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1.
Proc Natl Acad Sci U S A ; 121(28): e2309244121, 2024 Jul 09.
Artículo en Inglés | MEDLINE | ID: mdl-38968115

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/metabolismo
2.
Biochem J ; 479(13): 1487-1503, 2022 07 15.
Artículo en Inglés | MEDLINE | ID: mdl-35726684

RESUMEN

In oxygenic photosynthesis, the cytochrome b6f (cytb6f) complex links the linear electron transfer (LET) reactions occurring at photosystems I and II and generates a transmembrane proton gradient via the Q-cycle. In addition to this central role in LET, cytb6f also participates in a range of processes including cyclic electron transfer (CET), state transitions and photosynthetic control. Many of the regulatory roles of cytb6f are facilitated by auxiliary proteins that differ depending upon the species, yet because of their weak and transient nature the structural details of these interactions remain unknown. An apparent key player in the regulatory balance between LET and CET in cyanobacteria is PetP, a ∼10 kDa protein that is also found in red algae but not in green algae and plants. Here, we used cryogenic electron microscopy to determine the structure of the Synechocystis sp. PCC 6803 cytb6f complex in the presence and absence of PetP. Our structures show that PetP interacts with the cytoplasmic side of cytb6f, displacing the C-terminus of the PetG subunit and shielding the C-terminus of cytochrome b6, which binds the heme cn cofactor that is suggested to mediate CET. The structures also highlight key differences in the mode of plastoquinone binding between cyanobacterial and plant cytb6f complexes, which we suggest may reflect the unique combination of photosynthetic and respiratory electron transfer in cyanobacterial thylakoid membranes. The structure of cytb6f from a model cyanobacterial species amenable to genetic engineering will enhance future site-directed mutagenesis studies of structure-function relationships in this crucial ET complex.


Asunto(s)
Complejo de Citocromo b6f , Synechocystis , Microscopía por Crioelectrón , Complejo de Citocromo b6f/química , Complejo de Citocromo b6f/metabolismo , Complejo de Citocromo b6f/fisiología , Transporte de Electrón/fisiología , Fotosíntesis , Synechocystis/metabolismo , Synechocystis/fisiología , Tilacoides/genética , Tilacoides/metabolismo
3.
Int J Mol Sci ; 22(22)2021 Nov 19.
Artículo en Inglés | MEDLINE | ID: mdl-34830386

RESUMEN

The polypeptides encoded by the chloroplast ndh genes and some nuclear genes form the thylakoid NADH dehydrogenase (Ndh) complex, homologous to the mitochondrial complex I. Except for Charophyceae (algae related to higher plants) and a few Prasinophyceae, all eukaryotic algae lack ndh genes. Among vascular plants, the ndh genes are absent in epiphytic and in some species scattered among different genera, families, and orders. The recent identification of many plants lacking plastid ndh genes allows comparison on phylogenetic trees and functional investigations of the ndh genes. The ndh genes protect Angiosperms under various terrestrial stresses, maintaining efficient photosynthesis. On the edge of dispensability, ndh genes provide a test for the natural selection of photosynthesis-related genes in evolution. Variable evolutionary environments place Angiosperms without ndh genes at risk of extinction and, probably, most extant ones may have lost ndh genes recently. Therefore, they are evolutionary endpoints in phylogenetic trees. The low number of sequenced plastid DNA and the long lifespan of some Gymnosperms lacking ndh genes challenge models about the role of ndh genes protecting against stress and promoting leaf senescence. Additional DNA sequencing in Gymnosperms and investigations into the molecular mechanisms of their response to stress will provide a unified model of the evolutionary and functional consequences of the lack of ndh genes.


Asunto(s)
Cloroplastos/genética , NADH Deshidrogenasa/genética , Fotosíntesis/genética , Plastidios/genética , Carofíceas/genética , Genes del Cloroplasto/genética , Senescencia de la Planta/genética , Plastidios/metabolismo , Tilacoides/enzimología , Tilacoides/genética
4.
Int J Mol Sci ; 22(18)2021 Sep 13.
Artículo en Inglés | MEDLINE | ID: mdl-34576029

