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1.
Plant Physiol ; 187(3): 1325-1340, 2021 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-34618018

RESUMO

Thioredoxins (Trxs) are disulfide oxidoreductases that regulate many biological processes. The m-type thioredoxin (TrxA) is the only Trx present in all oxygenic photosynthetic organisms. Extensive biochemical and proteomic analyses have identified many TrxA target proteins in different photosynthetic organisms. However, the precise function of this essential protein in vivo is still poorly known. In this study, we generated a conditional Synechocystis sp. PCC 6803 mutant strain (STXA2) using an on-off promoter that is able to survive with only 2% of the TrxA level of the wild-type (WT) strain. STXA2 characterization revealed that TrxA depletion results in growth arrest and pronounced impairment of photosynthesis and the Calvin-Benson-Bassham (CBB) cycle. Analysis of the in vivo redox state of the bifunctional enzyme fructose-1,6-bisphosphatase/sedoheptulose-1,7-bisphosphatase showed higher levels of oxidation that affected enzyme activity in STXA2. This result implies that TrxA-mediated redox regulation of the CBB cycle is conserved in both cyanobacteria and chloroplasts, although the targets have different evolutionary origins. The STXA2 strain also accumulated more reactive oxygen species and was more sensitive to oxidative stress than the WT. Analysis of the in vivo redox state of 2-Cys peroxiredoxin revealed full oxidation, corresponding with TrxA depletion. Overall, these results indicate that depletion of TrxA in STXA2 greatly alters the cellular redox state, interfering with essential processes such as photosynthetic machinery operativity, carbon assimilation, and oxidative stress response. The TrxA regulatory role appears to be conserved along the evolution of oxygenic photosynthetic organisms.


Assuntos
Proteínas de Bactérias/metabolismo , Ciclo do Carbono , Tiorredoxinas de Cloroplastos/metabolismo , Estresse Oxidativo , Fotossíntese , Synechocystis/metabolismo , Synechocystis/enzimologia
2.
Plant J ; 108(1): 120-133, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34288193

RESUMO

Thioredoxins (TRXs) are well-known redox signalling players, which carry out post-translational modifications in target proteins. Chloroplast TRXs are divided into different types and have central roles in light energy uptake and the regulation of primary metabolism. The isoforms TRX m1, m2, and m4 from Arabidopsis thaliana are considered functionally related. Knowing their key position in the hub of plant metabolism, we hypothesized that the impairment of the TRX m signalling would not only have harmful consequences on chloroplast metabolism but also at different levels of plant development. To uncover the physiological and developmental processes that depend on TRX m signalling, we carried out a comprehensive study of Arabidopsis single, double, and triple mutants defective in the TRX m1, m2, and m4 proteins. As light and redox signalling are closely linked, we investigated the response to high light (HL) of the plants that are gradually compromised in TRX m signalling. We provide experimental evidence relating the lack of TRX m and the appearance of novel phenotypic features concerning mesophyll structure, stomata biogenesis, and stomatal conductance. We also report new data indicating that the isoforms of TRX m fine-tune the response to HL, including the accumulation of the protective pigment anthocyanin. These results reveal novel signalling functions for the TRX m and underline their importance for plant growth and fulfilment of the acclimation/response to HL conditions.


Assuntos
Arabidopsis/fisiologia , Tiorredoxinas de Cloroplastos/metabolismo , Transdução de Sinais , Antocianinas/metabolismo , Arabidopsis/genética , Arabidopsis/efeitos da radiação , Clorofila/metabolismo , Cloroplastos/metabolismo , Fluorescência , Luz , Mutação , Oxirredução , Folhas de Planta/genética , Folhas de Planta/fisiologia , Folhas de Planta/efeitos da radiação , Estômatos de Plantas/genética , Estômatos de Plantas/fisiologia , Estômatos de Plantas/efeitos da radiação , Isoformas de Proteínas
3.
J Exp Bot ; 72(13): 4949-4964, 2021 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-33963398

