RESUMEN
Diatoms are a diverse group of photosynthetic unicellular algae with a plastid of red-algal origin. As prolific primary producers in the ocean, diatoms fix as much carbon as all rainforests combined. The molecular mechanisms that contribute to the high photosynthetic productivity and ecological success of diatoms are however not yet fully understood. Using the model diatom Phaeodactylum tricornutum, here we show rhythmic transcript accumulation of plastid psaA, psbA, petB, and atpB genes as driven by a free running circadian clock. Treatment with the electron transport inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea overrides the circadian signal by markedly downregulating transcription of psaA, petB, and atpB genes but not the psbA gene. Changes in light quantity produce little change in plastid gene transcription while the effect of light quality seems modest with only the psaA gene responding in a pattern that is dependent on the redox state of the plastoquinone pool. The significance of these plastid transcriptional responses and the identity of the underlying genetic control systems are discussed with relevance to diatom photosynthetic acclimation.
Asunto(s)
Ritmo Circadiano/fisiología , Diatomeas/metabolismo , Regulación de la Expresión Génica/efectos de la radiación , Luz , Plastidios , Transcripción Genética/efectos de la radiación , Diatomeas/genética , Humanos , Oxidación-Reducción , ARN/fisiología , TemperaturaRESUMEN
Sigma factors are dissociable subunits of bacterial RNA polymerase that ensure efficient transcription initiation from gene promoters. Owing to their prokaryotic origin, chloroplasts possess a typical bacterial RNA polymerase together with its sigma factor subunit. The higher plant Arabidopsis thaliana contain as many as six sigma factors for the hundred or so of its chloroplast genes. The role of this relatively large number of transcription initiation factors for the miniature chloroplast genome, however, is not fully understood. Using two Arabidopsis T-DNA insertion mutants, we show that sigma factor 1 (SIG1) initiates transcription of a specific subset of chloroplast genes. We further show that the photosynthetic control of PSI reaction center gene transcription requires complementary regulation of the nuclear SIG1 gene at the transcriptional level. This SIG1 gene regulation is dependent on both a plastid redox signal and a light signal transduced by the phytochrome photoreceptor.
Asunto(s)
Aclimatación , Cloroplastos/metabolismo , Regulación de la Expresión Génica de las Plantas , Fotosíntesis , Proteínas de Plantas/metabolismo , Factor sigma/metabolismo , Arabidopsis , Proteínas de Plantas/genética , Factor sigma/genéticaRESUMEN
Two-component signal transduction systems mediate adaptation to environmental changes in bacteria, plants, fungi, and protists. Each two-component system consists of a sensor histidine kinase and a response regulator. Chloroplast sensor kinase (CSK) is a modified sensor histidine kinase found in chloroplasts-photosynthetic organelles of plants and algae. CSK regulates the transcription of chloroplast genes in response to changes in photosynthetic electron transport. In this study, the full-length and truncated forms of Arabidopsis CSK proteins were overexpressed and purified in order to characterise their kinase and redox sensing activities. Our results show that CSK contains a modified kinase catalytic domain that binds ATP with high affinity and forms a quinone adduct that may confer redox sensing activity.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Cloroplastos/metabolismo , Histidina Quinasa/metabolismo , Adenosina Trifosfato/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/fisiología , Cloroplastos/genética , Histidina Quinasa/genética , Histidina Quinasa/fisiología , Oxidación-Reducción , Fosforilación , Fotosíntesis , Proteínas Recombinantes , Alineación de Secuencia , Transducción de SeñalRESUMEN
Improving chilling tolerance in cold-sensitive crops, e.g. tomato, requires knowledge of the early molecular response to low temperature in these under-studied species. To elucidate early responding processes and regulators, we captured the transcriptional response at 30 minutes and 3 hours in the shoots and at 3 hours in the roots of tomato post-chilling from 24°C to 4°C. We used a pre-treatment control and a concurrent ambient temperature control to reveal that majority of the differential expression between cold and ambient conditions is due to severely compressed oscillation of a large set of diurnally regulated genes in both the shoots and roots. This compression happens within 30 minutes of chilling, lasts for the duration of cold treatment, and is relieved within 3 hours of return to ambient temperatures. Our study also shows that the canonical ICE1/CAMTA-to-CBF cold response pathway is active in the shoots, but not in the roots. Chilling stress induces synthesis of known cryoprotectants (trehalose and polyamines), in a CBF-independent manner, and induction of multiple genes encoding proteins of photosystems I and II. This study provides nuanced insights into the organ-specific response in a chilling sensitive plant, as well as the genes influenced by an interaction of chilling response and the circadian clock.
