas Modificadas Genéticamente , Unión Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , TransgenesAsunto(s)
Células Fotorreceptoras , Fitocromo/fisiología , Plantas Modificadas Genéticamente/efectos de la radiación , Solanum tuberosum/efectos de la radiación , Factores de Transcripción , Expresión Génica , Fenotipo , Fitocromo/biosíntesis , Fitocromo/genética , Fitocromo B , Componentes Aéreos de las Plantas/crecimiento & desarrollo , Componentes Aéreos de las Plantas/efectos de la radiación , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/crecimiento & desarrollo , Plantas Modificadas Genéticamente/metabolismo , Tolerancia a Radiación/genética , Solanum tuberosum/genética , Solanum tuberosum/crecimiento & desarrollo , Solanum tuberosum/metabolismo , Factores de Tiempo , Rayos Ultravioleta/efectos adversosRESUMEN
Xanthomonas oryzae pv. oryzae causes bacterial leaf blight, a serious disease of rice. We have identified a novel virulence deficient mutant (BXO1691) of X. oryzae pv. oryzae that has a Tn5 insertion in an open reading frame (phyA; putative phytase A) encoding a 373-amino acid (aa) protein containing a 28-aa predicted signal peptide. Extracellular protein profiles revealed that a 38-kDa band is absent in phyA mutants as compared with phyA+ strains. A BLAST search with phyA and its deduced polypeptide sequence indicated significant similarity with conserved hypothetical proteins in Xanthomonas axonopodis pv. citri and Xanthomonas campestris pv. campestris and limited homology to secreted phytases of Bacillus species. Homology modeling with a Bacillus phytase as the template suggests that the PhyA protein has a similar six-bladed beta-propeller architecture and exhibits conservation of certain critical active site residues. Phytases are enzymes that are involved in degradation of phytic acid (inositol hexaphosphate), a stored form of phosphate in plants. The phyA mutants exhibit a growth deficiency in media containing phytic acid as a sole phosphate source. Exogenous phosphate supplementation promotes migration of phyA X. oryzae pv. oryzae mutants in rice leaves. These results suggest that the virulence deficiency of phyA mutants is, at least in part, due to inability to use host phytic acid as a source of phosphate. phyA-like genes have not been previously reported to be involved in the virulence of any plant pathogenic bacterium.
Asunto(s)
Fosfatos/metabolismo , Ácido Fítico/metabolismo , Fitocromo/genética , Fitocromo/metabolismo , Xanthomonas/crecimiento & desarrollo , Xanthomonas/patogenicidad , Secuencia de Bases , Southern Blotting , Clonación Molecular , Escherichia coli/genética , Datos de Secuencia Molecular , Oryza/efectos de los fármacos , Oryza/crecimiento & desarrollo , Oryza/microbiología , Fragmentos de Péptidos/química , Fragmentos de Péptidos/aislamiento & purificación , Fitocromo A , Enfermedades de las Plantas/microbiología , Hojas de la Planta/efectos de los fármacos , Hojas de la Planta/crecimiento & desarrollo , Hojas de la Planta/microbiología , Plásmidos/genética , Eliminación de Secuencia , Virulencia/genética , Xanthomonas/genéticaRESUMEN
The possibility that reduced photomorphogenic responses could increase field crop yield has been suggested often, but experimental support is still lacking. Here, we report that ectopic expression of the Arabidopsis PHYB (phytochrome B) gene, a photoreceptor involved in detecting red to far-red light ratio associated with plant density, can increase tuber yield in field-grown transgenic potato (Solanum tuberosum) crops. Surprisingly, this effect was larger at very high densities, despite the intense reduction in the red to far-red light ratios and the concomitant narrowed differences in active phytochrome B levels between wild type and transgenics at these densities. Increased PHYB expression not only altered the ability of plants to respond to light signals, but they also modified the light environment itself. This combination resulted in larger effects of enhanced PHYB expression on tuber number and crop photosynthesis at high planting densities. The PHYB transgenics showed higher maximum photosynthesis in leaves of all strata of the canopy, and this effect was largely due to increased leaf stomatal conductance. We propose that enhanced PHYB expression could be used in breeding programs to shift optimum planting densities to higher levels.
