RESUMEN
Flavodiiron proteins (FLVs) catalyze the reduction of oxygen to water by using electrons from Photosystem I (PSI). In several photosynthetic organisms such as cyanobacteria, green algae, mosses and gymnosperms, FLV-dependent electron flow protects PSI from over-reduction and consequent damage especially under fluctuating light conditions. In this work we investigated biochemical and structural properties of FLVA and FLVB from the model moss Physcomitrium patens. The two proteins, expressed and purified from Escherichia coli, bind both iron and flavin cofactors and show NAD(P)H oxidase activity as well as oxygen reductase capacities. Moreover, the co-expression of both FLVA and FLVB, coupled to a tandem affinity purification procedure with two different affinity tags, enabled the isolation of the stable and catalytically active FLVA/B hetero tetrameric protein complex with cooperative nature. The multimeric organization was shown to be stabilized by inter-subunit disulfide bonds. This investigation provides valuable new information on the biochemical properties of FLVs, with new insights into their in vivo activity.
RESUMEN
Plants exposed to light fluctuations are protected from photodamage by non-photochemical quenching (NPQ), a reversible mechanism that enables dissipation of excess absorbed energy as heat, which is essential for plant fitness and crop productivity. In plants NPQ requires the presence of the membrane protein PsbS, which upon activation interacts with antenna proteins, inducing their dissipative conformation. Here, we exploited base editing (BE) in the moss Physcomitrium patens to introduce specific amino acid changes in vivo and assess their impact on PsbS activity, targeting transmembrane regions to investigate their role in essential protein-protein interactions. This approach enabled the recognition of residues essential for protein stability and the identification of a hydrophobic cluster of amino acids impacting PsbS activity. This work provides new information on the molecular mechanism of PsbS while also demonstrating the potential of BE approaches for in planta gene function analysis.
Asunto(s)
Luz , Fotosíntesis , Complejo de Proteína del Fotosistema II/metabolismo , Edición Génica , Complejos de Proteína Captadores de Luz/metabolismoRESUMEN
Plants rely on solar energy to synthesize ATP and NADPH for photosynthetic carbon fixation and all cellular need. Mitochondrial respiration is essential in plants, but this may be due to heterotrophic bottlenecks during plant development or because it is also necessary in photosynthetically active cells. In this study, we examined in vivo changes of cytosolic ATP concentration in response to light, employing a biosensing strategy in the moss Physcomitrium patens and revealing increased cytosolic ATP concentration caused by photosynthetic activity. Plants depleted of respiratory Complex I showed decreased cytosolic ATP accumulation, highlighting a critical role of mitochondrial respiration in light-dependent ATP supply of the cytosol. Consistently, targeting mitochondrial ATP production directly, through the construction of mutants deficient in mitochondrial ATPase (complex V), led to drastic growth reduction, despite only minor alterations in photosynthetic electron transport activity. Since P. patens is photoautotrophic throughout its development, we conclude that heterotrophic bottlenecks cannot account for the indispensable role of mitochondrial respiration in plants. Instead, our results support that mitochondrial respiration is essential for ATP provision to the cytosol in photosynthesizing cells. Mitochondrial respiration provides metabolic integration, ensuring supply of cytosolic ATP essential for supporting plant growth and development.
Asunto(s)
Adenosina Trifosfato , Bryopsida , Respiración de la Célula , Citosol , Mitocondrias , Fotosíntesis , Adenosina Trifosfato/metabolismo , Citosol/metabolismo , Mitocondrias/metabolismo , Bryopsida/metabolismo , Bryopsida/genética , Bryopsida/crecimiento & desarrollo , LuzRESUMEN
Light is the ultimate source of energy for photosynthetic organisms, but respiration is fundamental for supporting metabolism during the night or in heterotrophic tissues. In this work, we isolated Physcomitrella (Physcomitrium patens) plants with altered respiration by inactivating Complex I (CI) of the mitochondrial electron transport chain by independently targeting on two essential subunits. Inactivation of CI caused a strong growth impairment even in fully autotrophic conditions in tissues where all cells are photosynthetically active, demonstrating that respiration is essential for photosynthesis. CI mutants showed alterations in the stoichiometry of respiratory complexes while the composition of photosynthetic apparatus was substantially unaffected. CI mutants showed altered photosynthesis with high activity of both Photosystems I and II, likely the result of high chloroplast ATPase activity that led to smaller ΔpH formation across thylakoid membranes, decreasing photosynthetic control on cytochrome b6f in CI mutants. These results demonstrate that alteration of respiratory activity directly impacts photosynthesis in P. patens and that metabolic interaction between organelles is essential in their ability to use light energy for growth.
