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1.
J Plant Physiol ; 243: 153048, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31639536

RESUMO

Investigations of the luminal immunophilin AtCYP38 (cyclophilin 38) in Arabidopsis thaliana (At), the orthologue of the complex immunophilin TLP40 from Spinacia oleracea, revealed its involvement in photosystem II (PSII) repair and assembly, biogenesis of PSII complex, and cellular signalling. However, the main physiological roles of AtCYP38 and TLP40 are related to regulation of thylakoid PP2A-type phosphatase involved in PSII core protein dephosphorylation, and chaperone function during protein folding. Here we further investigate physiological roles of AtCYP38 and analyse the ultrastructure of chloroplasts from cyp38-2 plants. Transmission electron microscopy followed by quantitative micrography revealed modifications in thylakoid stacking. We also confirm that the depletion of AtCYP38 influences PSII performance, which leads to stunted phenotype of cyp38-2 plants.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Ciclofilinas/genética , Complexo de Proteína do Fotossistema II/metabolismo , Tilacoides/metabolismo , Arabidopsis/enzimologia , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Cloroplastos/ultraestrutura , Ciclofilinas/metabolismo , Microscopia Eletrônica de Transmissão
2.
Int J Mol Sci ; 20(17)2019 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-31470529

RESUMO

Ulmus pumila 'Jinye', the colorful leaf mutant of Ulmus pumila L., is widely used in landscaping. In common with most leaf color mutants, U. pumila 'Jinye' exhibits growth inhibition. In this study, U. pumila L. and U. pumila 'Jinye' were used to elucidate the reasons for growth inhibition at the physiological, cellular microstructural, and transcriptional levels. The results showed that the pigment (chlorophyll a, chlorophyll b, and carotenoids) content of U. pumila L. was higher than that of U. pumila 'Jinye', whereas U. pumila 'Jinye' had a higher proportion of carotenoids, which may be the cause of the yellow leaves. Examination of the cell microstructure and RNA sequencing analysis showed that the leaf color and growth inhibition were mainly due to the following reasons: first, there were differences in the structure of the thylakoid grana layer. U. pumila L. has a normal chloroplast structure and clear thylakoid grana slice layer structure, with ordered and compact thylakoids. However, U. pumila 'Jinye' exhibited the grana lamella stacking failures and fewer thylakoid grana slice layers. As the pigment carrier and the key location for photosynthesis, the close stacking of thylakoid grana could combine more chlorophyll and promote efficient electron transfer promoting the photosynthesis reaction. In addition, U. pumila 'Jinye' had a lower capacity for light energy absorption, transformation, and transportation, carbon dioxide (CO2) fixation, lipopolysaccharide biosynthesis, auxin synthesis, and protein transport. The genes related to respiration and starch consumption were higher than those of U. pumila L., which indicated less energy accumulation caused the growth inhibition of U. pumila 'Jinye'. Finally, compared with U. pumila 'Jinye', the transcription of genes related to stress resistance all showed an upward trend in U. pumila L. That is to say, U. pumila L. had a greater ability to resist adversity, which could maintain the stability of the intracellular environment and maintain normal progress of physiological metabolism. However, U. pumila 'Jinye' was more susceptible to changes in the external environment, which affected normal physiological metabolism. This study provides evidence for the main cause of growth inhibition in U. pumila 'Jinye', information for future cultivation, and information on the mutation mechanism for the breeding of colored leaf trees.


Assuntos
Cloroplastos/genética , Perfilação da Expressão Gênica/métodos , Mutação , Folhas de Planta/genética , Ulmus/genética , Carotenoides/metabolismo , Clorofila/metabolismo , Clorofila A/metabolismo , Cloroplastos/metabolismo , Cloroplastos/ultraestrutura , Cor , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Microscopia Eletrônica de Transmissão , Fotossíntese/genética , Pigmentos Biológicos/metabolismo , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , Tilacoides/genética , Tilacoides/metabolismo , Tilacoides/ultraestrutura , Ulmus/crescimento & desenvolvimento , Ulmus/metabolismo
3.
Biochim Biophys Acta Bioenerg ; 1860(12)2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31344362