RESUMEN

Natural genetic variation in photosynthesis is strictly associated with the remarkable adaptive plasticity observed amongst Arabidopsis thaliana accessions derived from environmentally distinct regions. Exploration of the characteristic features of the photosynthetic machinery could reveal the regulatory mechanisms underlying those traits. In this study, we performed a detailed characterisation and comparison of photosynthesis performance and spectral properties of the photosynthetic apparatus in the following selected Arabidopsis thaliana accessions commonly used in laboratories as background lines: Col-0, Col-1, Col-2, Col-8, Ler-0, and Ws-2. The main focus was to distinguish the characteristic disparities for every accession in photosynthetic efficiency that could be accountable for their remarkable plasticity to adapt. The biophysical and biochemical analysis of the thylakoid membranes in control conditions revealed differences in lipid-to-protein contribution, Chlorophyll-to-Carotenoid ratio (Chl/Car), and xanthophyll cycle pigment distribution among accessions. We presented that such changes led to disparities in the arrangement of the Chlorophyll-Protein complexes, the PSI/PSII ratio, and the lateral mobility of the thylakoid membrane, with the most significant aberrations detected in the Ler-0 and Ws-2 accessions. We concluded that selecting an accession suitable for specific research on the photosynthetic process is essential for optimising the experiment.


Asunto(s)
Arabidopsis/genética , Carotenoides , Clorofila/genética , Fotosíntesis/genética , Selección Genética/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/ultraestructura , Regulación de la Expresión Génica de las Plantas , Complejos Multiproteicos/genética , Complejos Multiproteicos/ultraestructura , Mutación/genética , Fenotipo , Tilacoides/genética , Tilacoides/ultraestructura
5.
Cells ; 10(9)2021 09 09.
Artículo en Inglés | MEDLINE | ID: mdl-34572012

RESUMEN

In Part I, by using 31P-NMR spectroscopy, we have shown that isolated granum and stroma thylakoid membranes (TMs), in addition to the bilayer, display two isotropic phases and an inverted hexagonal (HII) phase; saturation transfer experiments and selective effects of lipase and thermal treatments have shown that these phases arise from distinct, yet interconnectable structural entities. To obtain information on the functional roles and origin of the different lipid phases, here we performed spectroscopic measurements and inspected the ultrastructure of these TM fragments. Circular dichroism, 77 K fluorescence emission spectroscopy, and variable chlorophyll-a fluorescence measurements revealed only minor lipase- or thermally induced changes in the photosynthetic machinery. Electrochromic absorbance transients showed that the TM fragments were re-sealed, and the vesicles largely retained their impermeabilities after lipase treatments-in line with the low susceptibility of the bilayer against the same treatment, as reflected by our 31P-NMR spectroscopy. Signatures of HII-phase could not be discerned with small-angle X-ray scattering-but traces of HII structures, without long-range order, were found by freeze-fracture electron microscopy (FF-EM) and cryo-electron tomography (CET). EM and CET images also revealed the presence of small vesicles and fusion of membrane particles, which might account for one of the isotropic phases. Interaction of VDE (violaxanthin de-epoxidase, detected by Western blot technique in both membrane fragments) with TM lipids might account for the other isotropic phase. In general, non-bilayer lipids are proposed to play role in the self-assembly of the highly organized yet dynamic TM network in chloroplasts.


Asunto(s)
Lípidos/genética , Tilacoides/genética , Dicroismo Circular/métodos , Espectroscopía de Resonancia Magnética/métodos , Microscopía Electrónica/métodos , Fotosíntesis/genética
6.
Biochim Biophys Acta Biomembr ; 1863(12): 183758, 2021 12 01.
Artículo en Inglés | MEDLINE | ID: mdl-34480878

RESUMEN

Styrene maleic acid (SMA) polymers have proven to be very successful for the extraction of membrane proteins, forming SMA lipid particles (SMALPs), which maintain a lipid bilayer around the membrane protein. SMALP-encapsulated membrane proteins can be used for functional and structural studies. The SMALP approach allows retention of important protein-annular lipid interactions, exerts lateral pressure, and offers greater stability than traditional detergent solubilisation. However, SMA polymer does have some limitations, including a sensitivity to divalent cations and low pH, an absorbance spectrum that overlaps with many proteins, and possible restrictions on protein conformational change. Various modified polymers have been developed to try to overcome these challenges, but no clear solution has been found. A series of partially-esterified variants of SMA (SMA 2625, SMA 1440 and SMA 17352) has previously been shown to be highly effective for solubilisation of plant and cyanobacterial thylakoid membranes. It was hypothesised that the partial esterification of maleic acid groups would increase tolerance to divalent cations. Therefore, these partially-esterified polymers were tested for the solubilisation of lipids and membrane proteins, and their tolerance to magnesium ions. It was found that all partially esterified polymers were capable of solubilising and purifying a range of membrane proteins, but the yield of protein was lower with SMA 1440, and the degree of purity was lower for both SMA 1440 and SMA 17352. SMA 2625 performed comparably to SMA 2000. SMA 1440 also showed an increased sensitivity to divalent cations. Thus, it appears the interactions between SMA and divalent cations are more complex than proposed and require further investigation.


