RESUMEN
DNA is organized into chromatin-like structures that support the maintenance and regulation of genomes. A unique and poorly understood form of DNA organization exists in chloroplasts, which are organelles of endosymbiotic origin responsible for photosynthesis. Chloroplast genomes, together with associated proteins, form membrane-less structures known as nucleoids. The internal arrangement of the nucleoid, molecular mechanisms of DNA organization, and connections between nucleoid structure and gene expression remain mostly unknown. We show that Arabidopsis thaliana chloroplast nucleoids have a unique sequence-specific organization driven by DNA binding to the thylakoid membranes. DNA associated with the membranes has high protein occupancy, has reduced DNA accessibility, and is highly transcribed. In contrast, genes with low levels of transcription are further away from the membranes, have lower protein occupancy, and have higher DNA accessibility. Membrane association of active genes relies on the pattern of transcription and proper chloroplast development. We propose a speculative model that transcription organizes the chloroplast nucleoid into a transcriptionally active membrane-associated core and a less active periphery.
Asunto(s)
Arabidopsis , Cloroplastos , Tilacoides , Arabidopsis/genética , Arabidopsis/metabolismo , Cloroplastos/genética , Cloroplastos/metabolismo , Tilacoides/metabolismo , Tilacoides/genética , Tilacoides/ultraestructura , Regulación de la Expresión Génica de las Plantas , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Transcripción Genética , ADN de Cloroplastos/genética , ADN de Cloroplastos/metabolismoRESUMEN
Energy status and nutrients regulate photosynthetic protein expression. The unicellular green alga Chromochloris zofingiensis switches off photosynthesis in the presence of exogenous glucose (+Glc) in a process that depends on hexokinase (HXK1). Here, we show that this response requires that cells lack sufficient iron (-Fe). Cells grown in -Fe+Glc accumulate triacylglycerol (TAG) while losing photosynthesis and thylakoid membranes. However, cells with an iron supplement (+Fe+Glc) maintain photosynthesis and thylakoids while still accumulating TAG. Proteomic analysis shows that known photosynthetic proteins are most depleted in heterotrophy, alongside hundreds of uncharacterized, conserved proteins. Photosynthesis repression is associated with enzyme and transporter regulation that redirects iron resources to (a) respiratory instead of photosynthetic complexes and (b) a ferredoxin-dependent desaturase pathway supporting TAG accumulation rather than thylakoid lipid synthesis. Combining insights from diverse organisms from green algae to vascular plants, we show how iron and trophic constraints on metabolism aid gene discovery for photosynthesis and biofuel production.
Asunto(s)
Chlorophyta , Glucosa , Hierro , Metabolismo de los Lípidos , Fotosíntesis , Triglicéridos , Hierro/metabolismo , Glucosa/metabolismo , Triglicéridos/metabolismo , Chlorophyta/metabolismo , Chlorophyta/genética , Tilacoides/metabolismo , Proteómica , Hexoquinasa/metabolismo , Hexoquinasa/genética , Chlorophyceae/metabolismo , Chlorophyceae/genéticaRESUMEN
Photosystem II (PSII) catalyzes water oxidation and plastoquinone reduction by utilizing light energy. It is highly susceptible to photodamage under high-light conditions and the damaged PSII needs to be restored through a process known as the PSII repair cycle. The detailed molecular mechanism underlying the PSII repair process remains mostly elusive. Here, we report biochemical and structural features of a PSII-repair intermediate complex, likely arrested at an early stage of the PSII repair process in the green alga Chlamydomonas reinhardtii. The complex contains three protein factors associated with a damaged PSII core, namely Thylakoid Enriched Factor 14 (TEF14), Photosystem II Repair Factor 1 (PRF1), and Photosystem II Repair Factor 2 (PRF2). TEF14, PRF1 and PRF2 may facilitate the release of the manganese-stabilizing protein PsbO, disassembly of peripheral light-harvesting complexes from PSII and blockage of the QB site, respectively. Moreover, an α-tocopherol quinone molecule is located adjacent to the heme group of cytochrome b559, potentially fulfilling a photoprotective role by preventing the generation of reactive oxygen species.