Asunto(s)
Adenosina Trifosfatasas/genética , Bryopsida/genética , Proteínas de Cloroplastos/genética , Proteínas de Plantas/genética , Adenosina Trifosfatasas/metabolismo , Bryopsida/enzimología , Proteínas de Cloroplastos/metabolismo , Proteínas de Plantas/metabolismoRESUMEN
CRISPR-Cas9 has proven to be highly valuable for genome editing in plants, including the model plant Physcomitrium patens. However, the fact that most of the editing events produced using the native Cas9 nuclease correspond to small insertions and deletions is a limitation. CRISPR-Cas9 base editors enable targeted mutation of single nucleotides in eukaryotic genomes and therefore overcome this limitation. Here, we report two programmable base-editing systems to induce precise cytosine or adenine conversions in P. patens. Using cytosine or adenine base editors, site-specific single-base mutations can be achieved with an efficiency up to 55%, without off-target mutations. Using the APT gene as a reporter of editing, we could show that both base editors can be used in simplex or multiplex, allowing for the production of protein variants with multiple amino-acid changes. Finally, we set up a co-editing selection system, named selecting modification of APRT to report gene targeting (SMART), allowing up to 90% efficiency site-specific base editing in P. patens. These two base editors will facilitate gene functional analysis in P. patens, allowing for site-specific editing of a given base through single sgRNA base editing or for in planta evolution of a given gene through the production of randomly mutagenised variants using multiple sgRNA base editing.
Asunto(s)
Bryopsida , Bryopsida/genética , Sistemas CRISPR-Cas/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Edición Génica , Mutagénesis Sitio-DirigidaRESUMEN
In nature, photosynthetic organisms are exposed to highly dynamic environmental conditions where the excitation energy and electron flow in the photosynthetic apparatus need to be continuously modulated. Fluctuations in incident light are particularly challenging because they drive oversaturation of photosynthesis with consequent oxidative stress and photoinhibition. Plants and algae have evolved several mechanisms to modulate their photosynthetic machinery to cope with light dynamics, such as thermal dissipation of excited chlorophyll states (non-photochemical quenching, NPQ) and regulation of electron transport. The regulatory mechanisms involved in the response to light dynamics have adapted during evolution, and exploring biodiversity is a valuable strategy for expanding our understanding of their biological roles. In this work, we investigated the response to fluctuating light in Nannochloropsis gaditana, a eukaryotic microalga of the phylum Heterokonta originating from a secondary endosymbiotic event. Nannochloropsis gaditana is negatively affected by light fluctuations, leading to large reductions in growth and photosynthetic electron transport. Exposure to light fluctuations specifically damages photosystem I, likely because of the ineffective regulation of electron transport in this species. The role of NPQ, also assessed using a mutant strain specifically depleted of this response, was instead found to be minor, especially in responding to the fastest light fluctuations.