Asunto(s)
Lípidos/química , Maleatos/química , Proteínas de la Membrana/aislamiento & purificación , Poliestirenos/química , Tilacoides/química , Cationes , Cianobacterias/química , Esterificación , Membrana Dobles de Lípidos/química , Proteínas de la Membrana/química , Conformación Proteica , Tilacoides/genética
7.
Int J Mol Sci ; 22(16)2021 Aug 05.
Artículo en Inglés | MEDLINE | ID: mdl-34445127

RESUMEN

The common ice plant (Mesembryanthemum crystallinum L.) is a facultative crassulacean acid metabolism (CAM) plant, and its ability to recover from stress-induced CAM has been confirmed. We analysed the photosynthetic metabolism of this plant during the 72-h response period following salinity stress removal from three perspectives. In plants under salinity stress (CAM) we found a decline of the quantum efficiencies of PSII (Y(II)) and PSI (Y(I)) by 17% and 15%, respectively, and an increase in nonphotochemical quenching (NPQ) by almost 25% in comparison to untreated control. However, 48 h after salinity stress removal, the PSII and PSI efficiencies, specifically Y(II) and Y(I), elevated nonphotochemical quenching (NPQ) and donor side limitation of PSI (YND), were restored to the level observed in control (C3 plants). Swelling of the thylakoid membranes, as well as changes in starch grain quantity and size, have been found to be components of the salinity stress response in CAM plants. Salinity stress induced an over 3-fold increase in average starch area and over 50% decline of average seed number in comparison to untreated control. However, in plants withdrawn from salinity stress, during the first 24 h of recovery, we observed chloroplast ultrastructures closely resembling those found in intact (control) ice plants. Rapid changes in photosystem functionality and chloroplast ultrastructure were accompanied by the induction of the expression (within 24 h) of structural genes related to the PSI and PSII reaction centres, including PSAA, PSAB, PSBA (D1), PSBD (D2) and cp43. Our findings describe one of the most flexible photosynthetic metabolic pathways among facultative CAM plants and reveal the extent of the plasticity of the photosynthetic metabolism and related structures in the common ice plant.


Asunto(s)
Metabolismo Ácido de las Crasuláceas/genética , Mesembryanthemum/genética , Fotosíntesis/genética , Estrés Salino/genética , Cloroplastos/efectos de los fármacos , Cloroplastos/genética , Metabolismo Ácido de las Crasuláceas/efectos de los fármacos , Mesembryanthemum/efectos de los fármacos , Fotosíntesis/efectos de los fármacos , Plastidios/efectos de los fármacos , Plastidios/genética , Salinidad , Estrés Salino/efectos de los fármacos , Cloruro de Sodio/farmacología , Almidón/genética , Tilacoides/efectos de los fármacos , Tilacoides/genética
8.
Int J Mol Sci ; 22(15)2021 Jul 26.
Artículo en Inglés | MEDLINE | ID: mdl-34360743

RESUMEN

Under natural environments, light quality and quantity are extremely varied. To respond and acclimate to such changes, plants have developed a multiplicity of molecular regulatory mechanisms. Non-photochemical quenching of chlorophyll fluorescence (NPQ) and thylakoid protein phosphorylation are two mechanisms that protect vascular plants. To clarify the role of thylakoid protein phosphorylation in energy-dependent quenching of chlorophyll fluorescence (qE) in rice plants, we used a direct Western blot assay after BN-PAGE to detect all phosphoproteins by P-Thr antibody as well as by P-Lhcb1 and P-Lhcb2 antibodies. Isolated thylakoids in either the dark- or the light-adapted state from wild type (WT) and PsbS-KO rice plants were used for this approach to detect light-dependent interactions between PsbS, PSII, and LHCII proteins. We observed that the bands corresponding to the phosphorylated Lhcb1 and Lhcb2 as well as the other phosphorylated proteins were enhanced in the PsbS-KO mutant after illumination. The qE relaxation became slower in WT plants after 10 min HL treatment, which correlated with Lhcb1 and Lhcb2 protein phosphorylation in the LHCII trimers under the same experimental conditions. Thus, we concluded that light-induced phosphorylation of PSII core and Lhcb1/Lhcb2 proteins is enhanced in rice PsbS-KO plants which might be due to more reactive-oxygen-species production in this mutant.


