RESUMEN

Different modes of the phytochrome function are connected with its polymorphism, the major isoforms being phytochromes A and B (phyA and phyB). In its turn, phyA comprises two native species, phyA' and phyA'', whose precise nature and functions remain obscure. With the use of in situ fluorescence spectroscopy, we investigated their properties in a mutant of pea, phyA-3D, characterized by exaggerated photoresponses and impaired photodestruction of phyA. The mutation is a substitution of alanine by valine at the position 194 in phyA. The phyA-3DphyB and phyB mutants were also investigated. In dark-grown plants, all the lines had the content and properties of the two phyA species very similar to the wild type. However, a considerably more intense reduction in [phyA] without changes in the phyA'/phyA'' equilibrium was found in far-red grown mutant plants suggesting a hypersensitivity of phyA-3D with regard to its autoregulation. On the contrary, under red illumination, a higher stability of phyA-3D was observed confirming our earlier findings. This allows a conclusion that the A194V substitution in phyA-3D not only impairs its destruction but also enhances its signaling ability, suggesting a role of this locus in modulation of its activity.

Asunto(s)

Luz , Mutación , Fitocromo/genética , Fitocromo/efectos de la radiación , Pisum sativum , Mutación/genética , Mutación/efectos de la radiación , Fitocromo A , Proteínas de Plantas/genética , Proteínas de Plantas/efectos de la radiación , Raíces de Plantas/genética , Raíces de Plantas/efectos de la radiación , Isoformas de Proteínas/genética , Isoformas de Proteínas/efectos de la radiación

RESUMEN

Recent studies suggest that a common theme links the diverse elements of pineal photoneuroendocrine transduction--regulation via binding to 14-3-3 proteins. The elements include photoreception, neurotransmission, signal transduction and the synthesis of melatonin from tryptophan. We review general aspects of 14-3-3 proteins and their biological function as binding partners, and also focus on their roles in pineal photoneuroendocrine transduction.

Asunto(s)

Fototransducción/fisiología , Sistemas Neurosecretores/metabolismo , Glándula Pineal/metabolismo , Tirosina 3-Monooxigenasa/fisiología , Proteínas 14-3-3 , Animales , Arilamina N-Acetiltransferasa/química , Arilamina N-Acetiltransferasa/metabolismo , Luz , Melatonina/metabolismo , Modelos Moleculares , Norepinefrina/fisiología , Glándula Pineal/química , Relación Estructura-Actividad , Tirosina 3-Monooxigenasa/química , Tirosina 3-Monooxigenasa/clasificación , Tirosina 3-Monooxigenasa/metabolismo , Tirosina 3-Monooxigenasa/efectos de la radiación

RESUMEN

This paper describes the role 14-3-3 proteins play in vertebrate photoneuroendocrine transduction. 14-3-3 proteins form a complex with arylalkylamine N-acetyltransferase (AANAT), the enzyme which turns melatonin production on during the day and off at night. Complex formation is triggered at night by cAMP-dependent phosphorylation of the enzyme, and results in activation and protection against proteolysis. This enhances melatonin production >10-fold. Light exposure results in dephosphorylation of the enzyme and disassociation from 14-3-3, leading to destruction and a rapid drop in melatonin production and release and circulating levels.

Asunto(s)

Ritmo Circadiano/fisiología , Melatonina/fisiología , Sistemas Neurosecretores/fisiología , Transducción de Señal/fisiología , Tirosina 3-Monooxigenasa/fisiología , Proteínas 14-3-3 , Animales , Arilamina N-Acetiltransferasa/química , Arilamina N-Acetiltransferasa/metabolismo , Sitios de Unión , Luz , Modelos Moleculares , Conformación Proteica , Tirosina 3-Monooxigenasa/química