Asunto(s)
Chlamydomonas reinhardtii , Complejo de Proteína del Fotosistema II , Complejo de Proteína del Fotosistema II/metabolismo , Chlamydomonas reinhardtii/metabolismo , Chlamydomonas reinhardtii/genética , Tilacoides/metabolismo , Complejos de Proteína Captadores de Luz/metabolismo , Complejos de Proteína Captadores de Luz/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Grupo Citocromo b/metabolismo , Grupo Citocromo b/genética , Oxidación-Reducción , Especies Reactivas de Oxígeno/metabolismo , LuzRESUMEN
Photosystem II (PSII) functions were investigated in basil (Ocimum basilicum L.) plants sprayed with 1 mM salicylic acid (SA) under non-stress (NS) or mild drought-stress (MiDS) conditions. Under MiDS, SA-sprayed leaves retained significantly higher (+36%) chlorophyll content compared to NS, SA-sprayed leaves. PSII efficiency in SA-sprayed leaves under NS conditions, evaluated at both low light (LL, 200 µmol photons m-2 s-1) and high light (HL, 900 µmol photons m-2 s-1), increased significantly with a parallel significant decrease in the excitation pressure at PSII (1-qL) and the excess excitation energy (EXC). This enhancement of PSII efficiency under NS conditions was induced by the mechanism of non-photochemical quenching (NPQ) that reduced singlet oxygen (1O2) production, as indicated by the reduced quantum yield of non-regulated energy loss in PSII (ΦNO). Under MiDS, the thylakoid structure of water-sprayed leaves appeared slightly dilated, and the efficiency of PSII declined, compared to NS conditions. In contrast, the thylakoid structure of SA-sprayed leaves did not change under MiDS, while PSII functionality was retained, similar to NS plants at HL. This was due to the photoprotective heat dissipation by NPQ, which was sufficient to retain the same percentage of open PSII reaction centers (qp), as in NS conditions and HL. We suggest that the redox status of the plastoquinone pool (qp) under MiDS and HL initiated the acclimation response to MiDS in SA-sprayed leaves, which retained the same electron transport rate (ETR) with control plants. Foliar spray of SA could be considered as a method to improve PSII efficiency in basil plants under NS conditions, at both LL and HL, while under MiDS and HL conditions, basil plants could retain PSII efficiency similar to control plants.
Asunto(s)
Sequías , Ocimum basilicum , Complejo de Proteína del Fotosistema II , Hojas de la Planta , Ácido Salicílico , Estrés Fisiológico , Complejo de Proteína del Fotosistema II/metabolismo , Ácido Salicílico/farmacología , Ácido Salicílico/metabolismo , Ocimum basilicum/metabolismo , Ocimum basilicum/efectos de los fármacos , Hojas de la Planta/metabolismo , Hojas de la Planta/efectos de los fármacos , Clorofila/metabolismo , Fotosíntesis/efectos de los fármacos , Tilacoides/metabolismo , Tilacoides/efectos de los fármacos , LuzRESUMEN
Chlorophyll (Chl) plays a crucial role in photosynthesis, functioning as a photosensitizer. As an integral component of this process, energy absorbed by this pigment is partly emitted as red fluorescence. This signal can be readily imaged by fluorescence microscopy and provides a visualization of photosynthetic activity. However, due to limited resolution, signals cannot be assigned to specific subcellular/organellar membrane structures. By correlating fluorescence micrographs with transmission electron microscopy, researchers can identify sub-cellular compartments and membranes, enabling the monitoring of Chl distribution within thylakoid membrane substructures in cyanobacteria, algae, and higher plant single cells. Here, we describe a simple and effective protocol for correlative light-electron microscopy (CLEM) based on the autofluorescence of Chl and demonstrate its application to selected photosynthetic model organisms. Our findings illustrate the potential of this technique to identify areas of high Chl concentration and photochemical activity, such as grana regions in vascular plants, by mapping stacked thylakoids.
