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1.
Int J Mol Sci ; 22(11)2021 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-34072887

RESUMO

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.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Cloroplastos/genética , Metaloendopeptidases/genética , Tilacoides/genética , Arabidopsis/enzimologia , Cloroplastos/enzimologia , Regulação da Expressão Gênica de Plantas/genética , Mutação/genética , Transporte Proteico/genética , Proteólise , Tilacoides/enzimologia
2.
FEBS Lett ; 595(14): 1876-1885, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34060653

RESUMO

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.


Assuntos
Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/metabolismo , Guanosina Trifosfato/metabolismo , Proteínas de Membrana/metabolismo , Nucleosídeo-Trifosfatase/metabolismo , Synechocystis/enzimologia , Tilacoides/enzimologia , Trifosfato de Adenosina/química , Proteínas de Bactérias/genética , Clonagem Molecular , Ensaios Enzimáticos , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Guanosina Trifosfato/química , Hidrólise , Cinética , Proteínas de Membrana/genética , Microscopia Eletrônica , Nucleosídeo-Trifosfatase/genética , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Synechocystis/genética , Synechocystis/ultraestrutura , Tilacoides/genética , Tilacoides/ultraestrutura
3.
J Biol Chem ; 296: 100217, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33839679

RESUMO

Heme oxygenase (HO) converts heme to carbon monoxide, biliverdin, and free iron, products that are essential in cellular redox signaling and iron recycling. In higher plants, HO is also involved in the biosynthesis of photoreceptor pigment precursors. Despite many common enzymatic reactions, the amino acid sequence identity between plant-type and other HOs is exceptionally low (∼19.5%), and amino acids that are catalytically important in mammalian HO are not conserved in plant-type HOs. Structural characterization of plant-type HO is limited to spectroscopic characterization by electron spin resonance, and it remains unclear how the structure of plant-type HO differs from that of other HOs. Here, we have solved the crystal structure of Glycine max (soybean) HO-1 (GmHO-1) at a resolution of 1.06 Å and carried out the isothermal titration calorimetry measurements and NMR spectroscopic studies of its interaction with ferredoxin, the plant-specific electron donor. The high-resolution X-ray structure of GmHO-1 reveals several novel structural components: an additional irregularly structured region, a new water tunnel from the active site to the surface, and a hydrogen-bonding network unique to plant-type HOs. Structurally important features in other HOs, such as His ligation to the bound heme, are conserved in GmHO-1. Based on combined data from X-ray crystallography, isothermal titration calorimetry, and NMR measurements, we propose the evolutionary fine-tuning of plant-type HOs for ferredoxin dependency in order to allow adaptation to dynamic pH changes on the stroma side of the thylakoid membrane in chloroplast without losing enzymatic activity under conditions of fluctuating light.


Assuntos
Ferredoxinas/química , Heme Oxigenase-1/química , Heme/química , Ferro/química , Proteínas de Plantas/química , Soja/química , Sequência de Aminoácidos , Biliverdina/química , Biliverdina/metabolismo , Monóxido de Carbono/química , Monóxido de Carbono/metabolismo , Domínio Catalítico , Cloroplastos/química , Cloroplastos/enzimologia , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Ferredoxinas/genética , Ferredoxinas/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Heme/metabolismo , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Ligação de Hidrogênio , Ferro/metabolismo , Simulação de Acoplamento Molecular , Ressonância Magnética Nuclear Biomolecular , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Soja/enzimologia , Soja/genética , Tilacoides/química , Tilacoides/enzimologia
4.
Proc Natl Acad Sci U S A ; 118(4)2021 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-33479170

RESUMO

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.


Assuntos
Aminoácidos/química , Arabidopsis/enzimologia , Complexo de Proteína do Fotossistema II/química , Superóxidos/química , Tilacoides/enzimologia , alfa-Tocoferol/química , Aminoácidos/metabolismo , Arabidopsis/genética , Arabidopsis/efeitos da radiação , Sítios de Ligação , Radical Hidroxila/química , Radical Hidroxila/metabolismo , Transferases Intramoleculares/química , Transferases Intramoleculares/genética , Transferases Intramoleculares/metabolismo , Ferro/química , Ferro/metabolismo , Luz , Modelos Moleculares , Mutação , Oxirredução , Oxigênio/química , Oxigênio/metabolismo , Fotossíntese/fisiologia , Fotossíntese/efeitos da radiação , Complexo de Proteína do Fotossistema II/genética , Complexo de Proteína do Fotossistema II/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Superóxidos/metabolismo , Termodinâmica , Thermosynechococcus/enzimologia , Thermosynechococcus/genética , Thermosynechococcus/efeitos da radiação , Tilacoides/genética , Tilacoides/efeitos da radiação , alfa-Tocoferol/metabolismo
5.
Int J Mol Sci ; 21(22)2020 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-33207833