RESUMEN

The interactions of phytochrome A (phyA) and phytochrome B (phyB) in the photocontrol of vegetative and reproductive development in pea have been investigated using null mutants for each phytochrome. White-light-grown phyA phyB double mutant plants show severely impaired de-etiolation both at the seedling stage and later in development, with a reduced rate of leaf production and swollen, twisted internodes, and enlarged cells in all stem tissues. PhyA and phyB act in a highly redundant manner to control de-etiolation under continuous, high-irradiance red light. The phyA phyB double mutant shows no significant residual phytochrome responses for either de-etiolation or shade-avoidance, but undergoes partial de-etiolation in blue light. PhyB is shown to inhibit flowering under both long and short photoperiods and this inhibition is required for expression of the promotive effect of phyA. PhyA is solely responsible for the promotion of flowering by night-breaks with white light, whereas phyB appears to play a major role in detection of light quality in end-of-day light treatments, night breaks and day extensions. Finally, the inhibitory effect of phyB is not graft-transmissible, suggesting that phyB acts in a different manner and after phyA in the control of flower induction.

Asunto(s)

Células Fotorreceptoras , Fitocromo/fisiología , Pisum sativum/fisiología , Factores de Transcripción , Genes de Plantas , Luz , Datos de Secuencia Molecular , Mutación , Pisum sativum/crecimiento & desarrollo , Pisum sativum/efectos de la radiación , Fotoperiodo , Fitocromo/genética , Fitocromo A , Fitocromo B , Hojas de la Planta , Estructuras de las Plantas/crecimiento & desarrollo

RESUMEN

The daily rhythm in melatonin levels is controlled by cAMP through actions on the penultimate enzyme in melatonin synthesis, arylalkylamine N-acetyltransferase (AANAT; serotonin N-acetyltransferase, EC ). Results presented here describe a regulatory/binding sequence in AANAT that encodes a cAMP-operated binding switch through which cAMP-regulated protein kinase-catalyzed phosphorylation [RRHTLPAN --> RRHpTLPAN] promotes formation of a complex with 14-3-3 proteins. Formation of this AANAT/14-3-3 complex enhances melatonin production by shielding AANAT from dephosphorylation and/or proteolysis and by decreasing the K(m) for 5-hydroxytryptamine (serotonin). Similar switches could play a role in cAMP signal transduction in other biological systems.

Asunto(s)

Arilamina N-Acetiltransferasa/fisiología , Melatonina/fisiología , Glándula Pineal/fisiología , Tirosina 3-Monooxigenasa/fisiología , Proteínas 14-3-3 , Animales , N-Acetiltransferasa de Arilalquilamina , Células CHO , Cricetinae , Humanos , Transfección

RESUMEN

In fish, individual photoreceptor cells in the pineal organ and retina contain complete melatonin rhythm generating systems. In the pike and seabream, this includes a photodetector, circadian clock, and melatonin synthesis machinery; the trout lacks a functional clock. The melatonin rhythm is due in part to a nocturnal increase in the activity of the arylalkylamine N-acetyltransferase (AANAT) which is inhibited by light. Two AANATs have been identified in fish: AANAT1, more closely related to AANATs found in higher vertebrates, is specifically expressed in the retina; AANAT2 is specifically expressed in the pineal organ. We show that there is a physiological day/night rhythm in pineal AANAT2 protein in the pike, and that light exposure at midnight decreases the abundance of AANAT2 protein and activity. In culture, this decrease is blocked by inhibitors of the proteasomal degradation pathway. If glands are maintained under light at night, treatment with these inhibitors increases AANAT2 activity and protein. Organ culture studies with the trout and seabream also indicate that the light-induced decrease of AANAT2 activity is prevented when proteasomal proteolysis is blocked. A cAMP-dependent pathway protects AANAT2 protein from degradation. These results provide a clue to understanding how light regulates the daily rhythm in melatonin secretion in fish photoreceptor cells and provides evidence that proteasomal proteolysis is a conserved element in the regulation of AANAT in vertebrates.