RESUMO

Thylakoids are the place of the light-photosynthetic reactions. To gain maximal efficiency, these reactions are conditional to proper pigment-pigment and protein-protein interactions. In higher plants thylakoids, the interactions lead to a lateral asymmetry in localization of protein complexes (i.e. granal/stromal thylakoids) that have been defined as a domain-like structures characteristic by different biochemical composition and function (Albertsson P-Å. 2001,Trends Plant Science 6: 349-354). We explored this complex organization of thylakoid pigment-proteins at single cell level in the cyanobacterium Synechocystis sp. PCC 6803. Our 3D confocal images captured heterogeneous distribution of all main photosynthetic pigment-protein complexes (PPCs), Photosystem I (fluorescently tagged by YFP), Photosystem II and Phycobilisomes. The acquired images depicted cyanobacterial thylakoid membrane as a stable, mosaic-like structure formed by microdomains (MDs). These microcompartments are of sub-micrometer in sizes (~0.5-1.5 µm), typical by particular PPCs ratios and importantly without full segregation of observed complexes. The most prevailing MD is represented by MD with high Photosystem I content which allows also partial separation of Photosystems like in higher plants thylakoids. We assume that MDs stability (in minutes) provides optimal conditions for efficient excitation/electron transfer. The cyanobacterial MDs thus define thylakoid membrane organization as a system controlled by co-localization of three main PPCs leading to formation of thylakoid membrane mosaic. This organization might represent evolutional and functional precursor for the granal/stromal spatial heterogeneity in photosystems that is typical for higher plant thylakoids.


Assuntos
Proteínas de Bactérias/metabolismo , Microdomínios da Membrana/metabolismo , Tilacoides/metabolismo , Imagem Tridimensional , Microscopia Confocal , Fotossíntese/fisiologia , Complexo de Proteína do Fotossistema I/metabolismo , Complexo de Proteína do Fotossistema II/metabolismo , Ficobilissomas/metabolismo , Synechocystis
4.
Plant Sci ; 285: 230-238, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31203888

RESUMO

In higher plants, Fibrillins (FBNs) constitute a conserved plastid-lipid-associated (PAPs) protein family and modulate the metabolite transport and lipid metabolism in plastids of dicot species. However, FBNs have not functionally characterized in monocot species. In this study, the function of rice fibrillin 1 (OsFBN1) was investigated. The subcellular localization assay showed that the N-terminal chloroplast transport peptide (CTP) could facilitate the import of OsFBN1 into chloroplast. OsFBN1 specifically bound C18- and C20- fatty acids in vitro. Overexpressing OsFBN1 increased the tiller number but decreased the panicle length, grain-filling percent and JA levels compared to the wild type and RNAi silencing lines under heat stress. In addition, the overexpressing lines had more plastoglobules (PGs) than the wild type and RNAi silencing lines under both normal and heat stress conditions. Moreover, overexpressing OsFBN1 affected the transcription levels of OsAOS2 in JA synthesis, OsTHF1, OsABC1K7 and OsPsaE in thylakoid stability and photosynthesis, OsABC1-4 and OsSPS2 in ubiquinone-metabolism, OsHDR, OsDXR, and OsFPPS in isoprenoid metabolism. Collectively, these findings suggest the essential role of rice OsFBN1 in PG formation and lipid metabolism in chloroplasts, which coordinately regulate the growth and grain filling of the overexpressing lines under heat stress.


Assuntos
Cloroplastos/metabolismo , Ciclopentanos/metabolismo , Grão Comestível/metabolismo , Oryza/metabolismo , Oxilipinas/metabolismo , Proteínas de Plantas/metabolismo , Cloroplastos/ultraestrutura , Grão Comestível/crescimento & desenvolvimento , Resposta ao Choque Térmico , Metabolismo dos Lipídeos , Lipoproteínas/metabolismo , Microscopia Eletrônica de Transmissão , Oryza/genética , Oryza/fisiologia , Oryza/ultraestrutura , Fotossíntese , Proteínas de Plantas/fisiologia , Plantas Geneticamente Modificadas , Reação em Cadeia da Polimerase em Tempo Real , Tilacoides/metabolismo
5.
Plant Cell Physiol ; 60(8): 1811-1828, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31179502

RESUMO

Diatoms are unicellular algae and evolved by secondary endosymbiosis, a process in which a red alga-like eukaryote was engulfed by a heterotrophic eukaryotic cell. This gave rise to plastids of remarkable complex architecture and ultrastructure that require elaborate protein importing, trafficking, signaling and intracellular cross-talk pathways. Studying both plastids and mitochondria and their distinctive physiological pathways in organello may greatly contribute to our understanding of photosynthesis, mitochondrial respiration and diatom evolution. The isolation of such complex organelles, however, is still demanding, and existing protocols are either limited to a few species (for plastids) or have not been reported for diatoms so far (for mitochondria). In this work, we present the first isolation protocol for mitochondria from the model diatom Thalassiosira pseudonana. Apart from that, we extended the protocol so that it is also applicable for the purification of a high-quality plastids fraction, and provide detailed structural and physiological characterizations of the resulting organelles. Isolated mitochondria were structurally intact, showed clear evidence of mitochondrial respiration, but the fractions still contained residual cell fragments. In contrast, plastid isolates were virtually free of cellular contaminants, featured structurally preserved thylakoids performing electron transport, but lost most of their stromal components as concluded from Western blots and mass spectrometry. Liquid chromatography electrospray-ionization mass spectrometry studies on mitochondria and thylakoids, moreover, allowed detailed proteome analyses which resulted in extensive proteome maps for both plastids and mitochondria thus helping us to broaden our understanding of organelle metabolism and functionality in diatoms.