RESUMO

In plant grana thylakoid membranes Photosystem II (PSII) associates with a variable number of antenna proteins (LHCII) to form different types of supercomplexes (PSII-LHCII), whose organization is dynamically adjusted in response to light cues, with the C2S2 more abundant in high-light and the C2S2M2 in low-light. Paired PSII-LHCII supercomplexes interacting at their stromal surface from adjacent thylakoid membranes were previously suggested to mediate grana stacking. Here, we present the cryo-electron microscopy maps of paired C2S2 and C2S2M2 supercomplexes isolated from pea plants grown in high-light and low-light, respectively. These maps show a different rotational offset between the two supercomplexes in the pair, responsible for modifying their reciprocal interaction and energetic connectivity. This evidence reveals a different way by which paired PSII-LHCII supercomplexes can mediate grana stacking at diverse irradiances. Electrostatic stromal interactions between LHCII trimers almost completely overlapping in the paired C2S2 can be the main determinant by which PSII-LHCII supercomplexes mediate grana stacking in plants grown in high-light, whereas the mutual interaction of stromal N-terminal loops of two facing Lhcb4 subunits in the paired C2S2M2 can fulfil this task in plants grown in low-light. The high-light induced accumulation of the Lhcb4.3 protein in PSII-LHCII supercomplexes has been previously reported. Our cryo-electron microscopy map at 3.8 Å resolution of the C2S2 supercomplex isolated from plants grown in high-light suggests the presence of the Lhcb4.3 protein revealing peculiar structural features of this high-light-specific antenna important for photoprotection.


Assuntos
Complexos de Proteínas Captadores de Luz/metabolismo , Luz , Ervilhas/enzimologia , Complexo de Proteína do Fotossistema II/metabolismo , Tilacoides/enzimologia , Complexos de Proteínas Captadores de Luz/química , Complexo de Proteína do Fotossistema II/química
6.
Plant Cell ; 32(5): 1589-1609, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32169961

RESUMO

Protein folding is a complex cellular process often assisted by chaperones, but it can also be facilitated by interactions with lipids. Disulfide bond formation is a common mechanism to stabilize a protein. This can help maintain functionality amid changes in the biochemical milieu, including those relating to energy-transducing membranes. Plastidic Type I Signal Peptidase 1 (Plsp1) is an integral thylakoid membrane signal peptidase that requires an intramolecular disulfide bond for in vitro activity. We have investigated the interplay between disulfide bond formation, lipids, and pH in the folding and activity of Plsp1. By combining biochemical approaches with a genetic complementation assay using Arabidopsis thaliana plants, we provide evidence that interactions with lipids in the thylakoid membrane have reconstitutive chaperoning activity toward Plsp1. Further, the disulfide bridge appears to prevent an inhibitory conformational change resulting from proton motive force-mimicking pH conditions. Broader implications related to the folding of proteins in energy-transducing membranes are discussed.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Membranas Intracelulares/enzimologia , Chaperonas Moleculares/metabolismo , Força Próton-Motriz , Serina Endopeptidases/metabolismo , Tilacoides/enzimologia , Proteínas de Arabidopsis/química , Ritmo Circadiano/efeitos dos fármacos , Cisteína/metabolismo , Dissulfetos/metabolismo , Ditiotreitol/farmacologia , Estabilidade Enzimática , Escherichia coli/metabolismo , Genes Supressores , Bicamadas Lipídicas/metabolismo , Modelos Biológicos , Mutação/genética , Oxirredução , Conformação Proteica , Serina Endopeptidases/química
7.
Biochim Biophys Acta Bioenerg ; 1861(2): 148135, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31821793