Asunto(s)
Productos Agrícolas/crecimiento & desarrollo , Flores/crecimiento & desarrollo , Células Fotorreceptoras , Fitocromo/metabolismo , Solanum tuberosum/crecimiento & desarrollo , Factores de Transcripción , Arabidopsis/genética , Proteínas de Arabidopsis , Productos Agrícolas/efectos de la radiación , Flores/efectos de la radiación , Luz , Fitocromo/genética , Fitocromo B , Plantas Modificadas Genéticamente , Solanum tuberosum/efectos de la radiaciónAsunto(s)
Adenina/farmacología , Regulación de la Expresión Génica de las Plantas , Fotoperiodo , Células Fotorreceptoras , Fitocromo/fisiología , Reguladores del Crecimiento de las Plantas/metabolismo , Solanum tuberosum/genética , Factores de Transcripción , Adenina/análogos & derivados , Citocininas/biosíntesis , Cinetina , Luz , Fitocromo/genética , Fitocromo/metabolismo , Fitocromo B , Tubérculos de la Planta/efectos de los fármacos , Tubérculos de la Planta/crecimiento & desarrollo , Plantas Modificadas Genéticamente , Solanum tuberosum/crecimiento & desarrolloRESUMEN
Transgenic potatoes (Solanum tuberosum) with either increased (sense transformants) or reduced (antisense transformants) phytochrome A (phyA) levels were used, in combination with specific light treatments, to investigate the involvement of phyA in the perception of signals that entrain the circadian clock. Far-red or far-red plus red light treatments given during the night reset the circadian rhythm of leaf movements in wild-type plants and phyA over-expressors, but had little effect in phyA under-expressors. Far-red light was also able to reset the rhythm of leaf movement in wild-type Arabidopsis thaliana but was not effective in mutants without phyA. Blue light was necessary to reset the rhythm in phyA-deficient potato plants. Resetting of the rhythm by far-red plus red light was only slightly affected in transgenic plants with reduced levels of phytochrome B. The production of tubers was delayed by day extensions with far-red plus red light, but this effect was reduced in transgenic lines deficient in phyA. We conclude that phyA is involved in resetting the circadian clock controlling leaf movements and in photoperiod sensing in light-grown potato plants.
Asunto(s)
Ritmo Circadiano/fisiología , Fitocromo/fisiología , Solanum tuberosum/fisiología , Proteínas de Arabidopsis , Oscuridad , Luz , Oligodesoxirribonucleótidos Antisentido/farmacología , Fitocromo/genética , Fitocromo A , Hojas de la Planta/fisiología , Raíces de Plantas/fisiología , Análisis de Regresión , Estaciones del Año , Transducción de SeñalRESUMEN
Phytochrome in etiolated sprouts of wild type (WT) potato and its transgenic strains (DARA5 and DARA12) expressing Arabidopsis thaliana phytochrome B (phyB) was investigated using low-temperature (85 K) fluorescence spectroscopy and photochemistry. Phytochrome content, [Ptot], position of the Pr emission and excitation spectra, lambda(max), and extent of the Pr-->lumi-R, gamma1, and Pr-->Pfr, gamma2, phototransformations (at 85 and 273 K, respectively) were shown to vary in the transgenic lines and WT depending on tissue used (upper vs. lower parts of etiolated sprouts) and light-induced phytochrome depletion. Differences in the parameters between the transgenic lines and WT were detected which were interpreted in terms of the two phenomenological Pr types: a labile Pr' with gamma1 approximately 0.5 consisting of a major phytochrome A (phyA) fraction (phyA') and a relatively conserved Pr" with gamma1 = 0 comprising a minor phyA fraction (phyA") and phyB. Both DARA lines had higher [Pr"] as compared with WT in the lower parts of etiolated stems, especially after light-induced phytochrome depletion (residual phytochrome in DARA5 and DARA12 made up to one-third of its initial level vs. <5% in WT). These differences were associated with the expression of Arabidopsis phyB in the DARA lines and its higher light stability than that of phyA. Arabidopsis phyB expressed in potato was characterised by lambda(max) = 683/669 nm in the emission/excitation (absorption) spectra and gamma1 = 0. PhyB also revealed a relatively low gamma2 (approx. 0.5) and its early red drop as compared with the gamma2 wavelength dependence for phyA. This is believed to contribute to the lower signalling ability of phyB and to confine the region (red) of its physiological activity.
Asunto(s)
Células Fotorreceptoras , Fitocromo/metabolismo , Factores de Transcripción , Arabidopsis , Proteínas de Arabidopsis , Fluorescencia , Fotoquímica , Fitocromo/genética , Fitocromo A , Fitocromo B , Plantas Modificadas Genéticamente , Solanum tuberosumRESUMEN
Transgenic potato (Solanum tuberosum) plants expressing Arabidopsis phytochrome B were characterized morphologically and physiologically under white light in a greenhouse to explore their potential for improved photosynthesis and higher tuber yields. As expected, overexpression of functional phytochrome B caused pleiotropic effects such as semidwarfism, decreased apical dominance, a higher number of smaller but thicker leaves, and increased pigmentation. Because of increased numbers of chloroplasts in elongated palisade cells, photosynthesis per leaf area and in each individual plant increased. In addition, photosynthesis was less sensitive to photoinactivation under prolonged light stress. The beginning of senescence was not delayed, but deceleration of chlorophyll degradation extended the lifetime of photosynthetically active plants. Both the higher photosynthetic performance and the longer lifespan of the transgenic plants allowed greater biomass production, resulting in extended underground organs with increased tuber yields.