RESUMO

In plants, there is a complex interaction between carbon (C) and nitrogen (N) metabolism, and its coordination is fundamental for plant growth and development. Here, we studied the influence of thioredoxin (Trx) m on C and N partitioning using tobacco plants overexpressing Trx m from the chloroplast genome. The transgenic plants showed altered metabolism of C (lower leaf starch and soluble sugar accumulation) and N (with higher amounts of amino acids and soluble protein), which pointed to an activation of N metabolism at the expense of carbohydrates. To further delineate the effect of Trx m overexpression, metabolomic and enzymatic analyses were performed on these plants. These results showed an up-regulation of the glutamine synthetase-glutamate synthase pathway; specifically tobacco plants overexpressing Trx m displayed increased activity and stability of glutamine synthetase. Moreover, higher photorespiration and nitrate accumulation were observed in these plants relative to untransformed control plants, indicating that overexpression of Trx m favors the photorespiratory N cycle rather than primary nitrate assimilation. Taken together, our results reveal the importance of Trx m as a molecular mediator of N metabolism in plant chloroplasts.


Assuntos
Tiorredoxinas de Cloroplastos , Tabaco , Carbono/metabolismo , Tiorredoxinas de Cloroplastos/metabolismo , Cloroplastos/metabolismo , Regulação da Expressão Gênica de Plantas , Nitrogênio/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Tabaco/genética , Tabaco/metabolismo
4.
Plant J ; 107(2): 434-447, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33930214

RESUMO

Thioredoxins (TRXs) are ubiquitous disulfide oxidoreductases structured according to a highly conserved fold. TRXs are involved in a myriad of different processes through a common chemical mechanism. Plant TRXs evolved into seven types with diverse subcellular localization and distinct protein target selectivity. Five TRX types coexist in the chloroplast, with yet scarcely described specificities. We solved the crystal structure of a chloroplastic z-type TRX, revealing a conserved TRX fold with an original electrostatic surface potential surrounding the redox site. This recognition surface is distinct from all other known TRX types from plant and non-plant sources and is exclusively conserved in plant z-type TRXs. We show that this electronegative surface endows thioredoxin z (TRXz) with a capacity to activate the photosynthetic Calvin-Benson cycle enzyme phosphoribulokinase. The distinct electronegative surface of TRXz thereby extends the repertoire of TRX-target recognitions.


Assuntos
Proteínas de Algas/química , Tiorredoxinas de Cloroplastos/química , Proteínas de Algas/metabolismo , Chlamydomonas reinhardtii/metabolismo , Tiorredoxinas de Cloroplastos/metabolismo , Cloroplastos/metabolismo , Cristalografia , Oxirredução , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Eletricidade Estática
5.
Plant Cell ; 32(12): 3866-3883, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33037145

RESUMO

In addition to linear electron transport, photosystem I cyclic electron transport (PSI-CET) contributes to photosynthesis and photoprotection. In Arabidopsis (Arabidopsis thaliana), PSI-CET consists of two partially redundant pathways, one of which is the PROTON GRADIENT REGULATION5 (PGR5)/PGR5-LIKE PHOTOSYNTHETIC PHENOTYPE1 (PGRL1)-dependent pathway. Although the physiological significance of PSI-CET is widely recognized, the regulatory mechanism behind these pathways remains largely unknown. Here, we report on the regulation of the PGR5/PGRL1-dependent pathway by the m-type thioredoxins (Trx m). Genetic and phenotypic characterizations of multiple mutants indicated the physiological interaction between Trx m and the PGR5/PGRL1-dependent pathway in vivo. Using purified Trx proteins and ruptured chloroplasts, in vitro, we showed that the reduced form of Trx m specifically decreased the PGR5/PGRL1-dependent plastoquinone reduction. In planta, Trx m4 directly interacted with PGRL1 via disulfide complex formation. Analysis of the transgenic plants expressing PGRL1 Cys variants demonstrated that Cys-123 of PGRL1 is required for Trx m4-PGRL1 complex formation. Furthermore, the Trx m4-PGRL1 complex was transiently dissociated during the induction of photosynthesis. We propose that Trx m directly regulates the PGR5/PGRL1-dependent pathway by complex formation with PGRL1.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Tiorredoxinas de Cloroplastos/metabolismo , Proteínas de Membrana/metabolismo , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Plastoquinona/metabolismo , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Tiorredoxinas de Cloroplastos/genética , Cloroplastos/metabolismo , Dissulfetos/metabolismo , Transporte de Elétrons , Proteínas de Membrana/genética , Fotossíntese , Complexo de Proteínas do Centro de Reação Fotossintética/genética , Complexo de Proteína do Fotossistema I/metabolismo , Plantas Geneticamente Modificadas
6.
Plant Physiol ; 184(3): 1291-1302, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32917772

