RESUMEN

SUMMARY: In higher plants, multiple nuclear-encoded sigma factors activate select subsets of plastid gene promoters in a partially redundant manner. We analysed the light induction profiles of transcripts from six Arabidopsis sigma factor (AtSIG) genes in mature leaves, focusing on the effects of wavelength and intensity. Red-light illumination (660 nm) of dark-adapted plants strongly induced AtSIG1 transcripts, while blue-light illumination (470 nm) caused strong and rapid induction of AtSIG1 and AtSIG5 transcripts. The fluence response differed in blue-light-responsive rapid induction in AtSIG1 and AtSIG5. AtSIG1 transcripts increased to plateau with a threshold of 2 micromol m(-2) sec(-1) under all fluences examined (1-50 micromol m(-2) sec(-1)), and AtSIG5 transcripts were induced with a distinct two-phase profile, with the lower-fluence induction similar to that of AtSIG1 and further enhancement with increasing fluences greater than 10 micromol m(-2) sec(-1). Blue-light-receptor mutational analysis revealed that AtSIG5-specific two-phase induction is mediated through cryptochrome 1 and cryptochrome 2 at lower fluences and more significantly through cryptochrome 1 at higher fluences. In mature chloroplasts, the promoters of psbA and psbD are predominantly recognized by AtSIG5 among six sigma factors. Using a protoplast transient expression assay with AtSIG5-AtSIG1 chimeric genes, we present evidence that AtSIG5 contains determinants for activating the psbD blue-light-responsive promoter (BLRP) in region 4.2 rather than region 2.4. Amino acid scanning within AtSIG5 region 4.2 revealed that Asn484, but not Arg493, functions as a key residue for psbD BLRP activation. Arginine 493 may be involved in psbA promoter recognition.

Asunto(s)

Proteínas de Arabidopsis/genética , Arabidopsis/genética , Flavoproteínas/genética , Plastidios/genética , Factor sigma/genética , Arabidopsis/metabolismo , Células Cultivadas , Criptocromos , Análisis Mutacional de ADN , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Luz , Mutación , Complejo de Proteína del Fotosistema II/genética , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Regiones Promotoras Genéticas , Protoplastos/metabolismo , ARN de Planta/genética , Transcripción Genética

RESUMEN

A complex of photoreceptor phoborhodopsin (ppR; also called sensory rhodopsin II) and its cognate halobacterial transducer II (pHtrII) existing in the plasma membrane mediates the light signal to the cytoplasm in the earliest step of negative phototaxis in Natronomonas pharaonis. We have investigated the dynamics of the light-induced conformational changes of the ppR/pHtrII(1-159) complex formed in the presence of 0.1% n-dodecyl beta-d-maltoside (DDM) by a fluorescence resonance energy transfer (FRET) based method. Fluorescence donor and acceptor dyes were linked to cysteine residues genetically introduced at given positions in pHtrII and ppR. The light-induced FRET efficiency changes for various pairs of dye-labeled cysteine residues were determined to examine dynamics of movements of given residues in the transmembrane and the linker region including the HAMP domain in pHtrII induced by photoexcitation of ppR. Upon flash excitation of ppR, FRET efficiency changed depending on pairs of the labeled cysteine residues. The distances between V185 in ppR and the five given residues (102 through 141) in the pHtrII linker region estimated from the FRET efficiency increased by 0.3-0.8 A; on the other hand, the distances between S31 in ppR and the five residues in pHtrII decreased. The changes arose within 70 ms (the dead time of instrument) and decayed at a rate of 1.1 +/- 0.2 s. Azide significantly increased the decay rate of light-induced FRET efficiency changes by accelerating the decay of the M state of ppR. The decay rate of FRET efficiency changes coincided with the rate of recovery of the ppR to the initial state but not the decay of the M state. We conclude that the light-induced conformational change of pHtrII occurs before, at the formation or during the M state, and its relaxation is coupled tightly with the decay of the O state of ppR in the 1:1 complex formed in the DDM micelle.

Asunto(s)

Proteínas Arqueales/química , Proteínas Arqueales/metabolismo , Glucósidos/metabolismo , Halorrodopsinas/química , Halorrodopsinas/metabolismo , Luz , Rodopsinas Sensoriales/química , Rodopsinas Sensoriales/metabolismo , Termodinámica , Visión Ocular , Detergentes , Transferencia Resonante de Energía de Fluorescencia , Glucósidos/química , Micelas , Fotólisis , Conformación Proteica