RESUMO

In forests, understory plants are usually exposed to sunflecks on timescales of seconds or minutes. However, it is unclear how understory plants acclimate to fluctuating light. In this study, we compared chlorophyll fluorescence, PSI redox state and the electrochromic shift signal under fluctuating light between an understory plant Paris polyphylla (Liliaceae) and a light-demanding plant Bletilla striata (Orchidaceae). Within the first seconds after transition from low to high light, PSI was highly oxidized in P. polyphylla but was highly reduced in B. striata, although both species could not generate a sufficient trans-thylakoid proton gradient (ΔpH). Furthermore, the outflow of electrons from PSI to O2 was not significant in P. polyphylla, as indicated by the P700 redox kinetics upon dark-to-light transition. Therefore, the different responses of PSI to fluctuating light between P. polyphylla and B. striata could not be explained by ΔpH formation or alternative electron transport. In contrast, upon a sudden transition from low to high light, electron flow from PSII was much lower in P. polyphylla than in B. striata, suggesting that the rapid oxidation of PSI in P. polyphylla was largely attributed to the lower PSII activity. We propose, for the first time, that down-regulation of PSII activity is an important strategy used by some understory angiosperms to cope with sunflecks.


Assuntos
Luz , Melanthiaceae/enzimologia , Complexo de Proteína do Fotossistema II/metabolismo , Força Próton-Motriz/fisiologia , Tilacoides/enzimologia , Transporte de Elétrons/fisiologia , Orchidaceae/enzimologia , Oxigênio/metabolismo
8.
Biochim Biophys Acta Bioenerg ; 1861(2): 148141, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31825808

RESUMO

Hetero-oligomeric membrane protein complexes form the electron transport chain (ETC) of oxygenic photosynthesis. The ETC complexes undertake the light-driven vectorial electron and proton transport reactions, which generate energy-rich ATP and electron-rich NADPH molecules for carbon fixation. The rate of photosynthetic electron transport depends on the availability of photons and the relative abundance of electron transport complexes. The relative abundance of the two photosystems, critical for the quantum efficiency of photosynthesis in changing light quality conditions, has been determined successfully by optical methods. Due to the lack of spectroscopic signatures, however, relatively little is known about the stoichiometry of other non-photosystem complexes in plant photosynthetic membrane. Here we determine the ratios of all major thylakoid-bound ETC complexes in Arabidopsis by a label-free quantitative mass spectrometry technique. The calculated stoichiometries are consistent with known subunit composition of complexes and current estimates of photosystem and cytochrome b6f concentrations. The implications of these stoichiometries for photosynthetic light harvesting and the partitioning of electrons between the linear and cyclic electron transport pathways of photosynthesis are discussed.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Complexo Citocromos b6f/metabolismo , Fotossíntese/fisiologia , Tilacoides/enzimologia
9.
Proc Natl Acad Sci U S A ; 116(35): 17316-17322, 2019 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-31409711

RESUMO

A descendant of the red algal lineage, diatoms are unicellular eukaryotic algae characterized by thylakoid membranes that lack the spatial differentiation of stroma and grana stacks found in green algae and higher plants. While the photophysiology of diatoms has been studied extensively, very little is known about the spatial organization of the multimeric photosynthetic protein complexes within their thylakoid membranes. Here, using cryo-electron tomography, proteomics, and biophysical analyses, we elucidate the macromolecular composition, architecture, and spatial distribution of photosystem II complexes in diatom thylakoid membranes. Structural analyses reveal 2 distinct photosystem II populations: loose clusters of complexes associated with antenna proteins and compact 2D crystalline arrays of dimeric cores. Biophysical measurements reveal only 1 photosystem II functional absorption cross section, suggesting that only the former population is photosynthetically active. The tomographic data indicate that the arrays of photosystem II cores are physically separated from those associated with antenna proteins. We hypothesize that the islands of photosystem cores are repair stations, where photodamaged proteins can be replaced. Our results strongly imply convergent evolution between the red and the green photosynthetic lineages toward spatial segregation of dynamic, functional microdomains of photosystem II supercomplexes.