Asunto(s)
Clorofila/metabolismo , Fluorescencia , Complejos de Proteína Captadores de Luz/metabolismo , Luz , Oryza/metabolismo , Tilacoides/metabolismo , Clorofila/genética , Complejos de Proteína Captadores de Luz/genética , Oryza/genética , Fosforilación , Complejo de Proteína del Fotosistema II/genética , Complejo de Proteína del Fotosistema II/metabolismo , Tilacoides/genética
9.
Nat Plants ; 7(7): 979-988, 2021 07.
Artículo en Inglés | MEDLINE | ID: mdl-34140667

RESUMEN

In photosynthetic thylakoid membranes the proton motive force (pmf) not only drives ATP synthesis, in addition it is central to controlling and regulating energy conversion. As a consequence, dynamic fine-tuning of the two pmf components, electrical (Δψ) and chemical (ΔpH), is an essential element for adjusting photosynthetic light reactions to changing environmental conditions. Good evidence exists that the Δψ/ΔpH partitioning is controlled by thylakoid potassium and chloride ion transporters and channels. However, a detailed mechanistic understanding of how these thylakoid ion transporter/channels control pmf partitioning is lacking. Here, we combined functional measurements on potassium and chloride ion transporter and channel loss-of-function mutants with extended mathematical simulations of photosynthetic light reactions in thylakoid membranes to obtain detailed kinetic insights into the complex interrelationship between membrane energization and ion fluxes across thylakoid membranes. The data reveal that potassium and chloride fluxes in the thylakoid lumen determined by the K+/H+ antiporter KEA3 and the voltage-gated Cl- channel VCCN1/Best1 have distinct kinetic responses that lead to characteristic and light-intensity-dependent Δψ/ΔpH oscillations. These oscillations fine-tune photoprotective mechanisms and electron transport which are particularly important during the first minutes of illumination and under fluctuating light conditions. By employing the predictive power of the model, we unravelled the functional consequences of changes in KEA3 and VCCN1 abundance and regulatory/enzymatic parameters on membrane energization and photoprotection.


Asunto(s)
Arabidopsis/genética , Arabidopsis/fisiología , Transporte de Electrón/fisiología , Concentración de Iones de Hidrógeno , Fotosíntesis/fisiología , Fuerza Protón-Motriz/fisiología , Tilacoides/fisiología , Transporte de Electrón/genética , Variación Genética , Genotipo , Mutación , Fotosíntesis/genética , Fuerza Protón-Motriz/genética , Tilacoides/genética
10.
FEBS Lett ; 595(14): 1876-1885, 2021 07.
Artículo en Inglés | MEDLINE | ID: mdl-34060653

RESUMEN

IM30, the inner membrane-associated protein of 30 kDa, is conserved in cyanobacteria and chloroplasts. Although its exact physiological function is still mysterious, IM30 is clearly essential for thylakoid membrane biogenesis and/or dynamics. Recently, a cryptic IM30 GTPase activity has been reported, albeit thus far no physiological function has been attributed to this. Yet, it is still possible that GTP binding/hydrolysis affects formation of the prototypical large homo-oligomeric IM30 ring and rod structures. Here, we show that the Synechocystis sp. PCC 6803 IM30 protein in fact is an NTPase that hydrolyzes GTP and ATP, but not CTP or UTP, with about identical rates. While IM30 forms large oligomeric ring complexes, nucleotide binding and/or hydrolysis are clearly not required for ring formation.


Asunto(s)
Adenosina Trifosfato/metabolismo , Proteínas Bacterianas/metabolismo , Guanosina Trifosfato/metabolismo , Proteínas de la Membrana/metabolismo , Nucleósido-Trifosfatasa/metabolismo , Synechocystis/enzimología , Tilacoides/enzimología , Adenosina Trifosfato/química , Proteínas Bacterianas/genética , Clonación Molecular , Pruebas de Enzimas , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Guanosina Trifosfato/química , Hidrólisis , Cinética , Proteínas de la Membrana/genética , Microscopía Electrónica , Nucleósido-Trifosfatasa/genética , Unión Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Synechocystis/genética , Synechocystis/ultraestructura , Tilacoides/genética , Tilacoides/ultraestructura
11.
Int J Mol Sci ; 22(11)2021 May 31.
Artículo en Inglés | MEDLINE | ID: mdl-34072887

RESUMEN

FtsH metalloproteases found in eubacteria, animals, and plants are well-known for their vital role in the maintenance and proteolysis of membrane proteins. Their location is restricted to organelles of endosymbiotic origin, the chloroplasts, and mitochondria. In the model organism Arabidopsis thaliana, there are 17 membrane-bound FtsH proteases containing an AAA+ (ATPase associated with various cellular activities) and a Zn2+ metalloprotease domain. However, in five of those, the zinc-binding motif HEXXH is either mutated (FtsHi1, 2, 4, 5) or completely missing (FtsHi3), rendering these enzymes presumably inactive in proteolysis. Still, homozygous null mutants of the pseudo-proteases FtsHi1, 2, 4, 5 are embryo-lethal. Homozygous ftshi3 or a weak point mutant in FTSHi1 are affected in overall plant growth and development. This review will focus on the findings concerning the FtsHi pseudo-proteases and their involvement in protein import, leading to consequences in embryogenesis, seed growth, chloroplast, and leaf development and oxidative stress management.


Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Cloroplastos/genética , Metaloendopeptidasas/genética , Tilacoides/genética , Arabidopsis/enzimología , Cloroplastos/enzimología , Regulación de la Expresión Génica de las Plantas/genética , Mutación/genética , Transporte de Proteínas/genética , Proteolisis , Tilacoides/enzimología
12.
J Adv Res ; 30: 197-211, 2021 05.
Artículo en Inglés | MEDLINE | ID: mdl-34026296

RESUMEN

Introduction: Photosystem II (PSII) protein complex plays an essential role in the entire photosynthesis process. Various known and unknown protein factors are involved in the dynamics of the PSII complex that need to be characterized in crop plants for enhancing photosynthesis efficiency and productivity. Objectives: The experiments were conducted to decipher the regulatory proteins involved in PSII dynamics of rice crop. Methods: A novel rice regulatory protein PAP90 (PSII auxiliary protein ~90 kDa) was characterized by generating a loss-of-function mutant pap90. The mutation was characterized at molecular level followed by various experiments to analyze the morphological, physiological and biochemical processes of mutant under control and abiotic stresses. Results: The pap90 mutant showed reduced photosynthesis due to D1 protein instability that subsequently causes inadequate accumulation of thylakoid membrane complexes, especially PSII and decreases PSII functional efficiency. Expression of OsFtsH family genes and proteins were induced in the mutant, which are known to play a key role in D1 protein degradation and turnover. The reduced D1 protein accumulation in the mutant increased the production of reactive oxygen species (ROS). The accumulation of ROS along with the increased activity of antioxidant enzymes and induced expression of stress-associated genes and proteins in pap90 mutant contributed to its water-limited stress tolerance ability. Conclusion: We propose that PAP90 is a key auxiliary protein that interacts with D1 protein and maintains its stability, thereby promoting subsequent assembly of the PSII and associated membrane complexes.


Asunto(s)
Oryza/genética , Complejo de Proteína del Fotosistema II/genética , Proteínas de Plantas/genética , Regulación de la Expresión Génica de las Plantas , Luz , Mutación , Oryza/metabolismo , Fotosíntesis/genética , Complejo de Proteína del Fotosistema II/metabolismo , Proteínas de Plantas/metabolismo , Estabilidad Proteica , Especies Reactivas de Oxígeno/metabolismo , Estrés Fisiológico , Tilacoides/genética
13.
Plant Physiol ; 186(3): 1455-1472, 2021 07 06.
Artículo en Inglés | MEDLINE | ID: mdl-33856460

RESUMEN

Fatty acid photodecarboxylase (FAP) is one of the few enzymes that require light for their catalytic cycle (photoenzymes). FAP was first identified in the microalga Chlorella variabilis NC64A, and belongs to an algae-specific subgroup of the glucose-methanol-choline oxidoreductase family. While the FAP from C. variabilis and its Chlamydomonas reinhardtii homolog CrFAP have demonstrated in vitro activities, their activities and physiological functions have not been studied in vivo. Furthermore, the conservation of FAP activity beyond green microalgae remains hypothetical. Here, using a C. reinhardtii FAP knockout line (fap), we showed that CrFAP is responsible for the formation of 7-heptadecene, the only hydrocarbon of this alga. We further showed that CrFAP was predominantly membrane-associated and that >90% of 7-heptadecene was recovered in the thylakoid fraction. In the fap mutant, photosynthetic activity was not affected under standard growth conditions, but was reduced after cold acclimation when light intensity varied. A phylogenetic analysis that included sequences from Tara Ocean identified almost 200 putative FAPs and indicated that FAP was acquired early after primary endosymbiosis. Within Bikonta, FAP was retained in secondary photosynthetic endosymbiosis lineages but absent from those that lost the plastid. Characterization of recombinant FAPs from various algal genera (Nannochloropsis, Ectocarpus, Galdieria, Chondrus) provided experimental evidence that FAP photochemical activity was present in red and brown algae, and was not limited to unicellular species. These results thus indicate that FAP was conserved during the evolution of most algal lineages where photosynthesis was retained, and suggest that its function is linked to photosynthetic membranes.