Assuntos
Diatomáceas/metabolismo , Mitocôndrias/metabolismo , Plastídeos/metabolismo , Proteoma/metabolismo , Tilacoides/metabolismo
6.
Photosynth Res ; 142(1): 57-67, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31098930

RESUMO

Effect of water-soluble and stable sucrose-bound iron oxyhydroxide nanoparticles [Fe[III] sucrose complex (FSC)] on the efficiency of electron transport in the photosystem II membranes was studied. FSC significantly increases (by a factor 1.5) the rate of light-induced oxygen evolution in the presence of alternative electron acceptor 2,6-dichloro-p-benzoquinone (DCBQ). Without DCBQ, FSC only slightly (5%) provides the oxygen evolution. Electron transport supported by pair DCBQ + FSC is inhibited by diuron. Maximum of stimulating effect was recorded at Fe(III) concentration 5 µM. In the case of another benzoquinone electron acceptor (2-phenyl-p-benzoquinone and 2,3-dimethyl-p-benzoquinone) and 2,6-dichlorophenolindophenol, stimulating effect of FSC was not observed. Incubation of PSII membranes at different concentrations with FSC is accompanied by binding of Fe(III) by membrane components but only about 50% of iron can be extracted by membranes from Fe(III) solution at pH 6.5. This result implies the heterogeneity of FSC solution in a buffer. The heterogeneity depends on pH and decreases with its rising. At pH around 9.0 Fe(III), sucrose solution is homogeneous. The study of pH effect has shown that stimulation of electron transport is induced only by iron cations which can be bound by membranes. Not extractable iron pool cannot activate electron transfer from oxygen-evolving complex to DCBQ.


Assuntos
Transporte de Elétrons/efeitos dos fármacos , Compostos Férricos/farmacologia , Nanopartículas/química , Oxigênio/metabolismo , Fotossíntese , Complexo de Proteína do Fotossistema II/efeitos dos fármacos , Solubilidade , Spinacia oleracea/metabolismo , Sacarose/química , Tilacoides/metabolismo
7.
Biochim Biophys Acta Bioenerg ; 1860(6): 499-507, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-31055058

RESUMO

Plants have developed multiple self-regulatory mechanisms to efficiently function under varying sunlight conditions. At high light intensities, non-photochemical quenching (NPQ) is activated on a molecular level, safely dissipating an excess excitation as heat. The exact molecular mechanism for NPQ is still under debate, but it is widely agreed that the direct participation of the carotenoid pigments is involved, one of the proposed candidate being the zeaxanthin. In this work, we performed fluorescence measurements of violaxanthin- and zeaxanthin-enriched major light-harvesting complexes (LHCII), in ensemble and at the single pigment-protein complex level, where aggregation is prevented by immobilization of LHCIIs onto a surface. We show that a selective enrichment of LHCII with violaxanthin or zeaxanthin affects neither the ability of LHCII to switch into a dissipative conformation nor the maximal level of induced quenching. However, the kinetics of the fluorescence decrease due to aggregation on the timescale of seconds are different, prompting towards a modulatory effect of zeaxanthin in the dynamics of quenching.


Assuntos
Complexos de Proteínas Captadores de Luz/metabolismo , Zeaxantinas/metabolismo , Adaptação Fisiológica/fisiologia , Concentração de Íons de Hidrogênio , Luz , Complexos de Proteínas Captadores de Luz/química , Folhas de Planta , Conformação Proteica , Espectrometria de Fluorescência , Spinacia oleracea , Tilacoides/química , Tilacoides/metabolismo , Xantofilas/química , Xantofilas/metabolismo , Zeaxantinas/química
8.
Photosynth Res ; 141(3): 367-376, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31020482

RESUMO

Although the importance of nonphotochemical quenching (NPQ) on photosynthetic biomass production and crop yields is well established, the in vivo operation of the individual mechanisms contributing to overall NPQ is still a matter of controversy. In order to investigate the timescale and activation dynamics of specific quenching mechanisms, we have developed a technique called snapshot transient absorption (TA) spectroscopy, which can monitor molecular species involved in the quenching response with a time resolution of 30 s. Using intact thylakoid membrane samples, we show how conventional TA kinetic and spectral analyses enable the determination of the appropriate wavelength and time delay for snapshot TA experiments. As an example, we show how the chlorophyll-carotenoid charge transfer and excitation energy transfer mechanisms can be monitored based on signals corresponding to the carotenoid (Car) radical cation and Car S1 excited state absorption, respectively. The use of snapshot TA spectroscopy together with the previously reported fluorescence lifetime snapshot technique (Sylak-Glassman et al. in Photosynth Res 127:69-76, 2016) provides valuable information such as the concurrent appearance of specific quenching species and overall quenching of excited Chl. Furthermore, we show that the snapshot TA technique can be successfully applied to completely intact photosynthetic organisms such as live cells of Nannochloropsis. This demonstrates that the snapshot TA technique is a valuable method for tracking the dynamics of intact samples that evolve over time, such as the photosynthetic system in response to high-light exposure.