RESUMEN
State transitions and photosystem stoichiometry adjustment are two oxidation-reduction (redox)-regulated acclimatory responses in photosynthesis. State transitions are short-term adaptations that, in chloroplasts, involve reversible post-translational modification by phosphorylation of light-harvesting complex II (LHC II). Photosystem stoichiometry adjustments are long-term responses involving transcriptional regulation of reaction centre genes. Both responses are initiated by changes in light quality and are regulated by the redox state of plastoquinone (PQ). The LHC II kinase involved in the state 2 transition is a serine/threonine kinase known as STT7 in Chlamydomonas, and as STN7 in Arabidopsis. The phospho-LHC II phosphatase that produces the state 1 transition is a PP2C-type protein phosphatase currently termed both TAP38 and PPH1. In plants and algae, photosystem stoichiometry adjustment is governed by a modified two-component sensor kinase of cyanobacterial origin - chloroplast sensor kinase (CSK). CSK is a sensor of the PQ redox state. Chloroplast sigma factor 1 (SIG1) and plastid transcription kinase (PTK) are the functional partners of CSK in chloroplast gene regulation. We suggest a signalling pathway for photosystem stoichiometry adjustment. The signalling pathways of state transitions and photosystem stoichiometry adjustments are proposed to be distinct, with the two pathways sensing PQ redox state independently of each other.
Asunto(s)
Cloroplastos/química , Complejos de Proteína Captadores de Luz/química , Plantas/química , Transducción de Señal , Adaptación Fisiológica , Cloroplastos/enzimología , Cloroplastos/genética , Luz , Complejos de Proteína Captadores de Luz/metabolismo , Oxidación-Reducción , Fosfoproteínas Fosfatasas/metabolismo , Fosforilación , Fotosíntesis , Proteínas de Plantas/metabolismo , Plantas/enzimología , Plantas/genética , Plastoquinona/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transcripción GenéticaRESUMEN
Cyanobacteria employ two-component sensor-response regulator systems to monitor and respond to environmental challenges. The response regulators RpaA, RpaB, Rre1 and RppA are integral to circadian clock function and abiotic stress acclimation in cyanobacteria. RpaA, RpaB and Rre1 are known to interact with ferredoxin or thioredoxin, raising the possibility of their thiol regulation. Here, we report that Synechocystis sp. PCC 6803 Rre1, RpaA and RpaB exist as higher-order oligomers under oxidising conditions and that reduced thioredoxin A converts them to monomers. We further show that these response regulators contain redox-responsive cysteine residues with an Em7 around -300 mV. These findings suggest a direct thiol modulation of the activity of these response regulators, independent of their cognate sensor kinases.
Asunto(s)
Synechocystis , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cianobacterias/genética , Cianobacterias/metabolismo , Regulación Bacteriana de la Expresión Génica/genética , Oxidación-Reducción , Compuestos de Sulfhidrilo , Synechocystis/genética , Synechocystis/metabolismo , Tiorredoxinas/genética , Tiorredoxinas/metabolismoRESUMEN
Plant growth under spectrally-enriched low light conditions leads to adjustment in the relative abundance of the two photosystems in an acclimatory response known as photosystem stoichiometry adjustment. Adjustment of photosystem stoichiometry improves the quantum efficiency of photosynthesis but how this process perceives light quality changes and how photosystem amount is regulated remain largely unknown. By using a label-free quantitative mass spectrometry approach in Arabidopsis here we show that photosystem stoichiometry adjustment is primarily driven by the regulation of photosystem I content and that this forms the major thylakoid proteomic response under light quality. Using light and redox signaling mutants, we further show that the light quality-responsive accumulation of photosystem I gene transcripts and proteins requires phytochrome B photoreceptor but not plastoquinone redox signaling as previously suggested. In far-red light, the increased acceptor side limitation might deplete active photosystem I pool, further contributing to the adjustment of photosystem stoichiometry.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Luz , Oxidación-Reducción , Fotosíntesis/fisiología , Complejo de Proteína del Fotosistema I/metabolismo , Complejo de Proteína del Fotosistema II/metabolismo , Proteómica , Tilacoides/metabolismoRESUMEN
The extensive processing and protein-assisted stabilization of transcripts have been taken as evidence for a viewpoint that the control of gene expression had shifted entirely in evolution from transcriptional in the bacterial endosymbiont to posttranscriptional in the plastid. This suggestion is however at odds with many observations on plastid gene transcription. Chloroplasts of flowering plants and mosses contain two or more RNA polymerases with distinct promoter preference and division of labor for the coordinated synthesis of plastid RNAs. Plant and algal plastids further possess multiple nonredundant sigma factors that function as transcription initiation factors. The controlled accumulation of plastid sigma factors and modification of their activity by sigma-binding proteins and phosphorylation constitute additional transcriptional regulatory strategies. Plant and algal plastids also contain dedicated one- or two-component transcriptional regulators. Transcription initiation thus continues to form a critical control point at which varied developmental and environmental signals intersect with plastid gene expression.