Asunto(s)
Carboxiliasas/metabolismo , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , Ácidos Grasos/metabolismo , Microalgas/metabolismo , Procesos Fotoquímicos , Tilacoides/metabolismo , Ácidos Grasos/genética , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Variación Genética , Genotipo , Luz , Microalgas/genética , Mutación , Tilacoides/genética
14.
Int J Mol Sci ; 22(6)2021 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-33804002

RESUMEN

Antenna protein aggregation is one of the principal mechanisms considered effective in protecting phototrophs against high light damage. Commonly, it is induced, in vitro, by decreasing detergent concentration and pH of a solution of purified antennas; the resulting reduction in fluorescence emission is considered to be representative of non-photochemical quenching in vivo. However, little is known about the actual size and organization of antenna particles formed by this means, and hence the physiological relevance of this experimental approach is questionable. Here, a quasi-single molecule method, fluorescence correlation spectroscopy (FCS), was applied during in vitro quenching of LHCII trimers from higher plants for a parallel estimation of particle size, fluorescence, and antenna cluster homogeneity in a single measurement. FCS revealed that, below detergent critical micelle concentration, low pH promoted the formation of large protein oligomers of sizes up to micrometers, and therefore is apparently incompatible with thylakoid membranes. In contrast, LHCII clusters formed at high pH were smaller and homogenous, and yet still capable of efficient quenching. The results altogether set the physiological validity limits of in vitro quenching experiments. Our data also support the idea that the small, moderately quenching LHCII oligomers found at high pH could be relevant with respect to non-photochemical quenching in vivo.


Asunto(s)
Proteína con Homeodominio Antennapedia/genética , Complejos de Proteína Captadores de Luz/genética , Procesos Fototróficos/genética , Agregado de Proteínas/genética , Proteína con Homeodominio Antennapedia/química , Clorofila/química , Clorofila/genética , Clorofila/efectos de la radiación , Análisis por Conglomerados , Fluorescencia , Concentración de Iones de Hidrógeno , Luz/efectos adversos , Complejos de Proteína Captadores de Luz/química , Fotosíntesis/genética , Complejo de Proteína del Fotosistema II/genética , Complejo de Proteína del Fotosistema II/efectos de la radiación , Espectrometría de Fluorescencia , Tilacoides/química , Tilacoides/genética , Tilacoides/efectos de la radiación , Zeaxantinas/genética
15.
Plant Mol Biol ; 105(4-5): 513-523, 2021 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-33393067

RESUMEN

KEY MESSAGE: The thylakoid transit peptide of tobacco oxygen-evolving enhancer protein contains a minimal ten amino acid sequences for thylakoid lumen transports. This ten amino acids do not contain twin-arginine, which is required for typical chloroplast lumen translocation. Chloroplasts are intracellular organelles responsible for photosynthesis to produce organic carbon for all organisms. Numerous proteins must be transported from the cytosol to chloroplasts to support photosynthesis. This transport is facilitated by chloroplast transit peptides (TPs). Four chloroplast thylakoid lumen TPs were isolated from Nicotiana tabacum and were functionally analyzed as thylakoid lumen TPs. Typical chloroplast stroma-transit peptides and thylakoid lumen transit peptides (tTPs) are found in N. tabacum transit peptides (NtTPs) and the functions of these peptides are confirmed with TP-GFP fusion proteins under fluorescence microscopy and chloroplast fractionation, followed by Western blot analysis. During the functional analysis of tTPs, we uncovered the minimum 10 amino acid sequence is sufficient for thylakoid lumen transport. These ten amino acids can efficiently translocate GFP protein, even if they do not contain the twin-arginine residues required for the twin-arginine translocation (Tat) pathway, which is a typical thylakoid lumen transport. Further, thylakoid lumen transporting processes through the Tat pathway was examined by analyzing tTP sequence functions and we demonstrate that the importance of hydrophobic core for the tTP cleavage and target protein translocation.