Asunto(s)
Luz , Fotosíntesis/fisiología , Estramenopilos/metabolismo , Simbiosis/fisiología , Biodiversidad , Transporte de Electrón/fisiología , Estrés Oxidativo , Complejo de Proteína del Fotosistema I/metabolismo , Complejo de Proteína del Fotosistema I/efectos de la radiación , Plantas/metabolismo , Estramenopilos/crecimiento & desarrollo , Estramenopilos/efectos de la radiaciónRESUMEN
Photosynthetic electron transport is regulated by cyclic and pseudocyclic electron flow (CEF and PCEF) to maintain the balance between light availability and metabolic demands. CEF transfers electrons from photosystem I to the plastoquinone pool with two mechanisms, dependent either on PGR5/PGRL1 or on the type I NADH dehydrogenase-like (NDH) complex. PCEF uses electrons from photosystem I to reduce oxygen and in many groups of photosynthetic organisms, but remarkably not in angiosperms, it is catalyzed by flavodiiron proteins (FLVs). In this study, Physcomitrella patens plants depleted in PGRL1, NDH and FLVs in different combinations were generated and characterized, showing that all these mechanisms are active in this moss. Surprisingly, in contrast to flowering plants, Physcomitrella patens can cope with the simultaneous inactivation of PGR5- and NDH-dependent CEF but, when FLVs are also depleted, plants show strong growth reduction and photosynthetic activity is drastically reduced. The results demonstrate that mechanisms for modulation of photosynthetic electron transport have large functional overlap but are together indispensable to protect photosystem I from damage and they are an essential component for photosynthesis in any light regime.
Asunto(s)
Bryopsida , Complejo de Proteína del Fotosistema I , Bryopsida/metabolismo , Transporte de Electrón , Luz , Fotosíntesis , Complejo de Proteína del Fotosistema I/metabolismo , Desarrollo de la PlantaRESUMEN
Alternative electron pathways contribute to regulation of photosynthetic light reactions to adjust to metabolic demands in dynamic environments. The chloroplast NADH dehydrogenase-like (NDH) complex mediates the cyclic electron transport pathway around PSI in different cyanobacteria, algae, and plant species, but it is not fully conserved in all photosynthetic organisms. In order to assess how the physiological role of this complex changed during plant evolution, we isolated Physcomitrella patens lines knocked out for the NDHM gene that encodes a subunit fundamental for the activity of the complex. ndhm knockout mosses indicated high PSI acceptor side limitation upon abrupt changes in illumination. In P. patens, pseudo-cyclic electron transport mediated by flavodiiron proteins (FLVs) was also shown to prevent PSI over-reduction in plants exposed to light fluctuations. flva ndhm double knockout mosses had altered photosynthetic performance and growth defects under fluctuating light compared with the wild type and single knockout mutants. The results showed that while the contribution of NDH to electron transport is minor compared with FLV, NDH still participates in modulating photosynthetic activity, and it is critical to avoid PSI photoinhibition, especially when FLVs are inactive. The functional overlap between NDH- and FLV-dependent electron transport supports PSI activity and prevents its photoinhibition under light variations.
Asunto(s)
Bryopsida , Bryopsida/genética , Bryopsida/metabolismo , Cloroplastos/metabolismo , Transporte de Electrón , Luz , NADH Deshidrogenasa/genética , NADH Deshidrogenasa/metabolismo , Fotosíntesis , Complejo de Proteína del Fotosistema I/metabolismoRESUMEN
The regulation of photosynthesis is crucial to efficiently support the assimilation of carbon dioxide and to prevent photodamage. One key regulatory mechanism is the pseudo-cyclic electron flow (PCEF) mediated by class-C flavodiiron proteins (FLVs). These enzymes use electrons coming from Photosystem I (PSI) to reduce oxygen to water, preventing over-reduction in the acceptor side of PSI. FLVs are widely distributed among organisms performing oxygenic photosynthesis and they have been shown to be fundamental in many different conditions such as fluctuating light, sulfur deprivation and plant submersion. Moreover, since FLVs reduce oxygen they can help controlling the redox status of the cell and maintaining the microoxic environment essential for processes such as nitrogen fixation in cyanobacteria. Despite these important roles identified in various species, the genes encoding for FLV proteins have been lost in angiosperms where their activity could have been at least partially compensated by a more efficient cyclic electron flow (CEF). The present work reviews the information emerged on FLV function, analyzing recent structural data that suggest FLV could be regulated through a conformational change.