Asunto(s)
Arabidopsis/genética , Células Fotorreceptoras , Fitocromo/genética , Solanum tuberosum/genética , Factores de Transcripción , Proteínas de Arabidopsis , Clorofila/metabolismo , Expresión Génica , Genes de Plantas , Luz , Fenotipo , Fotosíntesis/genética , Fitocromo/metabolismo , Fitocromo B , Hojas de la Planta/crecimiento & desarrollo , Brotes de la Planta/crecimiento & desarrollo , Plantas Modificadas Genéticamente , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN de Planta/genética , ARN de Planta/metabolismo , Solanum tuberosum/crecimiento & desarrollo , Solanum tuberosum/metabolismoRESUMEN
How light signals are transduced by phytochromes is still poorly understood. Recent studies have provided evidence that a PAS domain protein, PIF3, physically interacts with phytochromes, plays a role in phytochrome signal transduction and might be a component of a novel signalling pathway in plants.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis/efectos de la radiación , Proteínas de Drosophila , Proteínas del Ojo , Células Fotorreceptoras de Invertebrados , Células Fotorreceptoras , Fitocromo/fisiología , Proteínas de Plantas/fisiología , Isoformas de Proteínas/fisiología , Transducción de Señal/efectos de la radiación , Factores de Transcripción , Arabidopsis/genética , Arabidopsis/fisiología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Núcleo Celular/metabolismo , Criptocromos , Flavoproteínas/fisiología , Luz , Sustancias Macromoleculares , Cebollas/citología , Cebollas/genética , Fotoquímica , Fitocromo/química , Fitocromo/genética , Fitocromo/efectos de la radiación , Fitocromo A , Fitocromo B , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/efectos de la radiación , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/efectos de la radiación , Receptores Acoplados a Proteínas G , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal/fisiología , TransfecciónRESUMEN
N-Terminal apoprotein fragments of oat phytochrome A (phyA) of 65 kDa (amino acids 1-595) and potato phyB of 66 kDa (1-596) were heterologously expressed in Escherichia coli and in the yeasts Saccharomyces cerevisiae and Pichia pastoris, and assembled with phytochromobilin (PthetaB; native chromophore) and phycocyanobilin (PCB). The phyA65 apoprotein from yeast showed a monoexponential assembly kinetics after an initial steep rise, whereas the corresponding apoprotein from E. coli showed only a slow monoexponential assembly. The phyB66 apoprotein incorporated either chromophore more slowly than the phyA65s, with biexponential kinetics. With all apoproteins, PthetaB was incorporated faster than PCB. The thermal stabilities of the Pfr forms of the N-terminal halves are similar to those known for the full-length recombinant phytochromes: oat phyA65 Pfr is highly stable, whereas potato phyB66 Pfr is rapidly converted into Pr. Thus, neither the C-terminal domain nor homodimer formation regulates this property. Rather, it is a characteristic of the phytochrome indicating its origin from mono- or dicots. The Pr to Pfr kinetics of the N-terminal phyA65 and phyB66 are different. The primary photoproduct I700 of phyA65-PCB decayed monoexponentially and the PthetaB analogue biexponentially, whereas the phyB66 I700 decayed monoexponentially irrespective of the chromophore incorporated. The formation of Pfr from Pr is faster with the N-terminal halves than with the full-length phytochromes, indicating an involvement of the C-terminal domain in the relatively slow protein conformational changes.