Assuntos
Processos Fotoquímicos , Análise Espectral , Difusão , Cinética , Spinacia oleracea/metabolismo , Termodinâmica , Tilacoides/metabolismo
9.
Biochim Biophys Acta Bioenerg ; 1860(6): 469-477, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-31029592

RESUMO

Photosystem I (PSI) is the potential target of photodamage under fluctuating light in angiosperms. However, the response of PSI to fluctuating light in young leaves has not yet been clarified. Furthermore, the photosynthetic regulation under fluctuating light in crassulacean acid metabolism (CAM) plants is little known. In this study, we measured PSI redox state and the electrochromic shift signal in the mature and young leaves of a CAM species Bryophyllum pinnatum. The mature leaves showed stronger capacity for photo-reduction of O2 mediated by the alternative electron flow (probably the water-water cycle) when compared with the young leaves. After an increase in light intensity, both the mature and young leaves showed insufficient proton gradient (ΔpH) across the thylakoid membranes within the first seconds. Meanwhile, PSI was highly oxidized in the mature leaves but was in a more reduced state in the young leaves. Furthermore, young leaves were more susceptible to PSI photoinhibition under fluctuating light. Therefore, in the mature leaves, the alternative electron flow significantly optimized the PSI redox state under fluctuating light at relatively low ΔpH. By comparison, in the young leaves, PSI redox state was largely determined by the buildup of ΔpH. Therefore, the major photoprotective mechanism responsible for safeguarding PSI under fluctuating light can be influenced by leaf developmental stages.


Assuntos
Kalanchoe/crescimento & desenvolvimento , Kalanchoe/fisiologia , Luz , Fotossíntese/fisiologia , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/fisiologia , ATPases de Cloroplastos Translocadoras de Prótons/metabolismo , Elétrons , Concentração de Íons de Hidrogênio , Oxirredução , Periodicidade , Complexo de Proteína do Fotossistema I/fisiologia , Prótons , Tilacoides/metabolismo , Água/metabolismo
10.
Nat Plants ; 5(4): 436-446, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30962530

RESUMO

Little is known about how the photosynthetic machinery is arranged in time and space during the biogenesis of thylakoid membranes. Using in situ cryo-electron tomography to image the three-dimensional architecture of the cyanobacterium Synechocystis, we observed that the tips of multiple thylakoids merge to form a substructure called the 'convergence membrane'. This high-curvature membrane comes into close contact with the plasma membrane at discrete sites. We generated subtomogram averages of 70S ribosomes and array-forming phycobilisomes, then mapped these structures onto the native membrane architecture as markers for protein synthesis and photosynthesis, respectively. This molecular localization identified two distinct biogenic regions in the thylakoid network: thylakoids facing the cytosolic interior of the cell that were associated with both marker complexes, and convergence membranes that were decorated by ribosomes but not phycobilisomes. We propose that the convergence membranes perform a specialized biogenic function, coupling the synthesis of thylakoid proteins with the integration of cofactors from the plasma membrane and the periplasmic space.


Assuntos
Membrana Celular/metabolismo , Synechocystis/metabolismo , Tilacoides/metabolismo , Membrana Celular/ultraestrutura , Microscopia Crioeletrônica , Tomografia com Microscopia Eletrônica , Ficobilissomas/metabolismo , Ficobilissomas/ultraestrutura , Ribossomos/metabolismo , Ribossomos/ultraestrutura , Synechocystis/ultraestrutura , Tilacoides/ultraestrutura
11.
PLoS One ; 14(4): e0214811, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30946768