Asunto(s)
Proteínas Bacterianas/metabolismo , Cianobacterias/metabolismo , Fijación del Nitrógeno/fisiología , Oxígeno/metabolismo , Fotosíntesis/fisiología , Complejo de Proteína del Fotosistema I/metabolismo , Proteínas Bacterianas/genética , Cianobacterias/genética , Oxidación-Reducción , Complejo de Proteína del Fotosistema I/genéticaRESUMEN
High-throughput RNA sequencing (RNA-seq) has recently become the method of choice to define and analyze transcriptomes. For the model moss Physcomitrella patens, although this method has been used to help analyze specific perturbations, no overall reference dataset has yet been established. In the framework of the Gene Atlas project, the Joint Genome Institute selected P. patens as a flagship genome, opening the way to generate the first comprehensive transcriptome dataset for this moss. The first round of sequencing described here is composed of 99 independent libraries spanning 34 different developmental stages and conditions. Upon dataset quality control and processing through read mapping, 28 509 of the 34 361 v3.3 gene models (83%) were detected to be expressed across the samples. Differentially expressed genes (DEGs) were calculated across the dataset to permit perturbation comparisons between conditions. The analysis of the three most distinct and abundant P. patens growth stages - protonema, gametophore and sporophyte - allowed us to define both general transcriptional patterns and stage-specific transcripts. As an example of variation of physico-chemical growth conditions, we detail here the impact of ammonium supplementation under standard growth conditions on the protonemal transcriptome. Finally, the cooperative nature of this project allowed us to analyze inter-laboratory variation, as 13 different laboratories around the world provided samples. We compare differences in the replication of experiments in a single laboratory and between different laboratories.
Asunto(s)
Bryopsida/genética , Conjuntos de Datos como Asunto , Genes de Plantas/genética , Mapeo Cromosómico , Genoma de Planta/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Transcriptoma/genéticaRESUMEN
MAIN CONCLUSION: Investigation of photosynthesis regulation in different plant groups exposed to variable conditions showed that all species have similar photosynthetic electron transport modulation while excess energy dissipation is species specific. Photosynthesis is regulated in response to dynamic environmental conditions to satisfy plant metabolic demands while also avoiding possible over-excitation of the electron transport chain and the generation of harmful reactive oxygen species. Photosynthetic organisms evolved several mechanisms to modulate light harvesting and electron transport efficiency to respond to conditions changing at different timescales, going from fast sun flecks to slow seasonal variations. These regulatory mechanisms changed during evolution of photosynthetic organisms, also adapting to various ecological niches, making the investigation of plant biodiversity highly valuable to uncover conserved traits and plasticity of photosynthetic regulation and complement studies on model species. In this work, a set of plants belonging to different genera of angiosperms, gymnosperms, ferns and lycophytes were investigated by monitoring their photosynthetic parameters in different seasons looking for common trends and differences. In all plants, analysed photosynthetic electron transport rate was found to be modulated by growth light intensity, ensuring a balance between available energy and photochemical capacity. Growth light also influenced the threshold where heat dissipation of excitation energy, a mechanism called non-photochemical quenching (NPQ), was activated. On the contrary, NPQ amplitude did not correlate with light intensity experienced by the plants but was a species-specific feature. The zeaxanthin-dependent component of NPQ, qZ, was found to be the most variable in different plants and its modulation influenced the intensity and the kinetic properties of the response.
Asunto(s)
Biodiversidad , Fotosíntesis/fisiología , Plantas/metabolismo , Transporte de Electrón , Ambiente , Luz , Complejo de Proteína del Fotosistema II/metabolismoRESUMEN
Contents Summary 105 I. Introduction 105 II. Diversity of molecular mechanisms for regulation of photosynthetic electron transport 106 III. Role of FLVs in the regulation of photosynthesis in eukaryotes 107 IV. Why were FLVs lost in angiosperms? 108 V. Conclusions 108 Acknowledgements 109 References 109 SUMMARY: Photosynthetic electron transport requires continuous modulation to maintain the balance between light availability and metabolic demands. Multiple mechanisms for the regulation of electron transport have been identified and are unevenly distributed among photosynthetic organisms. Flavodiiron proteins (FLVs) influence photosynthetic electron transport by accepting electrons downstream of photosystem I to reduce oxygen to water. FLV activity has been demonstrated in cyanobacteria, green algae and mosses to be important in avoiding photosystem I overreduction upon changes in light intensity. FLV-encoding sequences were nevertheless lost during evolution by angiosperms, suggesting that these plants increased the efficiency of other mechanisms capable of accepting electrons from photosystem I, making the FLV activity for protection from overreduction superfluous or even detrimental for photosynthetic efficiency.