RESUMO

In response to light, plants efficiently induce photosynthesis. Light activation of thiol enzymes by the thioredoxin (Trx) systems and cyclic electron transport by the PROTON GRADIENT REGULATION5 (PGR5)-dependent pathway contribute substantially to regulation of photosynthesis. Arabidopsis (Arabidopsis thaliana) mutants lacking f-type Trxs (trx f1f2) show delayed activation of carbon assimilation due to impaired photoreduction of Calvin-Benson cycle enzymes. To further study regulatory mechanisms that contribute to efficiency during the induction of photosynthesis, we analyzed the contributions of PSI donor- and acceptor-side regulation in the trx f1f2 mutant background. The cytochrome b 6 f complex is involved in PSI donor-side regulation, whereas PGR5-dependent PSI cyclic electron transport is required for both donor and acceptor functions. Introduction of the pgr1 mutation, which is conditionally defective in cytochrome b 6 f complex activity, into the trx f1f2 mutant background did not further affect the induction of photosynthesis, but the combined deficiency of Trx f and PGR5 severely impaired photosynthesis and suppressed plant growth under long-day conditions. In the pgr5 trx f1f2 mutant, the acceptor-side of PSI was almost completely reduced, and quantum yields of PSII and PSI hardly increased during the induction of photosynthesis. We also compared the photoreduction of thiol enzymes between the trx f1f2 and pgr5 trxf1f2 mutants. The pgr5 mutation did not result in further impaired photoreduction of Calvin-Benson cycle enzymes or ATP synthase in the trx f1f2 mutant background. These results indicated that acceptor-side limitations in the pgr5 trx f1f2 mutant suppress photosynthesis initiation, suggesting that PGR5 is required for efficient photosynthesis induction.


Assuntos
Arabidopsis/crescimento & desenvolvimento , Arabidopsis/genética , Arabidopsis/metabolismo , Tiorredoxinas de Cloroplastos/metabolismo , Transporte de Elétrons/fisiologia , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Complexo de Proteína do Fotossistema I/metabolismo , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Variação Genética , Genótipo , Mutação
7.
Plant J ; 104(3): 718-734, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32772439

RESUMO

Various regulatory mechanisms have evolved in plants to optimize photosynthetic activity under fluctuating light. Thioredoxins (TRX) are members of the regulatory network balancing activities of light and carbon fixation reactions in chloroplasts. We have studied the impact of two chloroplast TRX systems, the ferredoxin-dependent TRX reductase (FTR) and the NADPH-dependent TRX reductase C (NTRC) on regulation of photosynthesis by mutants lacking or overexpressing a component of either system. Plants were subjected to image-based phenotyping and chlorophyll fluorescence measurements that allow long-term monitoring of the development and photosynthetic activity of the rosettes, respectively. Our experiments demonstrate that NTRC and FTR systems respond differently to variation of light intensity. NTRC was an indispensable regulator of photosynthesis in young leaves, at light-intensity transitions and under low light intensities limiting photosynthesis, whereas steady-state exposure of plants to growth or higher light intensities diminished the need of NTRC in regulation of photosynthesis. In fluctuating light, overexpression of NTRC increased the quantum yield of Photosystem II (YII) at low light and stimulated the relaxation of non-photochemical quenching (NPQ) after high light exposure, indicating that overexpression of NTRC improves leaf capacity to convert light energy to chemical energy under these conditions. Overexpression of chimeric protein (NTR-TRXf) containing both the thioredoxin reductase and TRXf activity on an ntrc mutant background, did not completely recover either growth or steady-state photosynthetic activity, whereas OE-NTR-TRXf plants exposed to fluctuating light regained the wild-type level of Y(II) and NPQ.