RESUMO

Plastids perform many essential functions in plant metabolism including photosynthesis, synthesis of metabolites, and stress signaling. The most prominent type in green leaves is the chloroplast which contains thylakoids, plastoglobules, and starch. As these structures are closely linked to the metabolism of chloroplasts, changes during plant growth and development and during environmental stress situations are likely to occur. The aim of this study was to characterize changes in size and ultrastructure of chloroplast on cross-sections of leaves during high light stress, Botrytis infection, and dark induced senescence by quantitative transmission electron microscopy (TEM).The size of chloroplasts on cross sections of leaves decreased significantly when plants were subject to high light (49%), Botrytis infection (58%), and senescence (71%). The number of chloroplasts on cross sections of the palisade cell layer and spongy parenchyma, respectively, decreased significantly in plants exposed to high light conditions (48% and 29%), infected with Botrytis (48% and 46%), and during senescence (78% and 80%). Thylakoids on cross-sections of chloroplasts decreased significantly in plants exposed to high light (22%), inoculated with Botrytis cinerea (36%), and senescence (51%). This correlated with a massive increase in plastoglobules on cross-sections of chloroplasts of 88%, 2,306% and 19,617%, respectively. Starch contents on cross sections of chloroplasts were completely diminished in all three stress scenarios. These results demonstrate that the decrease in the number and size of chloroplasts is a reliable stress marker in plants during abiotic and biotic stress situations which can be easily detected with a light microscope. Further, lack of starch, the occurrence of large plastoglobules and decrease in thylakoids can also be regarded as reliable stress marker in plants which can be detected by TEM.


Assuntos
Plastídeos/ultraestrutura , Arabidopsis/metabolismo , Arabidopsis/microbiologia , Arabidopsis/ultraestrutura , Botrytis/patogenicidade , Cloroplastos/metabolismo , Cloroplastos/ultraestrutura , Luz , Microscopia Eletrônica de Transmissão , Doenças das Plantas/microbiologia , Folhas de Planta/metabolismo , Folhas de Planta/ultraestrutura , Plastídeos/metabolismo , Amido/metabolismo , Estresse Fisiológico , Tilacoides/metabolismo , Tilacoides/ultraestrutura
12.
RNA Biol ; 16(7): 906-917, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30990352

RESUMO

Understanding how viruses and subviral agents initiate disease is central to plant pathology. Whether RNA silencing mediates the primary lesion triggered by viroids (small non-protein-coding RNAs), or just intermediate-late steps of a signaling cascade, remains unsolved. While most variants of the plastid-replicating peach latent mosaic viroid (PLMVd) are asymptomatic, some incite peach mosaics or albinism (peach calico, PC). We have previously shown that two 21-nt small RNAs (PLMVd-sRNAs) containing a 12-13-nt PC-associated insertion guide cleavage, via RNA silencing, of the mRNA encoding a heat-shock protein involved in chloroplast biogenesis. To gain evidence supporting that such event is the initial lesion, and more specifically, that different chloroses have different primary causes, here we focused on a PLMVd-induced peach yellow mosaic (PYM) expressed in leaf sectors interspersed with others green. First, sequencing PLMVd-cDNAs from both sectors and bioassays mapped the PYM determinant at one nucleotide, a notion further sustained by the phenotype incited by other natural and artificial PLMVd variants. And second, sRNA deep-sequencing and RNA ligase-mediated RACE identified one PLMVd-sRNA with the PYM-associated change that guides cleavage, as predicted by RNA silencing, of the mRNA encoding a thylakoid translocase subunit required for chloroplast development. RT-qPCR showed lower accumulation of this mRNA in PYM-expressing tissues. Remarkably, PLMVd-sRNAs triggering PYM and PC have 5'-terminal Us, involving Argonaute 1 in what likely are the initial alterations eliciting distinct chloroses.


Assuntos
Interações Hospedeiro-Patógeno/genética , Doenças das Plantas/virologia , Vírus de Plantas/genética , Plastídeos/virologia , Polimorfismo de Nucleotídeo Único/genética , Replicação Viral/genética , Sequência de Aminoácidos , Sequência de Bases , Folhas de Planta/ultraestrutura , Folhas de Planta/virologia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Subunidades Proteicas/metabolismo , Prunus persica/ultraestrutura , Prunus persica/virologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Tilacoides/metabolismo
13.
Plant Cell Physiol ; 60(6): 1224-1238, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-30892620

RESUMO

Etioplasts developed in angiosperm cotyledon cells in darkness rapidly differentiate into chloroplasts with illumination. This process involves dynamic transformation of internal membrane structures from the prolamellar bodies (PLBs) and prothylakoids (PTs) in etioplasts to thylakoid membranes in chloroplasts. Although two galactolipids, monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), are predominant lipid constituents of membranes in both etioplasts and chloroplasts, their roles in the structural and functional transformation of internal membranes during etioplast-to-chloroplast differentiation are unknown. We previously reported that a 36% loss of MGDG by an artificial microRNA targeting major MGDG synthase (amiR-MGD1) only slightly affected PLB structures but strongly impaired PT formation and protochlorophyllide biosynthesis. Meanwhile, strong DGDG deficiency in a DGDG synthase mutant (dgd1) disordered the PLB lattice structure in addition to impaired PT development and protochlorophyllide biosynthesis. In this study, thylakoid biogenesis after PLB disassembly with illumination was strongly perturbed by amiR-MGD1. The amiR-MGD1 expression impaired the accumulation of Chl and the major light-harvesting complex II protein (LHCB1), which may inhibit rapid transformation from disassembled PLBs to the thylakoid membrane. As did amiR-MGD1 expression, dgd1 mutation impaired the accumulation of Chl and LHCB1 during etioplast-to-chloroplast differentiation. Furthermore, unlike in amiR-MGD1 seedlings, in dgd1 seedlings, disassembly of PLBs after illumination was retarded. Because DGDG but not MGDG prefers to form the bilayer lipid phase in membranes, the MGDG-to-DGDG ratio may strongly affect the transformation of PLBs to the thylakoid membrane during etioplast-to-chloroplast differentiation.