Asunto(s)
Evolución Biológica , Transporte de Electrón , Fotosíntesis/fisiología , Fenómenos Fisiológicos de las Plantas , Proteínas de Plantas/metabolismo , Magnoliopsida/fisiología , Complejo de Proteína del Fotosistema I/metabolismoRESUMEN
Non-photochemical quenching, NPQ, of chlorophyll fluorescence regulates the heat dissipation of chlorophyll excited states and determines the efficiency of the oxygenic photosynthetic systems. NPQ is regulated by a pH-sensing protein, responding to the chloroplast lumen acidification induced by excess light, coupled to an actuator, a chlorophyll/xanthophyll subunit where quenching reactions are catalyzed. In plants, the sensor is PSBS, while the two pigment-binding proteins Lhcb4 (also known as CP29) and LHCII are the actuators. In algae and mosses, stress-related light-harvesting proteins (LHCSR) comprise both functions of sensor and actuator within a single subunit. Here, we report on expressing the lhcsr1 gene from the moss Physcomitrella patens into several Arabidopsis thaliana npq4 mutants lacking the pH sensing PSBS protein essential for NPQ activity. The heterologous protein LHCSR1 accumulates in thylakoids of A. thaliana and NPQ activity can be partially restored. Complementation of double mutants lacking, besides PSBS, specific xanthophylls, allowed analyzing chromophore requirement for LHCSR-dependent quenching activity. We show that the partial recovery of NPQ is mostly due to the lower levels of Zeaxanthin in A. thaliana in comparison to P. patens. Complemented npq2npq4 mutants, lacking besides PSBS, Zeaxanthin Epoxidase, showed an NPQ recovery of up to 70% in comparison to A. thaliana wild type. Furthermore, we show that Lutein is not essential for the folding nor for the quenching activity of LHCSR1. In short, we have developed a system to study the function of LHCSR proteins using heterologous expression in a variety of A. thaliana mutants.
Asunto(s)
Arabidopsis/metabolismo , Bryopsida/genética , Complejos de Proteína Captadores de Luz/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Luz , Complejos de Proteína Captadores de Luz/genética , Mutación , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Procesos Fotoquímicos , Fotosíntesis , Plantas Modificadas Genéticamente , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tilacoides/genética , Tilacoides/metabolismo , Xantófilas/metabolismo , Zeaxantinas/metabolismoRESUMEN
Photosynthetic organisms support cell metabolism by harvesting sunlight and driving the electron transport chain at the level of thylakoid membranes. Excitation energy and electron flow in the photosynthetic apparatus is continuously modulated in response to dynamic environmental conditions. Alternative electron flow around photosystem I plays a seminal role in this regulation contributing to photoprotection by mitigating overreduction of the electron carriers. Different pathways of alternative electron flow coexist in the moss Physcomitrella patens, including cyclic electron flow mediated by the PGRL1/PGR5 complex and pseudo-cyclic electron flow mediated by the flavodiiron proteins FLV. In this work, we generated P. patens plants carrying both pgrl1 and flva knock-out mutations. A comparative analysis of the WT, pgrl1, flva, and pgrl1 flva lines suggests that cyclic and pseudo-cyclic processes have a synergic role in the regulation of photosynthetic electron transport. However, although both contribute to photosystem I protection from overreduction by modulating electron flow following changes in environmental conditions, FLV activity is particularly relevant in the first seconds after a light change whereas PGRL1 has a major role upon sustained strong illumination.