Asunto(s)
Regulación de la Expresión Génica de las Plantas/fisiología , Plastidios/metabolismo , Iniciación de la Transcripción Genética/fisiología , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plastidios/genéticaRESUMEN
Hetero-oligomeric membrane protein complexes form the electron transport chain (ETC) of oxygenic photosynthesis. The ETC complexes undertake the light-driven vectorial electron and proton transport reactions, which generate energy-rich ATP and electron-rich NADPH molecules for carbon fixation. The rate of photosynthetic electron transport depends on the availability of photons and the relative abundance of electron transport complexes. The relative abundance of the two photosystems, critical for the quantum efficiency of photosynthesis in changing light quality conditions, has been determined successfully by optical methods. Due to the lack of spectroscopic signatures, however, relatively little is known about the stoichiometry of other non-photosystem complexes in plant photosynthetic membrane. Here we determine the ratios of all major thylakoid-bound ETC complexes in Arabidopsis by a label-free quantitative mass spectrometry technique. The calculated stoichiometries are consistent with known subunit composition of complexes and current estimates of photosystem and cytochrome b6f concentrations. The implications of these stoichiometries for photosynthetic light harvesting and the partitioning of electrons between the linear and cyclic electron transport pathways of photosynthesis are discussed.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Complejo de Citocromo b6f/metabolismo , Fotosíntesis/fisiología , Tilacoides/enzimologíaRESUMEN
Photosynthetic efficiency depends on equal light energy conversion by two spectrally distinct, serially-connected photosystems. The redox state of the plastoquinone pool, located between the two photosystems, is a key regulatory signal that initiates acclimatory changes in the relative abundance of photosystems. The Chloroplast Sensor Kinase (CSK) links the plastoquinone redox signal with photosystem gene expression but the mechanism by which it monitors the plastoquinone redox state is unclear. Here we show that the purified Arabidopsis and Phaeodactylum CSK and the cyanobacterial CSK homologue, Histidine kinase 2 (Hik2), are iron-sulfur proteins. The Fe-S cluster of CSK is further revealed to be a high potential redox-responsive [3Fe-4S] center. CSK responds to redox agents with reduced plastoquinone suppressing its autokinase activity. Redox changes within the CSK iron-sulfur cluster translate into conformational changes in the protein fold. These results provide key insights into redox signal perception and propagation by the CSK-based chloroplast two-component system.
Asunto(s)
Cloroplastos/metabolismo , Histidina Quinasa/metabolismo , Hierro/metabolismo , Oxidación-Reducción , Azufre/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Activación Enzimática , Histidina Quinasa/química , Hierro/química , Fotosíntesis , Conformación Proteica , Proteínas Recombinantes , Análisis Espectral , Relación Estructura-Actividad , Azufre/químicaRESUMEN
The 2.5 Å structure of the cytochrome (cyt) b6 f complex provides a basis for control of the rate-limiting electron transfer step of oxygenic photosynthesis associated with the plastoquinol/quinone exchange pathway. Here, a structural change was made at a site containing two proline residues which border the intra-cyt pathway for plastoquinol/quinone exchange. The proline side chains confer a larger aperture for passage of plastoquinol/quinone. Change of these prolines to alanine in the cyanobacterium Synechococcus sp. PCC 7002 results in attenuation of this rate-limiting step, observed by a two-fold reduction in the rate of cell growth, O2 evolution, and plastoquinol-mediated reduction of cyt f. This study demonstrates modification by site-directed mutagenesis of photosynthetic energy transduction based on rational application of information in the atomic structure.