Assuntos
Membrana Celular/metabolismo , Cloroplastos/metabolismo , Galactolipídeos/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Galactosiltransferases/metabolismo , Tilacoides/metabolismo
14.
PLoS Genet ; 15(3): e1008047, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30875368

RESUMO

Large GTPases of the Dynamin Related Proteins (DRP) family shape lipid bilayers through membrane fission or fusion processes. Despite the highly organized photosynthetic membranes of thylakoids, a single DRP is known to be targeted inside the chloroplast. Fzl from the land plant Arabidopsis thaliana is inserted in the inner envelope and thylakoid membranes to regulate their morphology. Fzl may promote the fusion of thylakoids but this remains to be proven. Moreover, the physiological requirement for fusing thylakoids is currently unknown. Here, we find that the unicellular microalga Chlamydomonas reinhardtii encodes an Fzl ortholog (CrFzl) that is localized in the chloroplast where it is soluble. To explore its function, the CRISPR/Cas9 technology was employed to generate multiple CrFzl knock out strains. Phenotypic analyzes revealed a specific requirement of CrFzl for survival upon light stress. Consistent with this, strong irradiance lead to increased photoinhibition of photosynthesis in mutant cells. Fluorescence and electron microscopy analysis demonstrated that upon exposure to high light, CrFzl mutants show defects in chloroplast morphology but also large cytosolic vacuoles in close contact with the plastid. We further observe that strong irradiance induces an increased recruitment of the DRP to thylakoid membranes. Most importantly, we show that CrFzl is required for the fusion of thylakoids during mating. Together, our results suggest that thylakoids fusion may be necessary for resistance to light stress.


Assuntos
Proteínas de Algas/metabolismo , Chlamydomonas reinhardtii/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Tilacoides/metabolismo , Proteínas de Algas/genética , Sistemas CRISPR-Cas , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/efeitos da radiação , Cloroplastos/metabolismo , Dinaminas/genética , Dinaminas/metabolismo , GTP Fosfo-Hidrolases/genética , Técnicas de Inativação de Genes , Luz , Fusão de Membrana , Microscopia Eletrônica de Transmissão , Mutação , Processos Fototróficos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Estresse Fisiológico , Tilacoides/efeitos da radiação , Tilacoides/ultraestrutura
15.
Plant Sci ; 281: 251-260, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30824058

RESUMO

Fern spores are unicellular structures produced by the sporophyte generation that give rise to the haploid gametophyte. When released from the sporangium, spores are desiccation tolerant (DT) in the royal fern (Osmunda regalis) and contain fully developed chloroplasts. As a consequence, this type of spores is called chlorophyllous spores (CS). Upon transfer to germination conditions, CS initiate a process of imbibition that suppresses DT in 72 h, before the germination starts. In parallel to such change in DT, thylakoids undergo a profound remodelling in composition and function. Firstly, sustained quenching of chlorophyll fluorescence is relaxed, giving rise to photochemically active CS, while lipid composition shifts from that of a resting structure to a metabolically active cell. Basically trigalactolipids decreased in favour of monogalactolipids, with a parallel desaturation of fatty acids. Storage lipids such as triacylglycerol were quickly depleted. These results highlight the importance of the structure of thylakoids lipid as a key to protect membrane integrity during desiccation, together with the saturation of fatty acids and the constitutive chlorophyll quenching to prevent oxidative damage. The CS used here, in which the same cell shifts from DT to sensitive strategy in 72 h, reveal their potential as unicellular models for future studies on DT.