Asunto(s)
Clorofila , Tilacoides , Tilacoides/metabolismo , Tilacoides/ultraestructura , Clorofila/metabolismo , Fotosíntesis/fisiología , Microscopía Fluorescente/métodos , Microscopía Electrónica de Transmisión/métodosRESUMEN
BACKGROUND: The phosphorylation of the Light-Harvesting Complex of photosystem II (LHCII) driven by STATE TRANSITION 7 (STN7) kinase is a part of one of the crucial regulatory mechanisms of photosynthetic light reactions operating in fluctuating environmental conditions, light in particular. There are evidenced that STN7 can also be activated without light as well as in dark-chilling conditions. However, the biochemical mechanism standing behind this complex metabolic pathway has not been deciphered yet. RESULTS: In this work, we showed that dark-chilling induces light-independent LHCII phosphorylation in runner bean (Phaseolus coccineus L.). In dark-chilling conditions, we registered an increased reduction of the PQ pool which led to activation of STN7 kinase, subsequent LHCII phosphorylation, and possible LHCII relocation inside the thylakoid membrane. We also presented the formation of a complex composed of phosphorylated LHCII and photosystem I typically formed upon light-induced phosphorylation. Moreover, we indicated that the observed steps were preceded by the activation of the oxidative pentose phosphate pathway (OPPP) enzymes and starch accumulation. CONCLUSIONS: Our results suggest a direct connection between photosynthetic complexes reorganization and dark-chilling-induced activation of the thioredoxin system. The proposed possible pathway starts from the activation of OPPP enzymes and further NADPH-dependent thioredoxin reductase C (NTRC) activation. In the next steps, NTRC simultaneously activates ADP-glucose pyrophosphorylase and thylakoid membrane-located NAD(P)H dehydrogenase-like complex. These results in starch synthesis and electron transfer to the plastoquinone (PQ) pool, respectively. Reduced PQ pool activates STN7 kinase which phosphorylates LHCII. In this work, we present a new perspective on the mechanisms involving photosynthetic complexes while efficiently operating in the darkness. Although we describe the studied pathway in detail, taking into account also the time course of the following steps, the biological significance of this phenomenon remains puzzling.
Asunto(s)
Luz , Phaseolus , Phaseolus/fisiología , Phaseolus/metabolismo , Phaseolus/enzimología , Fosforilación , Tilacoides/metabolismo , Complejo de Proteína del Fotosistema I/metabolismo , Frío , Complejos de Proteína Captadores de Luz/metabolismo , Complejo de Proteína del Fotosistema II/metabolismo , Proteínas de Plantas/metabolismo , Almidón/metabolismo , Vía de Pentosa Fosfato/fisiología , Activación Enzimática , Fotosíntesis/fisiología , Estrés Fisiológico , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
The ability to harvest light effectively in a changing environment is necessary to ensure efficient photosynthesis and crop growth. One mechanism, known as qE, protects photosystem II (PSII) and regulates electron transfer through the harmless dissipation of excess absorbed photons as heat. This process involves reversible clustering of the major light-harvesting complexes of PSII (LHCII) in the thylakoid membrane and relies upon the ΔpH gradient and the allosteric modulator protein PsbS. To date, the exact role of PsbS in the qE mechanism has remained elusive. Here, we show that PsbS induces hydrophobic mismatch in the thylakoid membrane through dynamic rearrangement of lipids around LHCII leading to observed membrane thinning. We found that upon illumination, the thylakoid membrane reversibly shrinks from around 4.3 to 3.2 nm, without PsbS, this response is eliminated. Furthermore, we show that the lipid digalactosyldiacylglycerol (DGDG) is repelled from the LHCII-PsbS complex due to an increase in both the pKa of lumenal residues and in the dipole moment of LHCII, which allows for further conformational change and clustering in the membrane. Our results suggest a mechanistic role for PsbS as a facilitator of a hydrophobic mismatch-mediated phase transition between LHCII-PsbS and its environment. This could act as the driving force to sort LHCII into photoprotective nanodomains in the thylakoid membrane. This work shows an example of the key role of the hydrophobic mismatch process in regulating membrane protein function in plants.
Asunto(s)
Interacciones Hidrofóbicas e Hidrofílicas , Complejos de Proteína Captadores de Luz , Fotosíntesis , Complejo de Proteína del Fotosistema II , Tilacoides , Tilacoides/metabolismo , Tilacoides/química , Complejos de Proteína Captadores de Luz/metabolismo , Complejos de Proteína Captadores de Luz/química , Complejo de Proteína del Fotosistema II/metabolismo , Complejo de Proteína del Fotosistema II/química , Galactolípidos/metabolismo , Galactolípidos/química , LuzRESUMEN
Photosynthetic organisms have evolved an essential energy-dependent quenching (qE) mechanism to avoid any lethal damages caused by high light. While the triggering mechanism of qE has been well addressed, candidates for quenchers are often debated. This lack of understanding is because of the tremendous difficulty in measuring intact cells using transient absorption techniques. Here, we have conducted femtosecond pump-probe measurements to characterize this photophysical reaction using micro-sized cell fractions of the green alga Chlamydomonas reinhardtii that retain physiological qE function. Combined with kinetic modeling, we have demonstrated the presence of an ultrafast excitation energy transfer (EET) pathway from Chlorophyll a (Chl a) Qy to a carotenoid (car) S1 state, therefore proposing that this carotenoid, likely lutein1, is the quencher. This work has provided an easy-to-prepare qE active thylakoid membrane system for advanced spectroscopic studies and demonstrated that the energy dissipation pathway of qE is evolutionarily conserved from green algae to land plants.