Asunto(s)
Fragmentos de Péptidos/metabolismo , Células Fotorreceptoras , Fitocromo/metabolismo , Proteínas Recombinantes/metabolismo , Factores de Transcripción , Apoproteínas/biosíntesis , Apoproteínas/genética , Apoproteínas/metabolismo , Avena , Escherichia coli/genética , Isomerismo , Cinética , Peso Molecular , Fragmentos de Péptidos/biosíntesis , Fotólisis , Fitocromo/biosíntesis , Fitocromo/genética , Fitocromo A , Fitocromo B , Pichia/genética , Proteínas Recombinantes/biosíntesis , Saccharomyces cerevisiae/genética , Solanum tuberosum , Espectrofotometría , TemperaturaRESUMEN
In chloroplasts, carotenoids are essential pigments involved in photosynthesis. During-photomorphogenesis, a coordinated increase in the amounts of chlorophylls and carotenoids, in conjugation with other components, leads to the formation of a functional photosynthetic apparatus. To investigate the regulation of carotenoid biosynthesis during this process at the molecular level, GGPS, PSY and PDS cDNAs have been cloned from white mustard (Sinapis alba L). GGPS encodes a key enzyme in plastid isoprenoid metabolism, while the products of PSY and PDS catalyse the subsequent steps in carotenoid biosynthesis. Due to the low mRNA levels of the genes involved, the use of a RT-PCR protocol was necessary to measure gene expression during photomorphogenesis. With light, there is an up-regulation of PSY expression, the first gene within the carotenoid biosynthetic pathway, while PDS and GGPS expression levels remain constant. Treatment with different light qualities reveals a phytochrome-mediated regulation of PSY expression in developing white mustard seedlings. To obtain more detailed information on the light-regulation, Arabidopsis thaliana wild-type and phytochrome mutants were utilized. Continuous far-red and red light both increase the expression of PSY in wild-type seedlings, demonstrating that both light-labile and light-stable phytochromes are involved in PSY regulation. The response to far-red light is completely abolished in the phyA mutant, showing that PHYA mediates the increase in PSY transcript levels under these light conditions. In the phyB mutant, the red light response is normal, indicating that PSY expression is not controlled by PHYB but by other light-stable phytochromes. Measurement of chlorophylls and carotenoids under the same light regimes shows that the up-regulation of PSY expression does not necessarily result in an increase of the carotenoid content. Only those light conditions which allow chlorophyll biosynthesis lead to a significant increase of the carotenoid content. Therefore, it is proposed that up-regulation of PSY mRNA levels leads to an increased capacity for the formation of carotenoids. However, this only takes place under light conditions leading to protochlorophyllide photoconversion.
Asunto(s)
Transferasas Alquil y Aril/genética , Arabidopsis/enzimología , Carotenoides/biosíntesis , Luz , Planta de la Mostaza/enzimología , Oxidorreductasas/genética , Fitocromo/metabolismo , Plantas Medicinales , Transferasas Alquil y Aril/biosíntesis , Arabidopsis/genética , Arabidopsis/efectos de la radiación , Carotenoides/efectos de la radiación , Clonación Molecular , ADN Complementario/química , ADN de Plantas/química , Farnesiltransferasa , Geranilgeranil-Difosfato Geranilgeraniltransferasa , Planta de la Mostaza/genética , Planta de la Mostaza/efectos de la radiación , Mutagénesis Sitio-Dirigida , Oxidorreductasas/biosíntesis , Fitocromo/genética , ARN Mensajero/metabolismo , ARN de Planta/metabolismo , Semillas/enzimología , Semillas/genéticaRESUMEN
The cDNAs encoding full-length type A and B phytochromes (phyA and phyB, respectively) from potato were expressed in inducible yeast systems (Saccharomyces cerevisiae and Pichia pastoris). In addition, a deletion mutant of phyB (delta 1-74) was expressed. The apoproteins were reconstituted into chromoproteins by incorporation of the native chromophore, phytochromobilin (P phi B), and of phycocyanobilin (PCB). The incorporation of P phi B yielded chromoproteins with difference absorptions lambda max at 660 and 712 nm (Pr and Pfr, respectively) for phyA, and at 665 and 723 nm for phyB. All difference maxima of PCB phytochromes are blue-shifted by several nanometers with respect to those obtained with the P phi B chromophore. The deletion construct with PCB shows difference absorption maxima at 652 and 705 nm with the Pfr absorbance considerably reduced. Time-resolved kinetic analysis of a phyB-type phytochrome by nanosecond flash photolysis was performed for the first time. Recombinant full-length phyB afforded transient absorbance changes similar (but not identical) to those of phyA from Avena, whereas the kinetic behavior of these intermediates was very different. Contrary to phyA from Avena, the I700 intermediate from phyB reconstituted with either PCB or P phi B decayed following single exponential kinetics with a lifetime of 87 or 84 microseconds, respectively, at 10 degrees C. The formation of Pfr of PCB-containing recombinant phyB (phyB-PCB) could be fitted with three lifetimes of 9, 127, and 728 ms. The corresponding lifetimes of phyB-P phi B are 22.5, 343, and 2083 ms. Whereas for phyB-PCB all three millisecond lifetimes are related to the formation of Pfr, the 2 s component of phyB-P phi B is concomitant with a rapid recovery of Pr. For recombinant potato phyA and delta 1-74 phyB, no time-resolved data could be obtained due to the limited quantities available. As described for phytochromes of other dicotelydons, the Pfr forms of full-length phyA and PhyB of potato underwent rapid dark conversion to Pr.