Assuntos
Cloroplastos/metabolismo , Gleiquênias/metabolismo , Esporos/metabolismo , Cloroplastos/fisiologia , Gleiquênias/fisiologia , Germinação/fisiologia , Esporos/fisiologia , Tilacoides/metabolismo , Tilacoides/fisiologia
16.
Physiol Plant ; 166(1): 134-147, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30838662

RESUMO

To better understand the coordination between dark and light reactions during the transition from C3 to C4 photosynthesis, we optimized a method for separating thylakoids from mesophyll (MC) and bundle sheath cells (BSCs) across different plant species. We grew six Paniceae grasses including representatives from the C3 , C3 -C4 and C4 photosynthetic types and all three C4 biochemical subtypes [nicotinamide adenine dinucleotide phosphate-dependent malic enzyme (NADP-ME), nicotinamide adenine dinucleotide-dependent malic enzyme (NAD-ME) and phosphoenolpyruvate carboxykinase (PEPCK)] in addition to Zea mays under control conditions (1000 µmol quanta m-2  s-1 and 400 ppm of CO2 ). Proteomics analysis of thylakoids under native conditions, using blue native polyacrylamide gel electrophoresis followed by liquid chromatography-mass spectrometry (LC-MS), demonstrated the presence of subunits of all light-reaction-related complexes in all species and cell types. C4 NADP-ME species showed a higher photosystems I/II ratio and a clear accumulation of the NADH dehydrogenase-like complexes in BSCs, while Cytb6 f was more abundant in BSCs of C4 NAD-ME species. The C4 PEPCK species showed no clear differences between cell types. Our study presents, for the first time, a good separation between BSC and MC for a C3 -C4 intermediate grass which did not show noticeable differences in the distribution of the thylakoid complexes. For the NADP-ME species Panicum antidotale, growth at glacial CO2 (180 ppm of CO2 ) had no effect on the distribution of the light-reaction complexes, while growth at low light (200 µmol quanta m-2  s-1 ) promoted the accumulation of light-harvesting proteins in both cell types. These results add to our understanding of thylakoid distribution across photosynthetic types and subtypes, and introduce thylakoid distribution between the MC and BSC of a C3 -C4 intermediate species.


Assuntos
Células do Mesofilo/metabolismo , Poaceae/metabolismo , Tilacoides/metabolismo , Malato Desidrogenase/metabolismo , Fosfoenolpiruvato Carboxilase/metabolismo , Fotossíntese/fisiologia , Poaceae/fisiologia
17.
Plant Cell Physiol ; 60(6): 1374-1385, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-30847493

RESUMO

Two mutants sensitive to high light for growth and impaired in NDH-1 activity were isolated from a transposon-tagged library of Synechocystis sp. strain PCC 6803. Both mutants were tagged in the ssl3451 gene encoding a hypothetical protein, which shares a significant homology with the Arabidopsis (Arabidopsis thaliana) CHLORORESPIRATORY REDUCTION 42 (CRR42). In Arabidopsis, CRR42 associates only with an NDH-1 hydrophilic arm assembly intermediate (NAI) of about 400 kDa (NAI400), one of total three NAIs (NAI800, NAI500 and NAI400), and its deletion has little, if any, effect on accumulation of any NAIs in the stroma. In comparison, the ssl3451 product was localized mainly in the cytoplasm and associates with two NAIs of about 300 kDa (NAI300) and 130 kDa (NAI130). Deletion of Ssl3451 reduced the abundance of the NAI300 complex to levels no longer visible on gels and of the NAI130 complex to a low level, thereby impeding the assembly process of NDH-1 hydrophilic arm. Further, Ssl3451 interacts with another assembly factor Ssl3829 and they have a similar effect on accumulation of NAIs and NdhI maturation factor Slr1097 in the cytoplasm. We thus propose that Ssl3451 plays an important role in accumulation of the NAI300 and NAI130 complexes in the cytoplasm via its interacting protein Ssl3829.


Assuntos
Proteínas de Bactérias/metabolismo , Complexo de Proteína do Fotossistema I/metabolismo , Synechocystis/metabolismo , Citoplasma/metabolismo , Tilacoides/metabolismo
18.
Plant Cell Physiol ; 60(6): 1386-1399, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-30847494

RESUMO

NdbA, one of the three type 2 NAD(P)H dehydrogenases (NDH-2) in Synechocystis sp. PCC 6803 (hereafter Synechocystis) was here localized to the thylakoid membrane (TM), unique for the three NDH-2s, and investigated with respect to photosynthetic and cellular redox metabolism. For this purpose, a deletion mutant (ΔndbA) and a complementation strain overexpressing NdbA (ΔndbA::ndbA) were constructed. It is demonstrated that NdbA is expressed at very low level in the wild-type (WT) Synechocystis under photoautotrophic (PA) growth whilst substantially higher expression occurs under light-activated heterotrophic growth (LAHG). The absence of NdbA resulted in non-optimal growth of Synechocystis under LAHG and concomitantly enhanced the expression of photoprotection-related flavodiiron proteins and carbon acquisition-related proteins as well as various transporters, but downregulated a few iron homeostasis-related proteins. NdbA overexpression, on the other hand, promoted photosynthetic pigmentation and functionality of photosystem I under LAHG conditions while distinct photoprotective and carbon concentrating proteins were downregulated. NdbA overexpression also exerted an effect on the expression of many signaling and gene regulation proteins. It is concluded that the amount and function of NdbA in the TM has a capacity to modulate the redox signaling of gene expression, but apparently has a major physiological role in maintaining iron homeostasis under LAHG conditions. LC-MS/MS data are available via ProteomeXchange with identifier PXD011671.