Asunto(s)
Sustitución de Aminoácidos , Complejo de Citocromo b6f/química , Complejo de Citocromo b6f/genética , Synechococcus/metabolismo , Alanina/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Complejo de Citocromo b6f/metabolismo , Transporte de Electrón/efectos de los fármacos , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Oxígeno/metabolismo , Fotosíntesis/efectos de los fármacos , Plastoquinona/análogos & derivados , Plastoquinona/farmacología , Prolina/genética , Conformación Proteica/efectos de los fármacosRESUMEN
The halotolerant microalga Dunaliella salina has been widely studied for natural ß-carotene production. This work shows biochemical characterization of three newly isolated Dunaliellasalina strains, DF15, DF17, and DF40, compared with D. salina CCAP 19/30 and D. salina UTEX 2538 (also known as D. bardawil). Although all three new strains have been genetically characterized as Dunaliella salina strains, their ability to accumulate carotenoids and their capacity for photoprotection against high light stress are different. DF15 and UTEX 2538 reveal great potential for producing a large amount of ß-carotene and maintained a high rate of photosynthesis under light of high intensity; however, DF17, DF40, and CCAP 19/30 showed increasing photoinhibition with increasing light intensity, and reduced contents of carotenoids, in particular ß-carotene, suggesting that the capacity of photoprotection is dependent on the cellular content of carotenoids, in particular ß-carotene. Strong positive correlations were found between the cellular content of all-trans ß-carotene, 9-cis ß-carotene, all-trans α-carotene and zeaxanthin but not lutein in the D. salina strains. Lutein was strongly correlated with respiration in photosynthetic cells and strongly related to photosynthesis, chlorophyll and respiration, suggesting an important and not hitherto identified role for lutein in coordinated control of the cellular functions of photosynthesis and respiration in response to changes in light conditions, which is broadly conserved in Dunaliella strains. Statistical analysis based on biochemical data revealed a different grouping strategy from the genetic classification of the strains. The significance of these data for strain selection for commercial carotenoid production is discussed.
RESUMEN
Two-component signal transduction systems (TCSs) consist of sensor histidine kinases and response regulators. TCSs mediate adaptation to environmental changes in bacteria, plants, fungi and protists. Histidine kinase 2 (Hik2) is a sensor histidine kinase found in all known cyanobacteria and as chloroplast sensor kinase in eukaryotic algae and plants. Sodium ions have been shown to inhibit the autophosphorylation activity of Hik2 that precedes phosphoryl transfer to response regulators, but the mechanism of inhibition has not been determined. We report on the mechanism of Hik2 activation and inactivation probed by chemical cross-linking and size exclusion chromatography together with direct visualisation of the kinase using negative-stain transmission electron microscopy of single particles. We show that the functional form of Hik2 is a higher-order oligomer such as a hexamer or octamer. Increased NaCl concentration converts the active hexamer into an inactive tetramer. The action of NaCl appears to be confined to the Hik2 kinase domain.
Asunto(s)
Cianobacterias/enzimología , Histidina Quinasa/metabolismo , Multimerización de Proteína , Sodio/metabolismo , Cromatografía en Gel , Reactivos de Enlaces Cruzados/metabolismo , Histidina Quinasa/química , Histidina Quinasa/ultraestructura , Iones , Coloración Negativa , Dominios Proteicos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Cloruro de Sodio/farmacologíaRESUMEN
The green microalga Dunaliella salina survives in a wide range of salinities via mechanisms involving glycerol synthesis and degradation and is exploited for large amounts of nutraceutical carotenoids produced under stressed conditions. In this study, D. salina CCAP 19/30 was cultured in varying photoperiods and light intensities to study the relationship of light with different growth measurement parameters, with cellular contents of glycerol, starch and carotenoids, and with photosynthesis and respiration. Results show CCAP 19/30 regulated cell volume when growing under light/dark cycles: cell volume increased in the light and decreased in the dark, and these changes corresponded to changes in cellular glycerol content. The decrease in cell volume in the dark was independent of cell division and biological clock and was regulated by the photoperiod of the light/dark cycle. When the light intensity was increased to above 1000 µmol photons m(-2) s(-1), cells displayed evidence of photodamage. However, these cells also maintained the maximum level of photosynthesis efficiency and respiration possible, and the growth rate increased as light intensity increased. Significantly, the intracellular glycerol content also increased, >2-fold compared to the content in light intensity of 500 µmol photons m(-2) s(-1), but there was no commensurate increase in the pool size of carotenoids. These data suggest that in CCAP 19/30 glycerol stabilized the photosynthetic apparatus for maximum performance in high light intensities, a role normally attributed to carotenoids.