Assuntos
Organismos Aquáticos/enzimologia , Proteínas de Bactérias/química , Diatomáceas/enzimologia , Complexo de Proteína do Fotossistema II/química , Tilacoides/enzimologia , Proteínas de Bactérias/metabolismo , Complexo de Proteína do Fotossistema II/metabolismo
10.
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
11.
J Plant Physiol ; 232: 94-99, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30537617

RESUMO

The carrier of carbonic anhydrase (CA) activity was detected in gel among low molecular mass proteins from pea, spinach and Arabidopsis, after nondenaturing electrophoresis in PAAG of the dodecyl-ß-d-maltoside treated PSII membranes (the fragments of thylakoid membrane containing PSII complexes). The elimination of Mn-stabilizing protein PsbO by treatment of PSII membranes with salts, did not lead to a decrease in CA activity observed in the gel although it reduced the amount of this protein down to 25% compared to the original sample. The isolated protein PsbO did not demonstrated CA activity. The distinguished features of CA activity of PSII membranes were as follows: 1) resistance to heating, 2) high sensitivity to ethoxyzolamide, the specific inhibitor of CA, and 3) stimulation of this activity by acetazolamide, another specific inhibitor of CA at low concentration of the latter. CA activity was not stimulated by acetazolamide in the PSII membranes samples from Arabidopsis thaliana mutants with knocked out gene At4g20990 encoding αCA4 (according to the nomenclature by Fabre et al., 2007). Taking into account the above data and our previous findings that the energy-dependent part of nonphotochemical quenching of chlorophyll a fluorescence is highly suppressed in that mutant, we suppose that thylakoid membranes of higher plants contain in the vicinity of PSII complex a true CA belonging to the α family of CAs.


Assuntos
Anidrases Carbônicas/metabolismo , Complexo de Proteína do Fotossistema II/metabolismo , Arabidopsis/enzimologia , Arabidopsis/metabolismo , Arabidopsis/fisiologia , Clorofila A/metabolismo , Eletroforese em Gel de Poliacrilamida , Técnicas de Silenciamento de Genes , Immunoblotting , Complexo de Proteína do Fotossistema II/fisiologia , Tilacoides/enzimologia , Tilacoides/metabolismo
12.
New Phytol ; 222(1): 206-217, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30383301

RESUMO

Carbonic anhydrases (CAs) are involved in CO2 uptake and conversion, a fundamental process in photosynthetic organisms. Nevertheless, the mechanism underlying the regulation of CO2 uptake and intracellular conversion in cyanobacteria is largely unknown. We report the characterization of a previously unrecognized thylakoid-located CA Slr0051 (EcaB) from the cyanobacterium Synechocystis sp. PCC 6803, which possesses CA activity to regulate CO2 uptake. Inactivation of ecaB stimulated CO2 hydration in the thylakoids, suppressed by the classical CA inhibitor acetazolamide. Absence of ecaB increased the reduced state of the photosynthetic electron transport system, lowered the rate of photosynthetic O2 evolution at high light (HL) and pH, and decreased the cellular affinity for extracellular inorganic carbon. Furthermore, EcaB was upregulated in cells grown at limiting CO2 concentration or HL in tandem with CupA. EcaB is mainly located in the thylakoid membranes where it interacts with CupA and CupB involved in CO2 uptake by converting it to bicarbonate. We propose that modulation of the EcaB level and activity in response to CO2 changes, illumination or pH reversibly regulates its conversion to HCO3 by the two CO2 -uptake systems (CupA, CupB), dissipating the excess HCO3 - and alleviating photoinhibition, and thereby optimizes photosynthesis, especially under HL and alkaline conditions.


Assuntos
Dióxido de Carbono/metabolismo , Anidrases Carbônicas/metabolismo , Synechocystis/metabolismo , Tilacoides/enzimologia , Proteínas de Bactérias/metabolismo , Bicarbonatos/metabolismo , Biocatálise , Concentração de Íons de Hidrogênio , Luz , Mutação/genética , Fotossíntese/efeitos da radiação , Complexo de Proteína do Fotossistema II/metabolismo , Ligação Proteica , Synechocystis/crescimento & desenvolvimento , Tilacoides/metabolismo , Regulação para Cima/efeitos da radiação
13.
J Proteomics ; 192: 125-136, 2019 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-30170113