Assuntos
Proteínas de Bactérias/metabolismo , FMN Redutase/metabolismo , Synechocystis/metabolismo , Tilacoides/metabolismo , Transporte de Elétrons , Regulação da Expressão Gênica de Plantas , Luz , Microscopia Eletrônica de Transmissão , Fotossíntese , Synechocystis/enzimologia , Synechocystis/crescimento & desenvolvimento , Synechocystis/ultraestrutura , Tilacoides/enzimologia , Tilacoides/ultraestrutura
19.
Plant Physiol ; 179(4): 1779-1795, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30733257

RESUMO

The ecological success of diatoms, key contributors to photosynthesis, is partly based on their ability to perfectly balance efficient light harvesting and photoprotection. Diatoms contain higher numbers of antenna proteins than vascular plants for light harvesting and for photoprotection. These proteins are arranged in fucoxanthin-chlorophyll protein (FCP) complexes. The number of FCP complexes, their subunit composition, and their interactions in the thylakoid membranes remain elusive in different diatoms. We used the recently available genome sequence of the centric diatom Cyclotella cryptica to analyze gene sequences for putative light-harvesting proteins in C. meneghiniana, and to elucidate the FCP complex composition. We analyzed two pools of FCP complexes that were trimeric (FCPa) and nonameric (FCPb). FCPa was composed of four different trimeric subtypes. Two different nonameric FCPb complexes were present. All were distinguished by their polypeptide composition and partly by pigmentation. With use of a milder purification method, two fractions composed of different FCP complexes were isolated. One was enriched in FCPs incorporating the photoprotective subunit Lhcx1, such as the newly identified nonameric FCPb2 and the major trimeric FCPa4 complex, which are predetermined to be involved in energy-dependent nonphotochemical quenching. The other fraction contained mainly FCPs that were devoid of Lhcx1, FCPa3, and FCPb1. Both fractions also included small amounts of trimeric FCPa complexes with the centric diatom-specific Lhcx protein, Lhcx6_1, as subunit. Thus, the antenna organization of centric diatoms, as well as the distribution of different photoprotective Lhcx proteins, differs from that of other diatoms, as well as from plants.


Assuntos
Proteínas de Ligação à Clorofila/química , Diatomáceas/genética , Diatomáceas/metabolismo , Filogenia , Subunidades Proteicas/química , Tilacoides/metabolismo
20.
Physiol Plant ; 166(1): 288-299, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30793329

RESUMO

The PsbO protein is an essential extrinsic subunit of photosystem II, the pigment-protein complex responsible for light-driven water splitting. Water oxidation in photosystem II supplies electrons to the photosynthetic electron transfer chain and is accompanied by proton release and oxygen evolution. While the electron transfer steps in this process are well defined and characterized, the driving forces acting on the liberated protons, their dynamics and their destiny are all largely unknown. It was suggested that PsbO undergoes proton-induced conformational changes and forms hydrogen bond networks that ensure prompt proton removal from the catalytic site of water oxidation, i.e. the Mn4 CaO5 cluster. This work reports the purification and characterization of heterologously expressed PsbO from green algae Chlamydomonas reinhardtii and two isoforms from the higher plant Solanum tuberosum (PsbO1 and PsbO2). A comparison to the spinach PsbO reveals striking similarities in intrinsic protein fluorescence and CD spectra, reflecting the near-identical secondary structure of the proteins from algae and higher plants. Titration experiments using the hydrophobic fluorescence probe ANS revealed that eukaryotic PsbO proteins exhibit acid-base hysteresis. This hysteresis is a dynamic effect accompanied by changes in the accessibility of the protein's hydrophobic core and is not due to reversible oligomerization or unfolding of the PsbO protein. These results confirm the hypothesis that pH-dependent dynamic behavior at physiological pH ranges is a common feature of PsbO proteins and causes reversible opening and closing of their ß-barrel domain in response to the fluctuating acidity of the thylakoid lumen.


Assuntos
Chlamydomonas reinhardtii/metabolismo , Spinacia oleracea/metabolismo , Tilacoides/metabolismo , Ligações de Hidrogênio , Concentração de Íons de Hidrogênio , Complexo de Proteína do Fotossistema II/metabolismo
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