Assuntos
Arabidopsis/fisiologia , Tiorredoxinas de Cloroplastos/metabolismo , Fotossíntese/fisiologia , Folhas de Planta/fisiologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Tiorredoxinas de Cloroplastos/genética , Luz , Plantas Geneticamente Modificadas , Tiorredoxina Dissulfeto Redutase/genética , Tiorredoxina Dissulfeto Redutase/metabolismo
8.
Biochim Biophys Acta Bioenerg ; 1861(11): 148261, 2020 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-32659266

RESUMO

The activity of the molecular motor enzyme, chloroplast ATP synthase, is regulated in a redox-dependent manner. The γ subunit, CF1-γ, is the central shaft of this enzyme complex and possesses the redox-active cysteine pair, which is reduced by thioredoxin (Trx). In light conditions, Trx transfers the reducing equivalent obtained from the photosynthetic electron transfer system to the CF1-γ. Previous studies showed that the light-dependent reduction of CF1-γ is more rapid than those of other Trx target proteins in the stroma. Although there are multiple Trx isoforms in chloroplasts, it is not well understood as to which chloroplast Trx isoform primarily contributes to the reduction of CF1-γ, especially under physiological conditions. We therefore performed direct assessment of the CF1-γ reduction capacity of each of the Trx isoforms. The kinetic analysis of the reduction process showed no significant difference in the reduction efficiency between two major chloroplast Trxs, namely Trx-f and Trx-m. Based on the thorough analyses of the CF1-γ redox dynamics in Arabidopsis thaliana Trx mutant plants, we found that lack of Trx-f or Trx-m had no significant impact on the in vivo light-dependent reduction of CF1-γ. The results showed that CF1-γ can accept the reducing power from both Trx-f and Trx-m in chloroplasts.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , ATPases de Cloroplastos Translocadoras de Prótons/química , ATPases de Cloroplastos Translocadoras de Prótons/metabolismo , Tiorredoxinas de Cloroplastos/metabolismo , Cloroplastos/metabolismo , Fotossíntese , Transporte de Elétrons , Cinética , Oxirredução , Isoformas de Proteínas
9.
Int J Mol Sci ; 21(9)2020 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-32397215

RESUMO

Water deficit caused by osmotic stress and drought limits crop yield and tree growth worldwide. Screening and identifying candidate genes from stress-resistant species are a genetic engineering strategy to increase drought resistance. In this study, an increased concentration of mannitol resulted in elevated expression of thioredoxin f (KcTrxf) in the nonsecretor mangrove species Kandelia candel. By means of amino acid sequence and phylogenetic analysis, the mangrove Trx was classified as an f-type thioredoxin. Subcellular localization showed that KcTrxf localizes to chloroplasts. Enzymatic activity characterization revealed that KcTrxf recombinant protein possesses the disulfide reductase function. KcTrxf overexpression contributes to osmotic and drought tolerance in tobacco in terms of fresh weight, root length, malondialdehyde (MDA) content, and hydrogen peroxide (H2O2) production. KcTrxf was shown to reduce the stomatal aperture by enhancing K+ efflux in guard cells, which increased the water-retaining capacity in leaves under drought conditions. Notably, the abscisic acid (ABA) sensitivity was increased in KcTrxf-transgenic tobacco, which benefits plants exposed to drought by reducing water loss by promoting stomatal closure. KcTrxf-transgenic plants limited drought-induced H2O2 in leaves, which could reduce lipid peroxidation and retain the membrane integrity. Additionally, glutathione (GSH) contributing to reactive oxygen species (ROS) scavenging and transgenic plants are more efficient at regenerating GSH from oxidized glutathione (GSSG) under conditions of drought stress. Notably, KcTrxf-transgenic plants had increased glucose and fructose contents under drought stress conditions, presumably resulting from KcTrxf-promoted starch degradation under water stress. We conclude that KcTrxf contributes to drought tolerance by increasing the water status, by enhancing osmotic adjustment, and by maintaining ROS homeostasis in transgene plants.