Asunto(s)
Bryopsida/fisiología , Transporte de Electrón/fisiología , Proteínas del Complejo del Centro de Reacción Fotosintética/genética , Complejo de Proteína del Fotosistema I/metabolismo , Bryopsida/genética , Cloroplastos/metabolismo , Transporte de Electrón/genética , Luz , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Mutación , Fotosíntesis/genética , Fotosíntesis/fisiología , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , Complejo de Proteína del Fotosistema I/genética , Plantas Modificadas Genéticamente , Luz Solar , Tilacoides/metabolismoRESUMEN
Photosynthetic organisms support cell metabolism by harvesting sunlight to fuel the photosynthetic electron transport. The flow of excitation energy and electrons in the photosynthetic apparatus needs to be continuously modulated to respond to dynamics of environmental conditions, and Flavodiiron (FLV) proteins are seminal components of this regulatory machinery in cyanobacteria. FLVs were lost during evolution by flowering plants, but are still present in nonvascular plants such as Physcomitrella patens We generated P. patens mutants depleted in FLV proteins, showing their function as an electron sink downstream of photosystem I for the first seconds after a change in light intensity. flv knock-out plants showed impaired growth and photosystem I photoinhibition when exposed to fluctuating light, demonstrating FLV's biological role as a safety valve from excess electrons on illumination changes. The lack of FLVs was partially compensated for by an increased cyclic electron transport, suggesting that in flowering plants, the FLV's role was taken by other alternative electron routes.
Asunto(s)
Bryopsida/genética , Evolución Molecular , Fotosíntesis/genética , Proteínas de Plantas/genética , Bryopsida/crecimiento & desarrollo , Transporte de Electrón/genética , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Oxígeno/metabolismo , Luz SolarRESUMEN
The adaptation to dehydration and rehydration cycles represents a key step in the evolution of photosynthetic organisms and requires the development of mechanisms by which to sense external stimuli and translate them into signaling components. In this study, we used genetically encoded fluorescent sensors to detect specific transient increases in the Ca2+ concentration in the moss Physcomitrella patens upon dehydration and rehydration treatment. Observation of the entire plant in a single time-series acquisition revealed that various cell types exhibited different sensitivities to osmotic stress and that Ca2+ waves originated from the basal part of the gametophore and were directionally propagated towards the top of the plant. Under similar conditions, the vascular plant Arabidopsis thaliana exhibited Ca2+ waves that propagated at a higher speed than those of P. patens. Our results suggest that systemic Ca2+ propagation occurs in plants even in the absence of vascular tissue, even though the rates can be different.
Asunto(s)
Bryopsida/metabolismo , Señalización del Calcio , Arabidopsis/metabolismo , Bryopsida/citología , Bryopsida/fisiología , Calcio/análisis , Calcio/metabolismo , Calmodulina/metabolismo , Deshidratación , Transferencia Resonante de Energía de Fluorescencia , Proteínas Luminiscentes/metabolismo , Imagen Molecular/métodos , Presión Osmótica , Células Vegetales/metabolismo , Plantas Modificadas Genéticamente , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Two LHC-like proteins, Photosystem II Subunit S (PSBS) and Light-Harvesting Complex Stress-Related (LHCSR), are essential for triggering excess energy dissipation in chloroplasts of vascular plants and green algae, respectively. The mechanism of quenching was studied in Physcomitrella patens, an early divergent streptophyta (including green algae and land plants) in which both proteins are active. PSBS was localized in grana together with photosystem II (PSII), but LHCSR was located mainly in stroma-exposed membranes together with photosystem I (PSI), and its distribution did not change upon high-light treatment. The quenched conformation can be preserved by rapidly freezing the high-light-treated tissues in liquid nitrogen. When using green fluorescent protein as an internal standard, 77K fluorescence emission spectra on isolated chloroplasts allowed for independent assessment of PSI and PSII fluorescence yield. Results showed that both photosystems underwent quenching upon high-light treatment in the wild type in contrast to mutants depleted of LHCSR, which lacked PSI quenching. Due to the contribution of LHCII, P. patens had a PSI antenna size twice as large with respect to higher plants. Thus, LHCII, which is highly abundant in stroma membranes, appears to be the target of quenching by LHCSR.