RESUMEN

The photoreceptor phoborhodopsin (ppR; also called sensory rhodopsin II) forms a complex with its cognate the Halobacterial transducer II (pHtrII) in the membrane, through which changes in the environmental light conditions are transmitted to the cytoplasm in Natronomonas pharaonis to evoke negative phototaxis. We have applied a fluorescence resonance energy transfer (FRET)-based method for investigation of the light-induced conformational changes of the ppR/pHtrII complex. Several far-red dyes were examined as possible fluorescence donors or acceptors because of the absence of the spectral overlap of these dyes with all the photointermediates of ppR. The flash-induced changes of distances between the donor and an acceptor linked to cysteine residues which were genetically introduced at given positions in pHtrII(1-159) and ppR were determined from FRET efficiency changes. The dye-labeled complex was studied as solubilized in 0.1% n-dodecyl-beta-D-maltoside (DDM). The FRET-derived changes in distances from V78 and A79 in pHtrII to V185 in ppR were consistent with the crystal structure data (Moukhametzianov, R. et al. [2006] Nature, 440, 115-119). The distance from D102 in pHtrII linker region to V185 in ppR increased by 0.33 angstroms upon the flash excitation. These changes arose within 70 ms (the dead time of instrument) and decayed with a rate of 1.1 +/- 0.2 s. Thus, sub-angstrom-scale distance changes in the ppR/pHtrII complex were detected with this FRET-based method using far-red fluorescent dyes; this method should be a valuable tool to investigate conformation changes in the transducer, in particular its dynamics.

Asunto(s)

Proteínas Arqueales/química , Proteínas Arqueales/efectos de la radiación , Halorrodopsinas/química , Halorrodopsinas/efectos de la radiación , Rodopsinas Sensoriales/química , Rodopsinas Sensoriales/efectos de la radiación , Proteínas Arqueales/genética , Transferencia Resonante de Energía de Fluorescencia , Halobacteriaceae/química , Halobacteriaceae/genética , Halobacteriaceae/efectos de la radiación , Halorrodopsinas/genética , Complejos Multiproteicos , Fotoquímica , Conformación Proteica/efectos de la radiación , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/efectos de la radiación , Rodopsinas Sensoriales/genética

RESUMEN

The psbD blue light-responsive promoter (BLRP), whose activation has been considered to require strong blue light, is recognized only by SIG5 among six sigma factors of plastid RNA polymerase in Arabidopsis. We found SIG5 transcript accumulation was rapidly induced after a 30-min induction time by blue light (470 nm) with an intensity threshold of 5 micromol m(-2)s(-1) through cryptochromes. Besides this weak blue light, the psbD BLRP activation required the stronger light such as 50 micromol m(-2)s(-1) irrespective of blue or red light (660 nm). Thus, the two independent light signalings, the cryptochrome-mediated signaling to induce SIG5 transcription and the stronger light-dependent signaling, cooperate to activate the psbD BLRP.

Asunto(s)

Proteínas de Arabidopsis/genética , Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas/genética , Complejo de Proteína del Fotosistema II/genética , Regiones Promotoras Genéticas/genética , Arabidopsis/efectos de la radiación , Regulación de la Expresión Génica de las Plantas/efectos de la radiación , Cinética , Luz , Iluminación , Regiones Promotoras Genéticas/efectos de la radiación , Subunidades de Proteína/genética , Transcripción Genética/genética , Transcripción Genética/efectos de la radiación

RESUMEN

Light is one of the most important environmental factors regulating expression of photosynthesis genes. The plastid psbD gene encoding the photosystem II reaction center protein D2 is under the control of a unique blue light responsive promoter (BLRP) that is transcribed by a bacterial-type plastid RNA polymerase (PEP). Promoter recognition of PEP is mediated by one of the six nuclear-encoded sigma factors in Arabidopsis. The replacement of the plastid sigma factor associated with PEP may be the major mechanism for switching of plastid transcription pattern in response to environmental and developmental signals. This study demonstrates that AtSig5 is a unique sigma factor that is essential for psbD BLRP activity. A T-DNA insertional mutant with reduced AtSIG5 expression resulted in loss of primary transcripts from the psbD BLRP. Furthermore, transient overexpression of AtSig5 in dark-adapted protoplasts specifically elevated psbD and psbA transcription activities. On the other hand, overproduction of AtSig2 enhanced the transcription of psbA gene and trnE operon, but not psbD transcription. The AtSIG5 gene is phylogenetically distinct from other plastid sigma factors, and its expression is induced exclusively by blue light. We propose that AtSig5 acts as a mediator of blue light signaling that specifically activates the psbD BLRP in response to blue light in Arabidopsis.