Asunto(s)
Carotenoides , Chlamydomonas reinhardtii , Transferencia de Energía , Chlamydomonas reinhardtii/metabolismo , Carotenoides/metabolismo , Carotenoides/química , Tilacoides/metabolismo , Fotosíntesis , Complejos de Proteína Captadores de Luz/metabolismo , Complejos de Proteína Captadores de Luz/química , Complejos de Proteína Captadores de Luz/genética , Clorofila A/metabolismo , Clorofila A/química , Luz , Cinética , Clorofila/metabolismo , Chlamydomonas/metabolismoRESUMEN
It has been thoroughly documented, by using 31P-NMR spectroscopy, that plant thylakoid membranes (TMs), in addition to the bilayer (or lamellar, L) phase, contain at least two isotropic (I) lipid phases and an inverted hexagonal (HII) phase. However, our knowledge concerning the structural and functional roles of the non-bilayer phases is still rudimentary. The objective of the present study is to elucidate the origin of I phases which have been hypothesized to arise, in part, from the fusion of TMs (Garab et al. 2022 Progr Lipid Res 101,163). We take advantage of the selectivity of wheat germ lipase (WGL) in eliminating the I phases of TMs (Dlouhý et al. 2022 Cells 11: 2681), and the tendency of the so-called BBY particles, stacked photosystem II (PSII) enriched membrane pairs of 300-500 nm in diameter, to form large laterally fused sheets (Dunahay et al. 1984 BBA 764: 179). Our 31P-NMR spectroscopy data show that BBY membranes contain L and I phases. Similar to TMs, WGL selectively eliminated the I phases, which at the same time exerted no effect on the molecular organization and functional activity of PSII membranes. As revealed by sucrose-density centrifugation, magnetic linear dichroism spectroscopy and scanning electron microscopy, WGL disassembled the large laterally fused sheets. These data provide direct experimental evidence on the involvement of I phase(s) in the fusion of stacked PSII membrane pairs, and strongly suggest the role of non-bilayer lipids in the self-assembly of the TM system.
Asunto(s)
Complejo de Proteína del Fotosistema II , Tilacoides , Complejo de Proteína del Fotosistema II/metabolismo , Tilacoides/metabolismo , Espectroscopía de Resonancia Magnética , Lípidos/química , Fusión de Membrana/fisiologíaRESUMEN
A central goal of photoprotective energy dissipation processes is the regulation of singlet oxygen (1O2*) and reactive oxygen species in the photosynthetic apparatus. Despite the involvement of 1O2* in photodamage and cell signaling, few studies directly correlate 1O2* formation to nonphotochemical quenching (NPQ) or lack thereof. Here, we combine spin-trapping electron paramagnetic resonance (EPR) and time-resolved fluorescence spectroscopies to track in real time the involvement of 1O2* during photoprotection in plant thylakoid membranes. The EPR spin-trapping method for detection of 1O2* was first optimized for photosensitization in dye-based chemical systems and then used to establish methods for monitoring the temporal dynamics of 1O2* in chlorophyll-containing photosynthetic membranes. We find that the apparent 1O2* concentration in membranes changes throughout a 1 h period of continuous illumination. During an initial response to high light intensity, the concentration of 1O2* decreased in parallel with a decrease in the chlorophyll fluorescence lifetime via NPQ. Treatment of membranes with nigericin, an uncoupler of the transmembrane proton gradient, delayed the activation of NPQ and the associated quenching of 1O2* during high light. Upon saturation of NPQ, the concentration of 1O2* increased in both untreated and nigericin-treated membranes, reflecting the utility of excess energy dissipation in mitigating photooxidative stress in the short term (i.e., the initial â¼10 min of high light).