Asunto(s)

Arilamina N-Acetiltransferasa/genética , Cisteína Endopeptidasas/fisiología , Peces/metabolismo , Regulación Enzimológica de la Expresión Génica , Isoenzimas/genética , Complejos Multienzimáticos/fisiología , Glándula Pineal/enzimología , Animales , Arilamina N-Acetiltransferasa/metabolismo , Ritmo Circadiano , AMP Cíclico/fisiología , Femenino , Luz , Masculino , Técnicas de Cultivo de Órganos , Complejo de la Endopetidasa Proteasomal

RESUMEN

Arylalkylamine N-acetyltransferase (serotonin N-acetyltransferase, AANAT, EC ) is the penultimate enzyme in melatonin synthesis. As described here, a cell line (1E7) expressing human AANAT (hAANAT) has been developed to study the human enzyme. 1E7 hAANAT is detectable in immunoblots as a 23-kDa band and is immunocytochemically visualized in the cytoplasm. The specific concentration of hAANAT in homogenates is comparable to that of the night rat pineal gland. Kinetics of AANAT extracted from 1E7 cells are the same as those of bacterially expressed hAANAT; both preparations of hAANAT are equally sensitive to the inhibitor CoA-S-N-acetyltryptamine. Studies of cAMP regulation indicate that treatment with forskolin, dibutyryl cAMP, isobutylmethylxanthine, or isoproterenol activate cellular hAANAT within intact 1E7 cells approximately 8-fold without markedly increasing the abundance of AANAT protein or the activity of AANAT in broken cell preparations; and, that forskolin, isobutylmethylxanthine and isoproterenol elevate cyclic AMP production. These observations extend our understanding of cAMP regulation of AANAT activity, because it is currently thought that this only involves changes in the steady-state levels of AANAT protein. This previously unrecognized switching mechanism could function physiologically to control melatonin production without changing AANAT protein levels.

Asunto(s)

Arilamina N-Acetiltransferasa/metabolismo , AMP Cíclico/fisiología , Animales , N-Acetiltransferasa de Arilalquilamina , Arilamina N-Acetiltransferasa/antagonistas & inhibidores , Células COS , Extractos Celulares/análisis , Línea Celular , Colforsina/farmacología , Citoplasma/enzimología , Oscuridad , Activación Enzimática , Escherichia coli/genética , Humanos , Cinética , Melatonina/metabolismo , Glándula Pineal/metabolismo , Ratas , Ratas Sprague-Dawley , Triptaminas/farmacología

RESUMEN

Several novel allelic groups of tomato (Solanum lycopersicum L.) mutants with impaired photomorphogenesis have been identified after gamma-ray mutagenesis of phyA phyB1 double-mutant seed. Recessive mutants in one allelic group are characterized by retarded hook opening, increased hypocotyl elongation and reduced hypocotyl chlorophyll content under white light (WL). These mutants showed a specific impairment in response to blue light (BL) resulting from lesions in the gene encoding the BL receptor cryptochrome 1 (cry1). Phytochrome A and cry1 are identified as the major photoreceptors mediating BL-induced de-etiolation in tomato, and act under low and high irradiances, respectively. Phytochromes B1 and B2 also contribute to BL sensing, and the relative contribution of each of these four photoreceptors differs according to the light conditions and the specific process examined. Development of the phyA phyB1 phyB2 cry1 quadruple mutant under WL is severely impaired, and seedlings die before flowering. The quadruple mutant is essentially blind to BL, but experiments employing simultaneous irradiation with BL and red light suggest that an additional non-phytochrome photoreceptor may be active under short daily BL exposures. In addition to effects on de-etiolation, cry1 is active in older, WL-grown plants, and influences stem elongation, apical dominance, and the chlorophyll content of leaves and fruit. These results provide the first mutant-based characterization of cry1 in a plant species other than Arabidopsis.

Asunto(s)

Proteínas de Drosophila , Proteínas del Ojo , Flavoproteínas/fisiología , Luz , Mutación , Células Fotorreceptoras de Invertebrados , Fitocromo/fisiología , Solanum lycopersicum/fisiología , Alelos , Secuencia de Bases , Criptocromos , Cartilla de ADN , Flavoproteínas/genética , Complejos de Proteína Captadores de Luz , Solanum lycopersicum/genética , Proteínas del Complejo del Centro de Reacción Fotosintética/genética , Fitocromo/genética , Receptores Acoplados a Proteínas G