RESUMO

Chloroplast APX isoforms display controversial roles as H2O2 scavengers and signaling players in response to abiotic stress and conclusive results are lacking. We tested the hypothesis that thylakoidal APX displays an important role for drought tolerance, especially by regulating abundance of essential protein species. For this, OsApx8 RNAi-silenced rice (apx8) and non-transformed plants (NT) were exposed to mild water deficit. The drought-sensitivity in apx8 plants was revealed by decreases in shoot growth, relative water content and photosynthesis, which was accompanied by increased membrane damage, all compared to NT plants. This higher sensitivity of apx8 plants to mild drought stress was also related to a lower accumulation of important protein species involved in several metabolic processes, especially photosynthesis, photorespiration and redox metabolism. Despite apx8 plants have displayed an effective induction of compensatory antioxidant mechanisms in well-watered conditions, it was not enough to maintain H2O2 homeostasis and avoid oxidative and physiological disturbances under mild drought conditions. Thus, thylakoidal APX is involved in several phenotypic modifications at proteomic profile level, possibly via a H2O2-induced signaling mechanism. Consequently, this APX isoform is crucial for rice plants effectively cope with a mild drought condition. BIOLOGICAL SIGNIFICANCE: This work provides for the first time an integrative study involving proteomic, physiological and biochemical analyses directed to elucidation of thylakoidal APX roles for drought tolerance in rice plants. Our data reveal that this enzyme is crucial for maintaining of growth and photosynthesis under mild water deficit conditions. This essential role is related to maintaining of H2O2 homeostasis and accumulation of essential proteins involved in several important metabolic pathways. Remarkably, for drought resistance was essential the accumulation of proteins involved with metabolism of photosynthesis, signaling, carbohydrates, protein synthesis/degradation and stress. These results can contribute to understand the role of chloroplast ascorbate peroxidases in drought tolerance, highlighting the physiological importance of key proteins in this process.


Assuntos
Ascorbato Peroxidases/metabolismo , Oryza/enzimologia , Estresse Oxidativo , Proteínas de Plantas/metabolismo , Tilacoides/enzimologia , Desidratação , Peróxido de Hidrogênio/metabolismo , Fotossíntese
15.
Plant Physiol ; 178(3): 1065-1080, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30237207

RESUMO

Deg proteases are involved in protein quality control in prokaryotes. Of the three Arabidopsis (Arabidopsis thaliana) homologs, Deg1, Deg5, and Deg8, located in the thylakoid lumen, Deg1 forms a homohexamer, whereas Deg5 and Deg8 form a heterocomplex. Both Deg1 and Deg5-Deg8 were shown separately to degrade photosynthetic proteins during photoinhibition. To investigate whether Deg1 and Deg5-Deg8 are redundant, a full set of Arabidopsis Deg knockout mutants were generated and their phenotypes were compared. Under all conditions tested, deg1 mutants were affected more than the wild type and deg5 and deg8 mutants. Moreover, overexpression of Deg5-Deg8 could only partially compensate for the loss of Deg1. Comparative proteomics of deg1 mutants revealed moderate up-regulation of thylakoid proteins involved in photoprotection, assembly, repair, and housekeeping and down-regulation of those that form photosynthetic complexes. Quantification of protein levels in the wild type revealed that Deg1 was 2-fold more abundant than Deg5-Deg8. Moreover, recombinant Deg1 displayed higher in vitro proteolytic activity. Affinity enrichment assays revealed that Deg1 was precipitated with very few interacting proteins, whereas Deg5-Deg8 was associated with a number of thylakoid proteins, including D1, OECs, LHCBs, Cyt b 6 f, and NDH subunits, thus implying that Deg5-Deg8 is capable of binding substrates but is unable to degrade them efficiently. This work suggests that differences in protein abundance and proteolytic activity underlie the differential importance of Deg1 and Deg5-Deg8 protease complexes observed in vivo.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Proteostase , Serina Endopeptidases/metabolismo , Tilacoides/enzimologia , Arabidopsis/genética , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Técnicas de Inativação de Genes , Mutação , Fenótipo , Fotossíntese , Folhas de Planta/enzimologia , Folhas de Planta/genética , Folhas de Planta/fisiologia , Proteômica , Plântula/enzimologia , Plântula/genética , Plântula/fisiologia , Serina Endopeptidases/genética , Tilacoides/fisiologia
16.
Biochim Biophys Acta Bioenerg ; 1859(4): 292-299, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29410217