Asunto(s)
Fotosíntesis , Oxígeno Singlete , Tilacoides , Espectroscopía de Resonancia por Spin del Electrón/métodos , Oxígeno Singlete/metabolismo , Oxígeno Singlete/química , Tilacoides/metabolismo , Tilacoides/química , Detección de Spin/métodos , Clorofila/metabolismo , Clorofila/química , Spinacia oleracea/metabolismo , Spinacia oleracea/química , LuzRESUMEN
The determination of physiological tolerance ranges of photosynthetic species and of the biochemical mechanisms underneath are fundamental to identify target processes and metabolites that will inspire enhanced plant management and production for the future. In this context, the terrestrial green algae within the genus Prasiola represent ideal models due to their success in harsh environments (polar tundras) and their extraordinary ecological plasticity. Here we focus on the outstanding Prasiola antarctica and compare two natural populations living in very contrasting microenvironments in Antarctica: the dry sandy substrate of a beach and the rocky bed of an ephemeral freshwater stream. Specifically, we assessed their photosynthetic performance at different temperatures, reporting for the first time gnsd values in algae and changes in thylakoid metabolites in response to extreme desiccation. Stream population showed lower α-tocopherol content and thicker cell walls and thus, lower gnsd and photosynthesis. Both populations had high temperatures for optimal photosynthesis (around +20°C) and strong constitutive tolerance to freezing and desiccation. This tolerance seems to be related to the high constitutive levels of xanthophylls and of the cylindrical lipids di- and tri-galactosyldiacylglycerol in thylakoids, very likely related to the effective protection and stability of membranes. Overall, P. antarctica shows a complex battery of constitutive and plastic protective mechanisms that enable it to thrive under harsh conditions and to acclimate to very contrasting microenvironments, respectively. Some of these anatomical and biochemical adaptations may partially limit photosynthesis, but this has a great potential to rise in a context of increasing temperature.
Asunto(s)
Fotosíntesis , Tilacoides , Tilacoides/metabolismo , Regiones Antárticas , Fotosíntesis/fisiología , Chlorophyceae/fisiología , Chlorophyceae/metabolismo , Xantófilas/metabolismo , Adaptación Fisiológica/fisiología , Desecación , AclimataciónRESUMEN
The biological role of lipids goes far beyond the formation of a structural membrane bilayer platform for membrane proteins and controlling fluxes across the membranes. For example, in photosynthetic thylakoid membranes, lipids occupy well-defined binding niches within protein complexes and determine the structural organization of membrane proteins and their function by controlling generic physicochemical membrane properties. In this chapter, two-dimensional thin-layer chromatography (2D TLC) and gas chromatography (GC) techniques are presented for quantitative analysis of lipid classes and fatty acids in thylakoid membranes. In addition, lipid extraction methods from isolated thylakoid membranes and leaves are described together with a procedure for the derivatization of fatty acids to fatty acid methyl esters (FAME) that is required for GC analysis.
Asunto(s)
Ácidos Grasos , Fotosíntesis , Tilacoides , Tilacoides/metabolismo , Cromatografía en Capa Delgada/métodos , Cromatografía de Gases/métodos , Ácidos Grasos/metabolismo , Ácidos Grasos/química , Lípidos de la Membrana/metabolismo , Lípidos de la Membrana/química , Hojas de la Planta/metabolismo , Hojas de la Planta/química , Lípidos/química , Lípidos/aislamiento & purificación , Lípidos/análisisRESUMEN
The chloroplast thylakoid membrane is composed of membrane lipids and photosynthetic protein complexes, and the orchestration of thylakoid lipid biosynthesis and photosynthesis-associated protein accumulation is considered important for thylakoid development. Galactolipids consist of â¼80% of the thylakoid lipids, and their biosynthesis is fundamental for chloroplast development. We previously reported that the suppression of galactolipid biosynthesis decreased the expression of photosynthesis-associated nuclear-encoded genes (PhAPGs) and photosynthesis-associated plastid-encoded genes (PhAPGs). However, the mechanism for coordinative regulation between galactolipid biosynthesis in plastids and the expression of PhANGs and PhAPGs remains largely unknown. To elucidate this mechanism, we investigated the gene expression patterns in galactolipid-deficient Arabidopsis seedlings during the de-etiolation process. We found that galactolipids are crucial for inducing both the transcript accumulation of PhANGs and PhAPGs and the accumulation of plastid-encoded photosynthesis-associated proteins in developing chloroplasts. Genetic analysis indicates the contribution of the GENOMES UNCOUPLED1 (GUN1)-mediated plastid-to-nucleus signaling pathway to PhANG regulation in response to galactolipid levels. Previous studies suggested that the accumulation of GUN1 reflects the state of protein homeostasis in plastids and alters the PhANG expression level. Thus, we propose a model that galactolipid biosynthesis determines the protein homeostasis in plastids in the initial phase of de-etiolation and optimizes GUN1-dependent signaling to regulate the PhANG expression. This mechanism might contribute to orchestrating the biosynthesis of lipids and proteins for the biogenesis of functional chloroplasts in plants.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Cloroplastos , Galactolípidos , Regulación de la Expresión Génica de las Plantas , Fotosíntesis , Galactolípidos/metabolismo , Galactolípidos/biosíntesis , Fotosíntesis/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Cloroplastos/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Tilacoides/metabolismo , Plantones/genética , Plantones/metabolismo , Proteínas de Unión al ADNRESUMEN