Assuntos
Adaptação Fisiológica/efeitos dos fármacos , Tiorredoxinas de Cloroplastos/genética , Tiorredoxinas de Cloroplastos/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Rhizophoraceae/química , Tabaco/metabolismo , Ácido Abscísico/metabolismo , Ácido Abscísico/farmacologia , Sequência de Aminoácidos , Secas , Frutose/metabolismo , Glucose/metabolismo , Glutationa/metabolismo , Peróxido de Hidrogênio/metabolismo , Malondialdeído/metabolismo , Manitol/toxicidade , NADH NADPH Oxirredutases/metabolismo , Pressão Osmótica , Filogenia , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/metabolismo , Estômatos de Plantas/citologia , Estômatos de Plantas/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Análise de Sequência , Tabaco/efeitos dos fármacos , Regulação para Cima , Água/metabolismo
10.
J Biol Chem ; 295(1): 170-180, 2020 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-31776187

RESUMO

Calcium (Ca2+) and redox signaling enable cells to quickly adapt to changing environments. The signaling protein calredoxin (CRX) from the green alga Chlamydomonas reinhardtii is a chloroplast-resident thioredoxin having Ca2+-dependent activity and harboring a unique combination of an EF-hand domain connected to a typical thioredoxin-fold. Using small-angle X-ray scattering (SAXS), FRET, and NMR techniques, we found that Ca2+-binding not only induces a conformational change in the EF-hand domain, but also in the thioredoxin domain, translating into the onset of thioredoxin redox activity. Functional analyses of CRX with genetically altered EF-hands revealed that EF-hand 4 is important for mediating the communication between the two domains. Moreover, we crystallized a variant (C174S) of the CRX target protein peroxiredoxin 1 (PRX1) at 2.4 Å resolution, modeled the interaction complex of the two proteins, and analyzed it by cross-linking and MS analyses, revealing that the interaction interface is located close to the active sites of both proteins. Our findings shed light on the Ca2+ binding-induced changes in CRX structure in solution at the level of the overall protein and individual domains and residues.


Assuntos
Sinalização do Cálcio , Proteínas de Ligação ao Cálcio/metabolismo , Tiorredoxinas de Cloroplastos/metabolismo , Motivos EF Hand , Proteínas de Ligação ao Cálcio/química , Chlamydomonas reinhardtii , Tiorredoxinas de Cloroplastos/química , Simulação de Dinâmica Molecular , Ligação Proteica
11.
J Biol Chem ; 294(46): 17437-17450, 2019 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-31597700

RESUMO

Thioredoxin (Trx) is a redox-responsive protein that modulates the activities of its target proteins mostly by reducing their disulfide bonds. In chloroplasts, five Trx isoforms (Trx-f, Trx-m, Trx-x, Trx-y, and Trx-z) regulate various photosynthesis-related enzymes with distinct target selectivity. To elucidate the determinants of the target selectivity of each Trx isoform, here we investigated the residues responsible for target recognition by Trx-f, the most well-studied chloroplast-resident Trx. As reported previously, we found that positively-charged residues on the Trx-f surface are involved in the interactions with its targets. Moreover, several residues that are specifically conserved in Trx-f (e.g. Cys-126 and Thr-158) were also involved in interactions with target proteins. The validity of these residues was examined by the molecular dynamics simulation. In addition, we validated the impact of these key residues on target protein reduction by studying (i) Trx-m variants into which we introduced the key residues for Trx-f and (ii) Trx-like proteins, named atypical Cys His-rich Trx 1 (ACHT1) and ACHT2a, that also contain these key residues. These artificial or natural protein variants could reduce Trx-f-specific targets, indicating that the key residues for Trx-f are critical for Trx-f-specific target recognition. Furthermore, we demonstrate that ACHT1 and ACHT2a efficiently oxidize some Trx-f-specific targets, suggesting that its target selectivity also contributes to the oxidative regulation process. Our results reveal the key residues for Trx-f-specific target recognition and uncover ACHT1 and ACHT2a as oxidation factors of their target proteins, providing critical insight into redox regulation of photosynthesis.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Tiorredoxinas de Cloroplastos/metabolismo , Sequência de Aminoácidos , Arabidopsis/química , Proteínas de Arabidopsis/química , Tiorredoxinas de Cloroplastos/química , Sequência Conservada , Modelos Moleculares , Oxirredução , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo
12.
Plant Cell Physiol ; 60(7): 1504-1513, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-31038682