Asunto(s)

Proteínas de Arabidopsis/genética , Arabidopsis/genética , Complejo de Proteína del Fotosistema II/genética , Plastidios/genética , Factor sigma/genética , Factores de Transcripción/genética , Transcripción Genética , Arabidopsis/efectos de la radiación , Proteínas de Arabidopsis/metabolismo , Secuencia de Bases , Cartilla de ADN , ADN Bacteriano/genética , Escherichia coli/genética , Luz , Filogenia , Plantas Modificadas Genéticamente/genética , Plastidios/efectos de la radiación , Subunidades de Proteína/genética , Factor sigma/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética/efectos de la radiación

RESUMEN

Most plastid promoters recognized by bacteria-like plastid RNA polymerase (PEP) are similar to E. coli sigma(70)-type promoters comprising "-35" and "-10" elements. Among them, psbA promoter is unique in bearing additional elements between the conserved -35 and -10 elements. The psbA promoter activity is differentially maintained in the mature chloroplasts where the activity of most PEP promoters declines. Previously, we identified two types of PEP activities in wheat seedlings [Satoh et al. (1999) Plant J. 18: 407]; PEP present in the mature chloroplasts of the leaf tip (tip-type PEP) can initiate transcription from the -35-destructed psbA promoter, but the -35 element is essential for transcription by PEP present in immature chloroplasts of the leaf base (base-type PEP). To reveal which type of PEP functions in various types of plastids in tobacco, we analyzed the tobacco psbA promoter by means of a transplastomic approach. The promoter core context (-42 to +9) was sufficient for developmental regulation of the psbA promoter activity. The -35 promoter element was important for transcription initiation at the psbA promoter in all types of plastids, including chloroplasts in mature leaves, leucoplasts in roots, etioplasts in etiolated cotyledons. The conclusion is that the PEP bearing a promoter preference, similar to the wheat base-type PEP, functions dominantly in tobacco chloroplasts.

Asunto(s)

Cloroplastos/genética , Nicotiana/genética , Proteínas del Complejo del Centro de Reacción Fotosintética/genética , Regiones Promotoras Genéticas/genética , Secuencia de Bases , Luz , Datos de Secuencia Molecular , Proteínas del Complejo del Centro de Reacción Fotosintética/efectos de la radiación , Complejo de Proteína del Fotosistema II , Hojas de la Planta/genética , Hojas de la Planta/crecimiento & desarrollo , Hojas de la Planta/efectos de la radiación , Plantas Modificadas Genéticamente , Secuencias Reguladoras de Ácidos Nucleicos/genética , Homología de Secuencia de Ácido Nucleico , Transcripción Genética/efectos de la radiación

RESUMEN

The transcription of plastid gene psbD is under the control of the BLRP (blue-light-responsive promoter) recognized by plastid-encoded RNA polymerase, in which nuclear-encoded sigma factors play a crucial role in the promoter recognition. We examined the effects of light on mRNA levels of six different SIG genes in Arabidopsis and found that blue light extensively induced the accumulation of SIG5 transcripts, but red light did not. The blue light specificity was not observed in the accumulations of remaining five SIG genes. The blue light dependency of the SIG5 expression well explains the light-dependent behavior of the psbD BLRP.

Asunto(s)

Proteínas de Arabidopsis/genética , Arabidopsis/genética , Arabidopsis/efectos de la radiación , Plastidios/genética , Plastidios/efectos de la radiación , Factor sigma/genética , Arabidopsis/metabolismo , Regulación de la Expresión Génica de las Plantas/efectos de la radiación , Genes de Plantas/efectos de la radiación , Luz , Proteínas del Complejo del Centro de Reacción Fotosintética/genética , Complejo de Proteína del Fotosistema II , Regiones Promotoras Genéticas/efectos de la radiación , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN de Planta/genética , ARN de Planta/metabolismo

RESUMEN

Sigma factor binding proteins are involved in modifying the promoter preferences of the RNA polymerase in bacteria. We found the nuclear encoded protein (SibI) that is transported into chloroplasts and interacts specifically with the region 4 of Sig1 in Arabidopsis. SibI and its homologue, T3K9.5 are novel proteins, which are not homologous to any protein of known function. The expression of sibI was tissue specific, light dependent, and developmentally timed. We suggest the transcriptional regulation by sigma factor binding proteins to function in the plastids of higher plant.

Asunto(s)

Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas Nucleares/metabolismo , Factor sigma/metabolismo , Proteínas de Arabidopsis/genética , Secuencia de Bases , Northern Blotting , Cloroplastos/metabolismo , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , Regulación del Desarrollo de la Expresión Génica/efectos de la radiación , Regulación de la Expresión Génica de las Plantas/efectos de la radiación , Proteínas Fluorescentes Verdes , Luz , Proteínas Luminiscentes/genética , Datos de Secuencia Molecular , Proteínas Nucleares/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Unión Proteica/fisiología , Transporte de Proteínas/fisiología , ARN Mensajero/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Factor sigma/genética , Especificidad por Sustrato