In chloroplasts, insertion of proteins with multiple transmembrane domains (TMDs) into thylakoid membranes usually occurs in a co-translational manner. Here, we have characterized a thylakoid protein designated FPB1 (Facilitator of PsbB biogenesis1) which together with a previously reported factor PAM68 (Photosynthesis Affected Mutant68) is involved in assisting the biogenesis of CP47, a subunit of the Photosystem II (PSII) core. Analysis by ribosome profiling reveals increased ribosome stalling when the last TMD segment of CP47 emerges from the ribosomal tunnel in fpb1 and pam68. FPB1 interacts with PAM68 and both proteins coimmunoprecipitate with SecY/E and Alb3 as well as with some ribosomal components. Thus, our data indicate that, in coordination with the SecY/E translocon and the Alb3 integrase, FPB1 synergistically cooperates with PAM68 to facilitate the co-translational integration of the last two CP47 TMDs and the large loop between them into thylakoids and the PSII core complex.
Asunto(s)
Complejo de Proteína del Fotosistema II , Tilacoides , Cloroplastos/metabolismo , Complejo de Proteína del Fotosistema II/genética , Complejo de Proteína del Fotosistema II/metabolismo , Ribosomas/metabolismo , Tilacoides/metabolismoRESUMEN
Chlorophyll fluorescence is a ubiquitous tool in basic and applied plant science research. Various standard commercial instruments are available for characterization of photosynthetic material like leaves or microalgae, most of which integrate the overall fluorescence signals above a certain cut-off wavelength. However, wavelength-resolved (fluorescence signals appearing at different wavelengths having different time dependent decay) signals contain vast information required to decompose complex signals and processes into their underlying components that can untangle the photo-physiological process of photosynthesis. Hence, to address this we describe an advanced chlorophyll fluorescence spectrometer - ChloroSpec - allowing three-dimensional simultaneous detection of fluorescence intensities at different wavelengths in a time-resolved manner. We demonstrate for a variety of typical examples that most of the generally used fluorescence parameters are strongly wavelength dependent. This indicates a pronounced heterogeneity and a highly dynamic nature of the thylakoid and the photosynthetic apparatus under actinic illumination. Furthermore, we provide examples of advanced global analysis procedures integrating this three-dimensional signal and relevant information extracted from them that relate to the physiological properties of the organism. This conveniently obtained broad range of data can make ChloroSpec a new standard tool in photosynthesis research.
Asunto(s)
Clorofila , Fotosíntesis , Espectrometría de Fluorescencia , Clorofila/metabolismo , Espectrometría de Fluorescencia/métodos , Espectrometría de Fluorescencia/instrumentación , Fotosíntesis/fisiología , Hojas de la Planta/metabolismo , Fluorescencia , Tilacoides/metabolismoRESUMEN
Cyanobacteria inhabit areas with a broad range of light, temperature and nutrient conditions. The robustness of cyanobacterial cells, which can survive under different conditions, may depend on the resilience of photosynthetic activity. Cyanothece sp. PCC 8801 (Cyanothece), a freshwater cyanobacterium isolated from a Taiwanese rice field, had a higher repair activity of photodamaged photosystem II (PSII) under intense light than Synechocystis sp. PCC 6803 (Synechocystis), another freshwater cyanobacterium. Cyanothece contains myristic acid (14:0) as the major fatty acid at the sn-2 position of the glycerolipids. To investigate the role of 14:0 in the repair of photodamaged PSII, we used a Synechocystis transformant expressing a T-1274 encoding a lysophosphatidic acid acyltransferase (LPAAT) from Cyanothece. The wild-type and transformant cells contained 0.2 and 20.1 mol% of 14:0 in glycerolipids, respectively. The higher content of 14:0 in the transformants increased the fluidity of the thylakoid membrane. In the transformants, PSII repair was accelerated due to an enhancement in the de novo synthesis of D1 protein, and the production of singlet oxygen (1O2), which inhibited protein synthesis, was suppressed. The high content of 14:0 increased transfer of light energy received by phycobilisomes to PSI and CP47 in PSII and the content of carotenoids. These results indicated that an increase in 14:0 reduced 1O2 formation and enhanced PSII repair. The higher content of 14:0 in the glycerolipids may be required as a survival strategy for Cyanothece inhabiting a rice field under direct sunlight.