Asunto(s)
Fitocromo/genética , Proteínas de Plantas/genética , Solanum tuberosum/química , Biliverdina/análogos & derivados , Biliverdina/metabolismo , Western Blotting , Clonación Molecular , Expresión Génica/genética , Cinética , Estructura Molecular , Mutación/genética , Fotólisis , Ficobilinas , Ficocianina/metabolismo , Fitocromo/clasificación , Pichia/genética , Reacción en Cadena de la Polimerasa , Pirroles/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Saccharomyces cerevisiae/genética , Eliminación de Secuencia/genética , Espectrofotometría , TetrapirrolesRESUMEN
We sought to determine if phytochrome B (phyB)-mediated responses to the red light (R)/far-red light (FR) ratio are affected by phytochrome A (phyA) activity in light-grown seedlings of Arabidopsis thaliana. Pulses of FR delayed into the dark period were less effective than end-of-day (EOD) FR in promoting hypocotyl growth over a given period in darkness. White light minus blue light interposed instead of darkness between the end of the white-light photoperiod and the FR pulse was sufficient to maintain responsivity to the decrease in phyB in FR-light-absorbing form in wild-type (WT) seedlings, but not in the phyA mutant. Compared with EOD R, hourly R+FR pulses provided throughout the night caused a stronger promotion of stem growth than a single EOD R+FR pulse in WT Arabidopsis, cucumber, mustard, sunflower, tobacco, and tomato, but not in phyA Arabidopsis or in the aurea mutant of tomato. WT seedlings of Arabidopsis responded to a range of high EOD R/FR ratios, whereas the phyA mutant required stronger reductions in the EOD R/FR ratio. In sunlight, phyA seedlings of Arabidopsis showed no response to the "early warning" signals of neighboring vegetation, and hypocotyl-growth promotion occurred at higher plant densities than in the WT. Thus, under a series of light conditions, the sensitivity or responsivity to reductions in the R/FR ratio were larger in WT than in phyA seedlings. A product of phyA is therefore proposed to enhance the hypocotyl-growth response to decreases in phyB in FR-light-absorbing form in light grown seedlings.
Asunto(s)
Arabidopsis/fisiología , Células Fotorreceptoras , Fitocromo/metabolismo , Fitocromo/fisiología , Factores de Transcripción , Arabidopsis/efectos de los fármacos , Arabidopsis/efectos de la radiación , Proteínas de Arabidopsis , Cucumis sativus/fisiología , Oscuridad , Genes de Plantas , Hipocótilo , Luz , Iluminación , Solanum lycopersicum/fisiología , Planta de la Mostaza/fisiología , Fitocromo/genética , Fitocromo A , Fitocromo B , Plantas Medicinales , Plantas Tóxicas , Nicotiana/fisiologíaRESUMEN
Light-dependent NADPH:protochlorophyllide oxidoreductase (POR), a nuclear-encoded plastid-localized enzyme, catalyzes the photoreduction of protochlorophyllide (Pchlide) to chlorophyllide in higher plants, algae and cyanobacteria. Angiosperms require light for chlorophyll (Chl) biosynthesis and have recently been shown to contain two POR-encoding genes, PorA and PorB, that are differentially regulated by light and developmental state. PorA expression rapidly becomes undetectable after illumination of etiolated seedlings, whereas PorB expression persists throughout greening and in adult plants. In order to study the in vivo functions of Arabidopsis POR A and POR B we have abolished the expression of PorA through the use of the phytochrome A-mediated far-red high irradiance response. Wild-type seedlings grown in continuous far-red light (cFR) display the morphology of white light (WL)-grown seedlings, but contain only traces of Chl and do not green upon transfer to WL. This cFR-induced greening defect correlates with the absence of PorA mRNA, the putative POR A protein, phototransformable Pchlide-F655, and with strongly reduced POR enzymatic activity in plant extracts. In contrast, a cFR-grown phyA mutant expresses the PorA gene, accumulates Chl and visibly greens in WL. Furthermore, constitutive overexpression of POR A in cFR-grown transgenic Arabidopsis wild-type seedlings restores Chl accumulation and WL-induced greening. These data demonstrate that POR A is required for greening and that the availability of POR A limits Chl accumulation during growth in cFR. POR B apparently provides a means to sustain light-dependent Chl biosynthesis in fully greened, mature plants in the absence of phototransformable Pchlide-F655.