RESUMO

Cyanobacteria possess a sophisticated photosynthesis-based metabolism with admirable plasticity. This plasticity is possible via the deep regulation network, the thiol-redox regulations operated by thioredoxin (hereafter, Trx). In this context, we characterized the Trx-m1-deficient mutant strain of Anabaena sp., PCC 7120 (shortly named A.7120), cultivated under nitrogen limitation. Trx-m1 appears to coordinate the nitrogen response and its absence induces large changes in the proteome. Our data clearly indicate that Trx-m1 is crucial for the diazotrophic growth of A.7120. The lack of Trx-m1 resulted in a large differentiation of heterocysts (>20% of total cells), which were barely functional probably due to a weak expression of nitrogenase. In addition, heterocysts of the mutant strain did not display the usual cellular structure of nitrogen-fixative cells. This unveiled why the mutant strain was not able to grow under nitrogen starvation.


Assuntos
Anabaena/genética , Tiorredoxinas de Cloroplastos/fisiologia , Genes Bacterianos/fisiologia , Nitrogênio/deficiência , Anabaena/crescimento & desenvolvimento , Anabaena/metabolismo , Antioxidantes/metabolismo , Clorofila/metabolismo , Tiorredoxinas de Cloroplastos/genética , Cloroplastos/metabolismo , Genes Bacterianos/genética , Microscopia Eletrônica de Transmissão , Fotossíntese , Proteoma
13.
Biochem J ; 476(7): 1159-1172, 2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30988137

RESUMO

Photosynthesis is a highly regulated process in photoautotrophic cells. The main goal of the regulation is to keep the basic photosynthetic reactions, i.e. capturing light energy, conversion into chemical energy and production of carbohydrates, in balance. The rationale behind the evolution of strong regulation mechanisms is to keep photosynthesis functional under all conditions encountered by sessile plants during their lifetimes. The regulatory mechanisms may, however, also impair photosynthetic efficiency by overriding the photosynthetic reactions in controlled environments like crop fields or bioreactors, where light energy could be used for production of sugars instead of dissipation as heat and down-regulation of carbon fixation. The plant chloroplast has a high number of regulatory proteins called thioredoxins (TRX), which control the function of chloroplasts from biogenesis and assembly of chloroplast machinery to light and carbon fixation reactions as well as photoprotective mechanisms. Here, we review the current knowledge of regulation of photosynthesis by chloroplast TRXs and assess the prospect of improving plant photosynthetic efficiency by modification of chloroplast thioredoxin systems.


Assuntos
Tiorredoxinas de Cloroplastos/metabolismo , Fotossíntese/fisiologia , Plantas/metabolismo , Tiorredoxinas de Cloroplastos/genética , Cloroplastos/genética , Cloroplastos/metabolismo , Luz , Modelos Biológicos , Oxirredução , Estresse Oxidativo , Fotossíntese/genética , Plantas/genética , Plantas Geneticamente Modificadas , Tiorredoxina Dissulfeto Redutase/genética , Tiorredoxina Dissulfeto Redutase/metabolismo
14.
J Exp Bot ; 70(3): 1005-1016, 2019 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-30476130

RESUMO

The activity of the protein kinase STN7, involved in phosphorylation of the light-harvesting complex II (LHCII) proteins, has been reported as being co-operatively regulated by the redox state of the plastoquinone pool and the ferredoxin-thioredoxin (Trx) system. The present study aims to investigate the role of plastid Trxs in STN7 regulation and their impact on photosynthesis. For this purpose, tobacco plants overexpressing Trx f or m from the plastid genome were characterized, demonstrating that only Trx m overexpression was associated with a complete loss of LHCII phosphorylation that did not correlate with decreased STN7 levels. The absence of phosphorylation in Trx m-overexpressing plants impeded migration of LHCII from PSII to PSI, with the concomitant loss of PSI-LHCII complex formation. Consequently, the thylakoid ultrastructure was altered, showing reduced grana stacking. Moreover, the electron transport rate was negatively affected, showing an impact on energy-demanding processes such as the Rubisco maximum carboxylation capacity and ribulose 1,5-bisphosphate regeneration rate values, which caused a strong depletion in net photosynthetic rates. Finally, tobacco plants overexpressing a Trx m mutant lacking the reactive redox site showed equivalent physiological performance to the wild type, indicating that the overexpressed Trx m deactivates STN7 in a redox-dependent way.