RESUMEN

The role of phytochrome B2 (phyB2) in the control of photomorphogenesis in tomato (Solanum lycopersicum L.) has been investigated using recently isolated mutants carrying lesions in the PHYB2 gene. The physiological interactions of phytochrome A (phyA), phytochrome B1 (phyB1) and phyB2 have also been explored, using an isogenic series of all possible mutant combinations and several different phenotypic characteristics. The loss of phyB2 had a negligible effect on the development of white-light-grown wild-type or phyA-deficient plants, but substantially enhanced the elongated pale phenotype of the phyB1 mutant. This redundancy was also seen in the control of de-etiolation under continuous red light (R), where the loss of phyB2 had no detectable effect in the presence of phyB1. Under continuous R, phyA action was largely independent of phyB1 and phyB2 in terms of the control of hypocotyl elongation, but antagonized the effects of phyB1 in the control of anthocyanin synthesis, indicating that photoreceptors may interact differently to control different traits. Irradiance response curves for anthocyanin synthesis revealed that phyB1 and phyB2 together mediate all the detectable response to high-irradiance R, and, surprisingly, that the phyA-dependent low-irradiance component is also strongly reduced in the phyB1 phyB2 double mutant. This is not associated with a reduction in phyA protein content or responsiveness to continuous far-red light (FR), suggesting that phyB1 and phyB2 specifically influence phyA activity under low-irradiance R. Finally, the phyA phyB1 phyB2 triple mutant showed strong residual responsiveness to supplementary daytime FR, indicating that at least one of the two remaining phytochromes plays a significant role in tomato photomorphogenesis.

Asunto(s)

Células Fotorreceptoras , Fitocromo/fisiología , Solanum lycopersicum/crecimiento & desarrollo , Factores de Transcripción , Secuencia de Bases , Cartilla de ADN/genética , Genes de Plantas , Luz , Solanum lycopersicum/genética , Solanum lycopersicum/efectos de la radiación , Mutación , Fitocromo/genética , Fitocromo A , Fitocromo B

RESUMEN

The molecular processes underlying neural transmission are central issues in neurobiology. Here we describe a novel mechanism through which noradrenaline (NA) activates its target cells, using the mammalian pineal organ as a model. In this neuroendocrine transducer, NA stimulates arylalkylamine N:-acetyltransferase (AANAT; EC 2.3.1. 87), the key enzyme regulating the nocturnal melatonin production. In rodents, AANAT protein accumulates as a result of enhanced transcription, but in primates and ungulates, the AANAT mRNA level fluctuates only marginally, indicating that other mechanisms regulate AANAT protein and activity. These were investigated in cultured bovine pinealocytes. AANAT mRNA was readily detectable in unstimulated pinealocytes, and levels did not change following NA treatment. In contrast, NA increased AANAT protein levels in parallel with AANAT activity, apparently through a cyclic AMP-mediated mechanism. Immunocytochemistry revealed that the changes in AANAT protein levels occurred in virtually all pinealocytes. Inhibition of AANAT degradation by proteasomal proteolysis alone was found to switch-on enzyme activity by increasing AANAT protein levels five- to 10-fold. Accordingly, under unstimulated conditions AANAT protein is continually synthesized and immediately destroyed by proteasomal proteolysis. NA appears to act via cyclic AMP to protect AANAT from proteolytic destruction, resulting in accumulation of the protein. These findings show that tightly regulated control of proteasomal proteolysis of a specific protein alone can play a pivotal role in neural regulation.

Asunto(s)

Acetilcisteína/análogos & derivados , AMP Cíclico/metabolismo , Cisteína Endopeptidasas/metabolismo , Complejos Multienzimáticos/metabolismo , Neuronas/metabolismo , Norepinefrina/metabolismo , Glándula Pineal/metabolismo , Transducción de Señal/fisiología , Acetilcisteína/antagonistas & inhibidores , Acetilcisteína/metabolismo , Animales , Arilamina N-Acetiltransferasa/biosíntesis , Arilamina N-Acetiltransferasa/genética , Bucladesina/farmacología , Bovinos , Células Cultivadas , Colforsina/farmacología , AMP Cíclico/farmacología , Femenino , Masculino , Complejos Multienzimáticos/antagonistas & inhibidores , Neuronas/citología , Norepinefrina/farmacología , Péptido Hidrolasas/metabolismo , Glándula Pineal/citología , Inhibidores de Proteasas/farmacología , Complejo de la Endopetidasa Proteasomal , Inhibidores de la Síntesis de la Proteína/farmacología , Puromicina/farmacología , ARN Mensajero/metabolismo , Transducción de Señal/efectos de los fármacos , Estimulación Química