RESUMO

It is known, that the multi-subunit complex of photosystem II (PSII) and some of its single proteins exhibit carbonic anhydrase activity. Previously, we have shown that PSII depletion of HCO3-/CO2 as well as the suppression of carbonic anhydrase activity of PSII by a known inhibitor of α­carbonic anhydrases, acetazolamide (AZM), was accompanied by a decrease of electron transport rate on the PSII donor side. It was concluded that carbonic anhydrase activity was required for maximum photosynthetic activity of PSII but it was not excluded that AZM may have two independent mechanisms of action on PSII: specific and nonspecific. To investigate directly the specific influence of carbonic anhydrase inhibition on the photosynthetic activity in PSII we used another known inhibitor of α­carbonic anhydrase, trifluoromethanesulfonamide (TFMSA), which molecular structure and physicochemical properties are quite different from those of AZM. In this work, we show for the first time that TFMSA inhibits PSII carbonic anhydrase activity and decreases rates of both the photo-induced changes of chlorophyll fluorescence yield and the photosynthetic oxygen evolution. The inhibitory effect of TFMSA on PSII photosynthetic activity was revealed only in the medium depleted of HCO3-/CO2. Addition of exogenous HCO3- or PSII electron donors led to disappearance of the TFMSA inhibitory effect on the electron transport in PSII, indicating that TFMSA inhibition site was located on the PSII donor side. These results show the specificity of TFMSA action on carbonic anhydrase and photosynthetic activities of PSII. In this work, we discuss the necessity of carbonic anhydrase activity for the maximum effectiveness of electron transport on the donor side of PSII.


Assuntos
Anidrases Carbônicas/metabolismo , Elétrons , Mesilatos/farmacologia , Ervilhas/enzimologia , Fotossíntese/fisiologia , Complexo de Proteína do Fotossistema II/metabolismo , Acetazolamida/farmacologia , Bicarbonatos/metabolismo , Dióxido de Carbono/metabolismo , Inibidores da Anidrase Carbônica/farmacologia , Clorofila/metabolismo , Clorofila A , Transporte de Elétrons/efeitos dos fármacos , Transporte de Elétrons/efeitos da radiação , Concentração de Íons de Hidrogênio , Cinética , Luz , Oxigênio/metabolismo , Ervilhas/efeitos dos fármacos , Ervilhas/efeitos da radiação , Complexo de Proteína do Fotossistema II/antagonistas & inibidores , Tilacoides/efeitos dos fármacos , Tilacoides/enzimologia , Tilacoides/efeitos da radiação
17.
Subcell Biochem ; 87: 259-286, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29464563

RESUMO

In nature, plants are continuously exposed to varying environmental conditions. They have developed a wide range of adaptive mechanisms, which ensure their survival and maintenance of stable photosynthetic performance. Photosynthesis is delicately regulated at the level of the thylakoid membrane of chloroplasts and the regulatory mechanisms include a reversible formation of a large variety of specific protein-protein complexes, supercomplexes or even larger assemblies known as megacomplexes. Revealing their structures is crucial for better understanding of their function and relevance in photosynthesis. Here we focus our attention on the isolation and a structural characterization of various large protein supercomplexes and megacomplexes, which involve Photosystem II and Photosystem I, the key constituents of photosynthetic apparatus. The photosystems are often attached to other protein complexes in thylakoid membranes such as light harvesting complexes, cytochrome b 6 f complex, and NAD(P)H dehydrogenase. Structural models of individual supercomplexes and megacomplexes provide essential details of their architecture, which allow us to discuss their function as well as physiological significance.


Assuntos
Fotossíntese/fisiologia , Complexo de Proteína do Fotossistema I , Complexo de Proteína do Fotossistema II , Tilacoides/enzimologia , Complexo de Proteína do Fotossistema I/química , Complexo de Proteína do Fotossistema I/metabolismo , Complexo de Proteína do Fotossistema II/química , Complexo de Proteína do Fotossistema II/metabolismo
18.
Biochim Biophys Acta Bioenerg ; 1859(2): 129-136, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29174010

RESUMO

Light-induced Fourier transformed infrared (FTIR) difference spectroscopy is a powerful method to study the structures and reactions of redox cofactors involved in the photosynthetic electron transport chain. So far, most of the FTIR studies of the reactions of oxygenic photosynthesis have been performed using isolated photosystem I (PSI) and photosystem II (PSII) preparations, which, however, could be modified during isolation procedures. In this study, we developed a methodology to evaluate the photosynthetic activities of thylakoids using FTIR spectroscopy. FTIR difference spectra upon successive flashes using thylakoids from spinach exhibited signals typical of the S-state cycle at the Mn4CaO5 cluster and QB reactions in PSII with period-four and -two oscillations, respectively. Similar measurement in the presence of an artificial quinone as an exogenous electron acceptor showed features specific to the S-state cycle. Simulations of the oscillation patterns provided the quantum efficiencies of the S-state cycle and electron transfer in PSII. Moreover, FTIR measurement under continuous illumination on thylakoids in the presence of DCMU showed signals due to QA reduction and P700 oxidation simultaneously. From the relative amplitudes of marker bands of QA- and P700+, the molar ratio of photoactive PSII and PSI centers in thylakoids was estimated. FTIR analyses of the photo-reactions in thylakoids, which are more intact than isolated photosystems, will be useful in investigations of the photosynthetic mechanism especially by genetic modification of photosystem proteins.