Asunto(s)
Chlorophyta/crecimiento & desarrollo , Chlorophyta/efectos de la radiación , Luz , Fotoperiodo , Fotosíntesis/efectos de la radiación , Biomasa , Carotenoides/metabolismo , Respiración de la Célula/efectos de la radiación , Tamaño de la Célula/efectos de la radiación , Clorofila/metabolismo , Oscuridad , Fluorescencia , Glicerol/metabolismo , Oxígeno/metabolismo , Complejo de Proteína del Fotosistema II/metabolismo , Almidón/metabolismoRESUMEN
Two-component systems (TCSs) are ubiquitous signaling units found in prokaryotes. A TCS consists of a sensor histidine kinase and a response regulator protein as signal transducers. These regulatory systems mediate acclimation to various environmental changes by coupling environmental cues to gene expression. Hik2 is a sensor histidine kinase and its gene is found in all cyanobacteria. Hik2 is the homolog of Chloroplast Sensor Kinase (CSK), a protein involved in redox regulation of chloroplast gene expression during changes in light quality in plants and algae. Here we describe biochemical characterization of the signaling mechanism of Hik2 and its phosphotransferase activity. Results presented here indicate that Hik2 undergoes autophosphorylation on a conserved histidine residue, and becomes rapidly dephosphorylated by the action of response regulators Rre1 and RppA. We also show that the autophosphorylation of Hik2 is specifically inhibited by sodium ions.
RESUMEN
Photosynthetic electron transport regulates chloroplast gene transcription through the action of a bacterial-type sensor kinase known as chloroplast sensor kinase (CSK). CSK represses photosystem I (PS I) gene transcription in PS I light and thus initiates photosystem stoichiometry adjustment. In cyanobacteria and in non-green algae, CSK homologues co-exist with their response regulator partners in canonical bacterial two-component systems. In green algae and plants, however, no response regulator partner of CSK is found. Yeast two-hybrid analysis has revealed interaction of CSK with sigma factor 1 (SIG1) of chloroplast RNA polymerase. Here we present further evidence for the interaction between CSK and SIG1. We also show that CSK interacts with quinone. Arabidopsis SIG1 becomes phosphorylated in PS I light, which then specifically represses transcription of PS I genes. In view of the identical signalling properties of CSK and SIG1 and of their interactions, we suggest that CSK is a SIG1 kinase. We propose that the selective repression of PS I genes arises from the operation of a gene-regulatory phosphoswitch in SIG1. The CSK-SIG1 system represents a novel, rewired chloroplast-signalling pathway created by evolutionary tinkering. This regulatory system supports a proposal for the selection pressure behind the evolutionary stasis of chloroplast genes.
Asunto(s)
Arabidopsis/metabolismo , Evolución Biológica , Cloroplastos/metabolismo , Regulación de la Expresión Génica de las Plantas/fisiología , Células Procariotas/metabolismo , Secuencia de Aminoácidos , Arabidopsis/genética , Datos de Secuencia Molecular , Complejo de Proteína del Fotosistema I/fisiología , Transducción de SeñalRESUMEN
Chloroplast sensor kinase (CSK) is a bacterial-type sensor histidine kinase found in chloroplasts--photosynthetic plastids--in eukaryotic plants and algae. Using a yeast two-hybrid screen, we demonstrate recognition and interactions between: CSK, plastid transcription kinase (PTK), and a bacterial-type RNA polymerase sigma factor-1 (SIG-1). CSK interacts with itself, with SIG-1, and with PTK. PTK also interacts directly with SIG-1. PTK has previously been shown to catalyze phosphorylation of plastid-encoded RNA polymerase (PEP), suppressing plastid transcription nonspecifically. Phospho-PTK is inactive as a PEP kinase. Here, we propose that phospho-CSK acts as a PTK kinase, releasing PTK repression of chloroplast transcription, while CSK also acts as a SIG-1 kinase, blocking transcription specifically at the gene promoter of chloroplast photosystem I. Oxidation of the photosynthetic electron carrier plastoquinone triggers phosphorylation of CSK, inducing chloroplast photosystem II while suppressing photosystem I. CSK places photosystem gene transcription under the control of photosynthetic electron transport. This redox signaling pathway has its origin in cyanobacteria, photosynthetic prokaryotes from which chloroplasts evolved. The persistence of this mechanism in cytoplasmic organelles of photosynthetic eukaryotes is in precise agreement with the CoRR hypothesis for the function of organellar genomes: the plastid genome and its primary gene products are Co-located for Redox Regulation. Genes are retained in plastids primarily in order for their expression to be subject to this rapid and robust redox regulatory transcriptional control mechanism, whereas plastid genes also encode genetic system components, such as some ribosomal proteins and RNAs, that exist in order to support this primary, redox regulatory control of photosynthesis genes. Plastid genome function permits adaptation of the photosynthetic apparatus to changing environmental conditions of light quantity and quality.