RESUMEN

Although the physiological functions of phytochrome A (PhyA) are now known, the distribution of endogenous PhyA has not been examined. We have visualized endogenous PhyA apoprotein (PHYA) by immunolabeling cryosections of pea tissue, using PHYA-deficient mutants as negative controls. By this method, we examined the distribution of PHYA in different tissues and changes in its intracellular distribution in response to light. In apical hook cells of etiolated seedlings, PHYA immunolabeling was distributed diffusely in the cytosol. Exposure to continuous far-red (cFR) light caused a redistribution of the immunolabeling to the nucleus, first detectable after 1.5 hr and greatest at 4.5 hr. During this time, the amounts of spectrally active phytochrome and PHYA did not decline substantially. Exposure to continuous red (cR) light or to a brief pulse of red light also resulted in redistribution of immunolabeling to the nucleus, but this occurred much more rapidly and with a different pattern of intranuclear distribution than it did in response to cFR light. Exposures to cR light resulted in loss of immunolabeling, which was associated with PHYA degradation. These results indicate that the light-induced intracellular location of PHYA is wavelength dependent and imply that this is important for PhyA activity.

Asunto(s)

Núcleo Celular/metabolismo , Fitocromo/metabolismo , Pisum sativum/efectos de la radiación , Transporte Biológico , Oscuridad , Inmunohistoquímica , Pisum sativum/metabolismo , Fitocromo A

RESUMEN

Cryptochromes are blue light photoreceptors found in both plants and animals. They probably evolved from photolyases, which are blue/UV-light-absorbing photoreceptors involved in DNA repair. In seed plants, two different cryptochrome (CRY) genes have been found in Arabidopsis and one in Sinapis, while three genes have been found in the fern Adiantum. We report the characterisation of tomato CRY genes CRY1 and CRY2. They map to chromosomes 4 and 9, respectively, show relatively constitutive expression and encode proteins of 679 and 635 amino acids, respectively. These proteins show higher similarity to their Arabidopsis counterparts than to each other, suggesting that duplication between CRY1 and CRY2 is an ancient event in the evolution of seed plants. The seed plant cryptochromes form a group distinct from the fern cryptochromes, implying that only one gene was present in the common ancestor between these two groups of plants. Most intron positions in CRY genes from plants and ferns are highly conserved. Tomato cryl and cry2 proteins carry C-terminal domains 210 and 160 amino acids long, respectively. Several conserved motifs are found in these domains, some of which are common to both types of cryptochromes, while others are cryptochrome-type-specific.

Asunto(s)

Arabidopsis/genética , Proteínas de Drosophila , Proteínas del Ojo , Flavoproteínas/genética , Células Fotorreceptoras de Invertebrados , Solanum lycopersicum/genética , Secuencia de Aminoácidos , Proteínas de Arabidopsis , Mapeo Cromosómico , Criptocromos , ADN Complementario/química , ADN Complementario/genética , ADN de Plantas/química , ADN de Plantas/genética , Exones , Regulación de la Expresión Génica de las Plantas/efectos de la radiación , Genes de Plantas/genética , Intrones , Luz , Solanum lycopersicum/efectos de la radiación , Datos de Secuencia Molecular , Filogenia , Proteínas de Plantas/genética , Receptores Acoplados a Proteínas G , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido

RESUMEN

This study was conducted to determine the origin of the high variability in the mean nocturnal plasma melatonin concentration (MC) in sheep. Two extreme groups of 25 lambs each [low (L) and high (H)] were obtained by calculating their genetic value on the basis of the MC of their parents. The MC of lambs was significantly higher in the H group than in the L group (L: 189.7 +/- 24.4 vs. H: 344.1 +/- 33.0 pg/ml, P < 0.001). Within each group, 13 lambs were slaughtered during the day (D) and 12 lambs during the night (N). Pineal weight was significantly higher in the H group than in the L group (L: 83.5 +/- 6.7 vs. H: 119.1 +/- 9.2 mg, P < 0.01) but did not differ between D and N. The amount of melatonin released in vitro per milligram of pineal gland, the arylalkylamine N-acetyltransferase (AANAT) activity, the AANAT protein content, and the level of AANAT mRNA differed significantly between D and N but not with genetic group. Hydroxyindole O-methyltransferase activity did not differ significantly between D and N or between genetic groups. Therefore, the genetic difference in MC between the two groups of lambs was attributed to a difference in pineal size, not in enzymatic activity of the pinealocytes.