Assuntos
Fotossíntese , Complexo de Proteína do Fotossistema II/química , Spinacia oleracea/enzimologia , Tilacoides/enzimologia , Complexo de Proteína do Fotossistema II/genética , Complexo de Proteína do Fotossistema II/metabolismo , Espectroscopia de Infravermelho com Transformada de Fourier , Spinacia oleracea/genética , Tilacoides/genética
19.
Plant Physiol Biochem ; 122: 102-112, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29207281

RESUMO

Lipoxygenases (LOXs) are non-haem iron-containing dioxygenases that catalyse oxygenation of polyunsaturated fatty acids. This reaction is the first step in biosynthesis of oxylipins, which play important and diverse roles in stress response. In this study, we identified four LOX genes (PcLOXA, B, C, D) in chilling-sensitive runner bean (Phaseolus coccineus L.) plant and analyzed their expression patterns during long term dark-chilling (4 °C) stress and during day/night (21ºC/4 °C) temperature fluctuations. Three of the four identified LOX genes, namely PcLOXA, PcLOXB and PcLOXD, were induced by wounding stress, while only the PcLOXA was induced by dark-chilling of both detached (wounded) leaves and whole plants. We identified PcLOXA as a chloroplast-targeted LOX protein and investigated its expression during chilling stress in terms of abundance, localization inside chloroplasts and interactions with the thylakoid membranes. The analysis by immunogold electron microscopy has shown that more than 60% of detectable PcLOXA protein was associated with thylakoids, and dark-chilling of leaves resulted in increased amounts of this protein detected within grana margins of thylakoids. This effect was reversible under subsequent photo-activation of chilled leaves. PcLOXA binding to thylakoids is not mediated by the posttranslational modification but rather is based on direct interactions of the protein with membrane lipids; the binding strength increases under dark-chilling conditions.


Assuntos
Temperatura Baixa , Luz , Lipoxigenase/metabolismo , Phaseolus/enzimologia , Proteínas de Plantas/metabolismo , Tilacoides/enzimologia
20.
J Exp Bot ; 68(18): 5029-5043, 2017 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-28992187

RESUMO

Membrane proteins that are imported into chloroplasts must be accurately routed in order to establish and maintain the highly differentiated membranes characteristic of these organelles. Little is known about the targeting information or pathways involved, especially in the case of proteins with multiple transmembrane domains. We have studied targeting of the SCY components of the two SEC translocases in chloroplasts. SCY1 and SCY2 share a similar, highly conserved structure with 10 transmembrane domains, but are targeted to different membranes: the thylakoids and inner envelope, respectively. We used protoplast transfections and a confocal microscopy imaging assay in combination with a domain-swapping approach to investigate sorting pathways and identify important targeting elements in these proteins. We show that the N-terminal region of SCY1 contains targeting determinants that allow SCY1 to be recruited to the signal-recognition particle pathway. In addition, substituting the N-terminal region of SCY1 for the N-terminal region of SCY2 causes SCY2 to be displaced out of the inner envelope. The region of SCY2 that contains transmembrane domains 3 and 4 is necessary for localization to the inner envelope and may serve as a membrane anchor, enhancing the integration of other transmembrane domains via either stop-transfer or post-import mechanisms.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Proteínas de Cloroplastos/metabolismo , Proteínas de Membrana/metabolismo , Canais de Translocação SEC/metabolismo , Sequência de Aminoácidos , Arabidopsis/citologia , Arabidopsis/genética , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Proteínas de Cloroplastos/genética , Cloroplastos/enzimologia , Genes Reporter , Modelos Biológicos , Transporte Proteico , Protoplastos , Proteínas Recombinantes de Fusão , Canais de Translocação SEC/genética , Alinhamento de Sequência , Partícula de Reconhecimento de Sinal , Tilacoides/enzimologia
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