Asunto(s)
Aminoácidos/metabolismo , Proteínas de Cloroplastos/metabolismo , Cloroplastos/metabolismo , Nicotiana/metabolismo , Oxígeno/metabolismo , Tilacoides/metabolismo , Secuencia de Aminoácidos , Aminoácidos/genética , Proteínas de Cloroplastos/genética , Cloroplastos/genética , Elementos de Facilitación Genéticos/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Membranas Intracelulares/metabolismo , Microscopía Confocal , Filogenia , Proteínas de Plantas/clasificación , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Señales de Clasificación de Proteína/genética , Transporte de Proteínas , Homología de Secuencia de Aminoácido , Tilacoides/genética , Nicotiana/clasificación , Nicotiana/genética
16.
Proc Natl Acad Sci U S A ; 118(4)2021 01 26.
Artículo en Inglés | MEDLINE | ID: mdl-33479170

RESUMEN

Photosystem II (PSII) is an intrinsic membrane protein complex that functions as a light-driven water:plastoquinone oxidoreductase in oxygenic photosynthesis. Electron transport in PSII is associated with formation of reactive oxygen species (ROS) responsible for oxidative modifications of PSII proteins. In this study, oxidative modifications of the D1 and D2 proteins by the superoxide anion (O2•-) and the hydroxyl (HO•) radicals were studied in WT and a tocopherol cyclase (vte1) mutant, which is deficient in the lipid-soluble antioxidant α-tocopherol. In the absence of this antioxidant, high-resolution tandem mass spectrometry was used to identify oxidation of D1:130E to hydroxyglutamic acid by O2•- at the PheoD1 site. Additionally, D1:246Y was modified to either tyrosine hydroperoxide or dihydroxyphenylalanine by O2•- and HO•, respectively, in the vicinity of the nonheme iron. We propose that α-tocopherol is localized near PheoD1 and the nonheme iron, with its chromanol head exposed to the lipid-water interface. This helps to prevent oxidative modification of the amino acid's hydrogen that is bonded to PheoD1 and the nonheme iron (via bicarbonate), and thus protects electron transport in PSII from ROS damage.


Asunto(s)
Aminoácidos/química , Arabidopsis/enzimología , Complejo de Proteína del Fotosistema II/química , Superóxidos/química , Tilacoides/enzimología , alfa-Tocoferol/química , Aminoácidos/metabolismo , Arabidopsis/genética , Arabidopsis/efectos de la radiación , Sitios de Unión , Radical Hidroxilo/química , Radical Hidroxilo/metabolismo , Transferasas Intramoleculares/química , Transferasas Intramoleculares/genética , Transferasas Intramoleculares/metabolismo , Hierro/química , Hierro/metabolismo , Luz , Modelos Moleculares , Mutación , Oxidación-Reducción , Oxígeno/química , Oxígeno/metabolismo , Fotosíntesis/fisiología , Fotosíntesis/efectos de la radiación , Complejo de Proteína del Fotosistema II/genética , Complejo de Proteína del Fotosistema II/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Superóxidos/metabolismo , Termodinámica , Thermosynechococcus/enzimología , Thermosynechococcus/genética , Thermosynechococcus/efectos de la radiación , Tilacoides/genética , Tilacoides/efectos de la radiación , alfa-Tocoferol/metabolismo
17.
Plant J ; 106(1): 113-132, 2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33372269

RESUMEN

The chloroplast signal recognition particle 54 kDa (CpSRP54) protein is a member of the CpSRP pathway known to target proteins to thylakoid membranes in plants and green algae. Loss of CpSRP54 in the marine diatom Phaeodactylum tricornutum lowers the accumulation of a selection of chloroplast-encoded subunits of photosynthetic complexes, indicating a role in the co-translational part of the CpSRP pathway. In contrast to plants and green algae, absence of CpSRP54 does not have a negative effect on the content of light-harvesting antenna complex proteins and pigments in P. tricornutum, indicating that the diatom CpSRP54 protein has not evolved to function in the post-translational part of the CpSRP pathway. Cpsrp54 KO mutants display altered photophysiological responses, with a stronger induction of photoprotective mechanisms and lower growth rates compared to wild type when exposed to increased light intensities. Nonetheless, their phenotype is relatively mild, thanks to the activation of mechanisms alleviating the loss of CpSRP54, involving upregulation of chaperones. We conclude that plants, green algae, and diatoms have evolved differences in the pathways for co-translational and post-translational insertion of proteins into the thylakoid membranes.