Asunto(s)

Variación Genética , Melatonina/sangre , Melatonina/genética , Glándula Pineal/anatomía & histología , Glándula Pineal/enzimología , Acetilserotonina O-Metiltransferasa/genética , Acetilserotonina O-Metiltransferasa/metabolismo , Animales , Arilamina N-Acetiltransferasa/genética , Arilamina N-Acetiltransferasa/metabolismo , Ritmo Circadiano/genética , Expresión Génica/fisiología , Masculino , ARN Mensajero/análisis , Radioinmunoensayo , Ovinos

RESUMEN

Gibberellin (GA) 20-oxidase (GA 20-ox) and GA 3beta-hydroxylase (GA 3beta-hy) are enzymes that catalyze the late steps in the formation of active GAs, and are potential control points in the regulation of GA biosynthesis by light. We have investigated the photoregulation of the GA 20-ox and GA 3beta-hy transcript levels in pea (Pisum sativum L.). The GA 20-ox transcript level was higher in light-grown seedlings than in etiolated seedlings, whereas GA 3beta-hy mRNA accumulation was higher in etiolated seedlings. However, transfer of etiolated seedlings to light led to a 5-fold increase in the expression of both transcripts 4 h after transfer. GA 20-ox mRNA accumulation is regulated by both phytochromes A and B. Transfer to light also resulted in a 6-fold decrease in GA(1) levels within 2 h. These results suggest that the light-induced drop in GA(1) level is not achieved through regulation of GA 20-ox and GA 3beta-hy mRNA accumulation. The application of exogenous GA(1) to apical buds of etiolated seedlings prior to light treatments inhibited the light-induced accumulation of both GA 20-ox and GA 3beta-hy mRNA, suggesting that negative feedback regulation is an important mechanism in the regulation of GA 20-ox and GA 3beta-hy mRNA accumulation during de-etiolation of pea seedlings.

Asunto(s)

Regulación de la Expresión Génica de las Plantas , Oxigenasas de Función Mixta/genética , Células Fotorreceptoras , Pisum sativum/enzimología , Pisum sativum/genética , Factores de Transcripción , Transcripción Genética , Oscuridad , Regulación Enzimológica de la Expresión Génica/efectos de la radiación , Regulación de la Expresión Génica de las Plantas/efectos de la radiación , Luz , Pisum sativum/crecimiento & desarrollo , Fitocromo/metabolismo , Fitocromo A , Fitocromo B

RESUMEN

The nocturnal increase in circulating melatonin in vertebrates is regulated by 10- to 100-fold increases in pineal serotonin N-acetyltransferase (AA-NAT) activity. Changes in the amount of AA-NAT protein were shown to parallel changes in AA-NAT activity. When neural stimulation was switched off by either light exposure or L-propranolol-induced beta-adrenergic blockade, both AA-NAT activity and protein decreased rapidly. Effects of L-propranolol were blocked in vitro by dibutyryl adenosine 3',5'-monophosphate (cAMP) or inhibitors of proteasomal proteolysis. This result indicates that adrenergic-cAMP regulation of AA-NAT is mediated by rapid reversible control of selective proteasomal proteolysis. Similar proteasome-based mechanisms may function widely as selective molecular switches in vertebrate neural systems.