Assuntos
Tiorredoxinas de Cloroplastos/genética , Regulação da Expressão Gênica de Plantas , Fotossíntese/genética , Proteínas de Plantas/genética , Proteínas Serina-Treonina Quinases/genética , Tabaco/genética , Tiorredoxinas de Cloroplastos/metabolismo , Cloroplastos/enzimologia , Oxirredução , Complexo de Proteína do Fotossistema II/metabolismo , Proteínas de Plantas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Tabaco/enzimologia , Tabaco/metabolismo
15.
Sci Rep ; 8(1): 6666, 2018 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-29703909

RESUMO

Changes in the redox state of the photosynthetic electron transport chain act as a signal to trigger acclimation responses to environmental cues and thioredoxin has been suggested to work as a key factor connecting the redox change with transcriptional regulation in the cyanobacterium Synechocystis sp. PCC 6803. We screened for redox-dependent transcription factors interacting with thioredoxin M (TrxM) and isolated the GntR-type transcription factor Sll1961 previously reported to be involved in acclimation responses of the photosynthetic machinery. Biochemical analyses using recombinant Sll1961 proteins of wild type and mutants of three cysteine residues, C124, C229 and C307, revealed that an intramolecular disulfide bond is formed between C229 and C307 under oxidizing conditions and TrxM can reduce it by attacking C307. Sll1961 exists in a dimeric form of about 80 kDa both under reducing and oxidizing conditions. C124 can form an intermolecular disulfide bond but it is not essential for dimerization. Based on these observations, tertiary structure models of the Sll1961 homodimer and the Sll1961-TrxM complex were constructed.


Assuntos
Tiorredoxinas de Cloroplastos/metabolismo , Mapeamento de Interação de Proteínas , Synechocystis/metabolismo , Fatores de Transcrição/metabolismo , Dissulfetos/metabolismo , Modelos Moleculares , Oxirredução , Fotossíntese , Ligação Proteica , Conformação Proteica , Multimerização Proteica
16.
Nat Commun ; 9(1): 50, 2018 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-29298981

RESUMO

Activation of the plastid-encoded RNA polymerase is tightly controlled and involves a network of phosphorylation and, as yet unidentified, thiol-mediated events. Here, we characterize PLASTID REDOX INSENSITIVE2, a redox-regulated protein required for full PEP-driven transcription. PRIN2 dimers can be reduced into the active monomeric form by thioredoxins through reduction of a disulfide bond. Exposure to light increases the ratio between the monomeric and dimeric forms of PRIN2. Complementation of prin2-2 with different PRIN2 protein variants demonstrates that the monomer is required for light-activated PEP-dependent transcription and that expression of the nuclear-encoded photosynthesis genes is linked to the activity of PEP. Activation of PEP during chloroplast development likely is the source of a retrograde signal that promotes nuclear LHCB expression. Thus, regulation of PRIN2 is the thiol-mediated mechanism required for full PEP activity, with PRIN2 monomerization via reduction by TRXs providing a mechanistic link between photosynthetic electron transport and activation of photosynthetic gene expression.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Tiorredoxinas de Cloroplastos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Plântula/enzimologia , Sequência de Aminoácidos , Transporte de Elétrons , Fotossíntese , Plastídeos/metabolismo , Transcrição Genética
17.
Plant Cell Rep ; 37(2): 279-291, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29080907