Asunto(s)
Proteínas de Cloroplastos/metabolismo , Cloroplastos/metabolismo , Diatomeas/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Chlorophyta/genética , Chlorophyta/metabolismo , Proteínas de Cloroplastos/genética , Cloroplastos/genética , Diatomeas/genética , Edición Génica , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Tilacoides/genética , Tilacoides/metabolismo
18.
Open Biol ; 10(9): 200144, 2020 09.
Artículo en Inglés | MEDLINE | ID: mdl-32931722

RESUMEN

Non-photochemical quenching (NPQ) is an important photoprotective mechanism in plants and algae. Although the process is extensively studied, little is known about its relationship with ultrastructural changes of the thylakoid membranes. In order to better understand this relationship, we studied the effects of illumination on the organization of thylakoid membranes in Monstera deliciosa leaves. This evergreen species is known to exhibit very large NPQ and to possess giant grana with dozens of stacked thylakoids. It is thus ideally suited for small-angle neutron scattering measurements (SANS)-a non-invasive technique, which is capable of providing spatially and statistically averaged information on the periodicity of the thylakoid membranes and their rapid reorganizations in vivo. We show that NPQ-inducing illumination causes a strong decrease in the periodic order of granum thylakoid membranes. Development of NPQ and light-induced ultrastructural changes, as well as the relaxation processes, follow similar kinetic patterns. Surprisingly, whereas NPQ is suppressed by diuron, it impedes only the relaxation of the structural changes and not its formation, suggesting that structural changes do not cause but enable NPQ. We also demonstrate that the diminishment of SANS peak does not originate from light-induced redistribution and reorientation of chloroplasts inside the cells.


Asunto(s)
Araceae/química , Membranas Intracelulares/química , Neutrones , Hojas de la Planta/química , Dispersión del Ángulo Pequeño , Tilacoides/genética , Difracción de Rayos X , Membranas Intracelulares/metabolismo , Luz , Tilacoides/metabolismo
19.
Nat Plants ; 6(9): 1179-1191, 2020 09.
Artículo en Inglés | MEDLINE | ID: mdl-32895528

RESUMEN

The thylakoid membranes of cyanobacteria form a complex intracellular membrane system with a distinctive proteome. The sites of biogenesis of thylakoid proteins remain uncertain, as do the signals that direct thylakoid membrane-integral proteins to the thylakoids rather than to the plasma membrane. Here, we address these questions by using fluorescence in situ hybridization to probe the subcellular location of messenger RNA molecules encoding core subunits of the photosystems in two cyanobacterial species. These mRNAs cluster at thylakoid surfaces mainly adjacent to the central cytoplasm and the nucleoid, in contrast to mRNAs encoding proteins with other locations. Ribosome association influences the distribution of the photosynthetic mRNAs on the thylakoid surface, but thylakoid affinity is retained in the absence of ribosome association. However, thylakoid association is disrupted in a mutant lacking two mRNA-binding proteins, which probably play roles in targeting photosynthetic proteins to the thylakoid membrane.


Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Membrana Celular/metabolismo , Cianobacterias/genética , Cianobacterias/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Membrana Celular/genética , Hibridación Fluorescente in Situ , Transporte de Proteínas/genética , Tilacoides/genética , Tilacoides/metabolismo
20.
Transgenic Res ; 29(3): 295-305, 2020 06.
Artículo en Inglés | MEDLINE | ID: mdl-32318934

RESUMEN

Chloroplast transformation has many potential advantages for the production of recombinant proteins in plants. However, it has been reported that chloroplast expression of many proteins, such as human epidermal growth factor (hEGF), results hindered by post-transcriptional mechanisms. hEGF degradation has been related to the redox potential of the stroma and protein misfolding. To solve this problem, we proposed the redirection of hEGF into the thylakoid lumen where the environment could improve disulfide bonds formation stabilizing the functional conformation of the protein. We generated transplastomic tobacco plants targeting hEGF protein to the thylakoid lumen by adding a transit peptide (Str). Following this approach, we could detect thylakoid lumen-targeted hEGF by western blotting while stromal accumulation of hEGF remained undetectable. Southern blot analysis confirmed the integration of the transgene through homologous recombination into the plastome. Northern blot analysis showed similar levels of egf transcripts in the EGF and StrEGF lines. These results suggest that higher stability of the hEGF peptide in the thylakoid lumen is the primary cause of the increased accumulation of the recombinant protein observed in StrEGF lines. They also highlight the necessity of exploring different sub-organellar destinations to improve the accumulation levels of a specific recombinant protein in plastids.


Asunto(s)
Cloroplastos/metabolismo , Factor de Crecimiento Epidérmico/metabolismo , Nicotiana/metabolismo , Plantas Modificadas Genéticamente/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Tilacoides/metabolismo , Transgenes , Cloroplastos/genética , Factor de Crecimiento Epidérmico/genética , Humanos , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/crecimiento & desarrollo , Plastidios/genética , Plastidios/metabolismo , Proteínas Recombinantes de Fusión/genética , Tilacoides/genética , Nicotiana/genética , Nicotiana/crecimiento & desarrollo
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