Asunto(s)

Arilamina N-Acetiltransferasa/metabolismo , Cisteína Endopeptidasas/metabolismo , Melatonina/biosíntesis , Complejos Multienzimáticos/metabolismo , Glándula Pineal/metabolismo , Agonistas Adrenérgicos beta/farmacología , Antagonistas Adrenérgicos beta/farmacología , Animales , Bucladesina/farmacología , AMP Cíclico/metabolismo , Inhibidores de Cisteína Proteinasa/farmacología , Isoproterenol/farmacología , Luz , Glándula Pineal/citología , Glándula Pineal/efectos de los fármacos , Glándula Pineal/enzimología , Propranolol/farmacología , Complejo de la Endopetidasa Proteasomal , Ratas , Receptores Adrenérgicos beta/fisiología

RESUMEN

In garden pea (Pisum sativum L.), a long-day plant, long photoperiods promote flowering by reducing the synthesis or transport of a graft-transmissible inhibitor of flowering. Previous physiological studies have indicated that this promotive effect is predominantly achieved through a response that requires long exposures to light and for which far-red (FR) light is the most effective. These characteristics implicate the action of phytochrome A (phyA). To investigate this matter further, we screened ethylmethane sulfonate-mutagenized pea seedlings for FR-unresponsive, potentially phyA-deficient mutants. Two allelic, recessive mutants were isolated and were designated fun1 for FR unresponsive. The fun1-1 mutant is specifically deficient in the PHYA apoprotein and has a seedling phenotype indistinguishable from wild type when grown under white light. However, fun1-1 plants grown to maturity under long photoperiods show a highly pleiotropic phenotype, with short internodes, thickened stems, delayed flowering and senescence, longer peduncles, and higher seed yield. This phenotype results in large part from an inability of fun1-1 to detect day extensions. These results establish a crucial role for phyA in the control of flowering in pea, and show that phyA mediates responses to both red and FR light. Furthermore, grafting and epistasis studies with fun1 and dne, a mutant deficient in the floral inhibitor, show that the roles of phyA in seedling deetiolation and in day-length detection are genetically separable and that the phyA-mediated promotion of flowering results from a reduction in the synthesis or transport of the floral inhibitor.

RESUMEN

A remarkably constant feature of vertebrate physiology is a daily rhythm of melatonin in the circulation, which serves as the hormonal signal of the daily light/dark cycle: melatonin levels are always elevated at night. The biochemical basis of this hormonal rhythm is one of the enzymes involved in melatonin synthesis in the pineal gland-the melatonin rhythm-generating enzyme-serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AA-NAT, E.C. 2.3.1.87). In all vertebrates, enzyme activity is high at night. This reflects the influences of internal circadian clocks and of light. The dynamics of this enzyme are remarkable. The magnitude of the nocturnal increase in enzyme activity ranges from 7- to 150-fold on a species-to-species basis among vertebrates. In all cases the nocturnal levels of AA-NAT activity decrease very rapidly following exposure to light. A major advance in the study of the molecular basis of these changes was the cloning of cDNA encoding the enzyme. This has resulted in rapid progress in our understanding of the biology and structure of AA-NAT and how it is regulated. Several constant features of this enzyme have become apparent, including structural features, tissue distribution, and a close association of enzyme activity and protein. However, some remarkable differences among species in the molecular mechanisms involved in regulating the enzyme have been discovered. In sheep, AA-NAT mRNA levels show relatively little change over a 24-hour period and changes in AA-NAT activity are primarily regulated at the protein level. In the rat, AA-NAT is also regulated at a protein level; however, in addition, AA-NAT mRNA levels exhibit a 150-fold rhythm, which reflects cyclic AMP-dependent regulation of expression of the AA-NAT gene. In the chicken, cyclic AMP acts primarily at the protein level and a rhythm in AA-NAT mRNA is driven by a noncyclic AMP-dependent mechanism linked to the clock within the pineal gland. Finally, in the trout, AA-NAT mRNA levels show little change and activity is regulated by light acting directly on the pineal gland. The variety of mechanisms that have evolved among vertebrates to achieve the same goal-a rhythm in melatonin-underlines the important role melatonin plays as the hormonal signal of environmental lighting in vertebrates.