Asunto(s)
Luz , Ácido Mirístico , Complejo de Proteína del Fotosistema II , Synechocystis , Tilacoides , Complejo de Proteína del Fotosistema II/metabolismo , Synechocystis/metabolismo , Synechocystis/genética , Ácido Mirístico/metabolismo , Tilacoides/metabolismo , Fotosíntesis , Aciltransferasas/metabolismo , Aciltransferasas/genética , Oxígeno Singlete/metabolismoRESUMEN
Plant photosynthesis contains two functional modules, the light-driven reactions in the thylakoid membrane and the carbon-fixing reactions in the chloroplast stroma. In nature, light availability for photosynthesis often undergoes massive and rapid fluctuations. Efficient and productive use of such variable light supply requires an instant crosstalk and rapid synchronization of both functional modules. Here, we show that this communication involves the stromal exposed C-terminus of the thylakoid K+-exchange antiporter KEA3, which regulates the ΔpH across the thylakoid membrane and therefore pH-dependent photoprotection. By combining in silico, in vitro, and in vivo approaches, we demonstrate that the KEA3 C-terminus senses the energy state of the chloroplast in a pH-dependent manner and regulates transport activity in response. Together our data pinpoint a regulatory feedback loop by which the stromal energy state orchestrates light capture and photoprotection via multi-level regulation of KEA3.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Tilacoides/metabolismo , Protones , Antiportadores/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Fotosíntesis/fisiología , Cloroplastos/metabolismo , LuzRESUMEN
The emergence of thylakoid membranes in cyanobacteria is a key event in the evolution of all oxygenic photosynthetic cells, from prokaryotes to eukaryotes. Recent analyses show that they could originate from a unique lipid phase transition rather than from a supposed vesicular budding mechanism. Emergence of thylakoids coincided with the great oxygenation event, more than two billion years ago. The acquisition of semi-autonomous organelles, such as the mitochondrion, the chloroplast, and, more recently, the chromatophore, is a critical step in the evolution of eukaryotes. They resulted from primary endosymbiotic events that seem to share general features, i.e., an acquisition of a bacterium/cyanobacteria likely via a phagocytic membrane, a genome reduction coinciding with an escape of genes from the organelle to the nucleus, and, finally, the appearance of an active system translocating nuclear-encoded proteins back to the organelles. An intense mobilization of foreign genes of bacterial origin, via horizontal gene transfers, plays a critical role. Some third partners, like Chlamydia, might have facilitated the transition from cyanobacteria to the early chloroplast. This chapter further details our current understanding of primary endosymbiosis, focusing on primary chloroplasts, thought to have appeared over a billion years ago, and the chromatophore, which appeared around a hundred years ago.
Asunto(s)
Cromatóforos , Cianobacterias , Tilacoides/metabolismo , Cloroplastos/genética , Cloroplastos/metabolismo , Fotosíntesis/genética , Cianobacterias/genética , Cianobacterias/metabolismo , Eucariontes , Simbiosis/genéticaRESUMEN
Plant cell chloroplasts are bounded by a two-membrane envelope. Their photosynthetic function is based on the development of an operational large internal membrane network, called the thylakoids, and on enzymatic processes present in the chloroplast matrix, called the stroma. Thylakoid membranes are distinct from the chloroplast envelope, and their biogenesis is dependent on biosynthetic and transport activities specific of the chloroplast envelope. Starting with the isolation of intact chloroplasts, the method presents the separation by differential centrifugation of the three compartments. A protocol is detailed for leaves of spinach, Arabidopsis or pea.