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Bryopsida/metabolismo , Complejos de Proteína Captadores de Luz/metabolismo , Complejo de Proteína del Fotosistema I/metabolismo , Complejo de Proteína del Fotosistema II/metabolismo , Proteínas de Plantas/metabolismo , Estrés Fisiológico , Bryopsida/efectos de los fármacos , Bryopsida/efectos de la radiación , Bryopsida/ultraestructura , Catálisis/efectos de los fármacos , Clorofila/metabolismo , Cloroplastos/efectos de los fármacos , Cloroplastos/metabolismo , Cloroplastos/ultraestructura , Digitonina/farmacología , Glucósidos/farmacología , Luz , Microdominios de Membrana/efectos de los fármacos , Microdominios de Membrana/metabolismo , Microdominios de Membrana/efectos de la radiación , Procesos Fotoquímicos/efectos de los fármacos , Espectrometría de Fluorescencia , Estrés Fisiológico/efectos de los fármacos , Estrés Fisiológico/efectos de la radiación , Termodinámica , Tilacoides/metabolismo , Tilacoides/efectos de la radiación , Tilacoides/ultraestructuraRESUMEN
Photo-reduction of O2 to water mediated by flavodiiron proteins (FDPs) represents a safety valve for the photosynthetic electron transport chain in fluctuating light. So far, the FDP-mediated O2 photo-reduction has been evidenced only in cyanobacteria and the moss Physcomitrella; however, a recent phylogenetic analysis of transcriptomes of photosynthetic organisms has also revealed the presence of FDP genes in several nonflowering plant groups. What remains to be clarified is whether the FDP-dependent O2 photo-reduction is actually operational in these organisms. We have established a simple method for the monitoring of FDP-mediated O2 photo-reduction, based on the measurement of redox kinetics of P700 (the electron donor of photosystem I) upon dark-to-light transition. The O2 photo-reduction is manifested as a fast re-oxidation of P700. The validity of the method was verified by experiments with transgenic organisms, namely FDP knock-out mutants of Synechocystis and Physcomitrella and transgenic Arabidopsis plants expressing FDPs from Physcomitrella. We observed the fast P700 re-oxidation in representatives of all green plant groups excluding angiosperms. Our results provide strong evidence that the FDP-mediated O2 photo-reduction is functional in all nonflowering green plant groups. This finding suggests a major change in the strategy of photosynthetic regulation during the evolution of angiosperms.
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Cianobacterias/metabolismo , Cycadopsida/metabolismo , Flavoproteínas/metabolismo , Cianobacterias/efectos de la radiación , Cycadopsida/efectos de la radiación , Transporte de Electrón , Cinética , Luz , Oxidación-Reducción , Fotosíntesis/efectos de la radiación , FilogeniaRESUMEN
Photosystem I (PSI) is a pigment protein complex catalyzing the light-driven electron transport from plastocyanin to ferredoxin in oxygenic photosynthetic organisms. Several PSI subunits are highly conserved in cyanobacteria, algae and plants, whereas others are distributed differentially in the various organisms. Here we characterized the structural and functional properties of PSI purified from the heterokont alga Nannochloropsis gaditana, showing that it is organized as a supercomplex including a core complex and an outer antenna, as in plants and other eukaryotic algae. Differently from all known organisms, the N. gaditana PSI supercomplex contains five peripheral antenna proteins, identified by proteome analysis as type-R light-harvesting complexes (LHCr4-8). Two antenna subunits are bound in a conserved position, as in PSI in plants, whereas three additional antennae are associated with the core on the other side. This peculiar antenna association correlates with the presence of PsaF/J and the absence of PsaH, G and K in the N. gaditana genome and proteome. Excitation energy transfer in the supercomplex is highly efficient, leading to a very high trapping efficiency as observed in all other PSI eukaryotes, showing that although the supramolecular organization of PSI changed during evolution, fundamental functional properties such as trapping efficiency were maintained.