Asunto(s)

Arilamina N-Acetiltransferasa/metabolismo , Melatonina/sangre , Glándula Pineal/enzimología , Secuencia de Aminoácidos , Animales , Arilamina N-Acetiltransferasa/química , Arilamina N-Acetiltransferasa/genética , Secuencia de Bases , Evolución Biológica , Humanos , Datos de Secuencia Molecular , ARN Mensajero/metabolismo , Especificidad de la Especie

RESUMEN

In vertebrates, the circadian rhythm in the activity of serotonin N-acetyltransferase [arylalkylamine N-acetyltransferase (AA-NAT); EC 2.3.1.87] drives the daily rhythm in circulating melatonin. We have discovered that expression of the AA-NAT gene in the rat pineal gland is essentially turned off during the day and turned on at night, resulting in a more than 150-fold rhythm. Expression is regulated by a photoneural system that acts through an adrenergic-cAMP mechanism in pinealocytes, probably involving cAMP response element-binding protein phosphorylation. Turning off AA-NAT expression appears to involve de novo synthesis of a protein that attenuates transcription. A approximately 10-fold night/day rhythm in AA-NAT messenger RNA occurs in the retina, and AA-NAT messenger RNA is also detected at low levels in the brain.

Asunto(s)

Arilamina N-Acetiltransferasa/biosíntesis , Encéfalo/enzimología , Ritmo Circadiano , Melatonina/biosíntesis , Glándula Pineal/enzimología , Transcripción Genética , Agonistas Adrenérgicos beta/farmacología , Antagonistas Adrenérgicos beta/farmacología , Secuencia de Aminoácidos , Animales , Arilamina N-Acetiltransferasa/química , Secuencia de Consenso , ADN Complementario , Oscuridad , Luz , Masculino , Datos de Secuencia Molecular , Especificidad de Órganos , Fenilefrina/farmacología , Glándula Pineal/fisiología , Reacción en Cadena de la Polimerasa , Propranolol/farmacología , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Receptores Adrenérgicos beta/fisiología , Homología de Secuencia de Aminoácido , Ovinos

RESUMEN

The adrenergic transmitter norepinephrine (NE) dramatically increases the prominence of only two out of the hundreds of [35S]methionine-labeled pineal proteins resolved by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). One of these regulated proteins is AIP 37/6 (37 kDa, pI approximately 6). The labeling of this protein is increased approximately 100-fold by NE. In the study presented here the identity of AIP 37/6 was investigated. The results of microsequencing, immunochemical analysis of 2D-PAGE blots and size exclusion chromatography indicate that AIP 37/6 is an isoform of cytosolic malate dehydrogenase (cMDH; approximately 36.3 kDa; pI approximately 6.5). Associated studies indicate that this isoform is phosphorylated whereas the bulk of cMDH is not. Cotranslational phosphorylation of cMDH is discussed.

Asunto(s)

Malato Deshidrogenasa/metabolismo , Glándula Pineal/metabolismo , Glándula Pineal/fisiología , Proteínas/metabolismo , Animales , Autorradiografía , Cicloheximida/farmacología , Femenino , Inmunohistoquímica , Masculino , Metionina/farmacología , Fosforilación , Ratas , Ratas Sprague-Dawley

RESUMEN

We isolated a new pea mutant that was selected on the basis of pale color and elongated internodes in a screen under white light. The mutant was designated pcd1 for phytochrome chromophore deficient. Light-grown pcd1 plants have yellow-green foliage with a reduced chlorophyll (Chl) content and an abnormally high Chl a/Chl b ratio. Etiolated pcd1 seedlings are developmentally insensitive to far-red light, show a reduced response to red light, and have no spectrophotometrically detectable phytochrome. The phytochrome A apoprotein is present at the wild-type level in etiolated pcd1 seedlings but is not depleted by red light treatment. Crude phytochrome preparations from etiolated pcd1 tissue also lack spectral activity but can be assembled with phycocyanobilin, an analog of the endogenous phytochrome chromophore phytochromobilin, to yield a difference spectrum characteristic of an apophytochrome-phycocyanobilin adduct. These results indicate that the pcd1-conferred phenotype results from a deficiency in phytochrome chromophore synthesis. Furthermore, etioplast preparations from pcd1 seedlings can metabolize biliverdin (BV) IX[alpha] but not heme to phytochromobilin, indicating that pcd1 plants are severely impaired in their ability to convert heme to BV IX[alpha]. This provides clear evidence that the conversion of heme to BV IX[alpha] is an enzymatic process in higher plants and that it is required for synthesis of the phytochrome chromophore and hence for normal photomorphogenesis.