RESUMO

KEY MESSAGE: M-type thioredoxins are required to regulate zeaxanthin epoxidase activity and to maintain the steady-state level of the proton motive force, thereby influencing NPQ properties under low-light conditions in Arabidopsis. Non-photochemical quenching (NPQ) helps protect photosynthetic organisms from photooxidative damage via the non-radiative dissipation of energy as heat. Energy-dependent quenching (qE) is a major constituent of NPQ. However, the mechanism underlying the regulation of qE is not well understood. In this study, we demonstrate that the m-type thioredoxins TRX-m1, TRX-m2, and TRX-m4 (TRX-ms) interact with the xanthophyll cycle enzyme zeaxanthin epoxidase (ZE) and are required for maintaining the redox-dependent stabilization of ZE by regulating its intermolecular disulfide bridges. Reduced ZE activity and accumulated zeaxanthin levels were observed under TRX-ms deficiency. Furthermore, concurrent deficiency of TRX-ms resulted in a significant increase in proton motive force (pmf) and acidification of the thylakoid lumen under low irradiance, perhaps due to the significantly reduced ATP synthase activity under TRX-ms deficiency. The increased pmf, combined with acidification of the thylakoid lumen and the accumulation of zeaxanthin, ultimately contribute to the elevated stable qE in VIGS-TRX-m2m4/m1 plants under low-light conditions. Taken together, these results indicate that TRX-ms are involved in regulating NPQ-dependent photoprotection in Arabidopsis.


Assuntos
Arabidopsis/metabolismo , Clorofila/metabolismo , Tiorredoxinas de Cloroplastos/metabolismo , Luz , Fotossíntese/efeitos da radiação , Xantofilas/metabolismo , Proteínas de Arabidopsis/metabolismo , Oxirredução , Oxirredutases/metabolismo , Ligação Proteica , Força Próton-Motriz/efeitos da radiação , Tilacoides/metabolismo , Zeaxantinas/metabolismo
18.
Philos Trans R Soc Lond B Biol Sci ; 372(1730)2017 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-28808108

RESUMO

Thioredoxins (TRXs) are protein oxidoreductases that control the structure and function of cellular proteins by cleavage of a disulphide bond between the side chains of two cysteine residues. Oxidized thioredoxins are reactivated by thioredoxin reductases (TR) and a TR-dependent reduction of TRXs is called a thioredoxin system. Thiol-based redox regulation is an especially important mechanism to control chloroplast proteins involved in biogenesis, in regulation of light harvesting and distribution of light energy between photosystems, in photosynthetic carbon fixation and other biosynthetic pathways, and in stress responses of plants. Of the two plant plastid thioredoxin systems, the ferredoxin-dependent system relays reducing equivalents from photosystem I via ferredoxin and ferredoxin-thioredoxin reductase (FTR) to chloroplast proteins, while NADPH-dependent thioredoxin reductase (NTRC) forms a complete thioredoxin system including both reductase and thioredoxin domains in a single polypeptide. Chloroplast thioredoxins transmit environmental light signals to biochemical reactions, which allows fine tuning of photosynthetic processes in response to changing environmental conditions. In this paper we focus on the recent reports on specificity and networking of chloroplast thioredoxin systems and evaluate the prospect of improving photosynthetic performance by modifying the activity of thiol regulators in plants.This article is part of the themed issue 'Enhancing photosynthesis in crop plants: targets for improvement'.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/fisiologia , Tiorredoxinas de Cloroplastos/genética , Produtos Agrícolas/fisiologia , Proteínas de Arabidopsis/metabolismo , Tiorredoxinas de Cloroplastos/metabolismo , Fotossíntese
19.
New Phytol ; 215(1): 240-255, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28248438

RESUMO

Rice is vulnerable to cold stress. Seedlings are very sensitive to cold stress and this harms global rice production. The effects of cold on chloroplast development are well known, but little is known about the underlying molecular mechanisms. Here, we isolated a temperature-sensitive virescent (tsv) mutant that is extremely sensitive to cold stress. It displayed defective chloroplasts, decreased chlorophyll and zero survivorship under cold stress. We isolated and identified TSV by map-based cloning and rescue experiments, combined with genetic, cytological and molecular biological analyses. We found that TSV, a putative plastidic oxidoreductase, is a new type of virescent protein. A mutation in tsv causes premature termination of the gene product. The activity of plastid-encoded RNA polymerase (PEP) and the expression of genes participating in chlorophyll synthesis were severely reduced in the tsv mutant under cold stress, but not at normal temperatures. TSV expression was induced by low temperatures. Strikingly, TSV interacted with OsTrxZ (a subunit of PEP in chloroplasts) and enhanced OsTrxZ stability under low temperatures. We demonstrated that TSV protects rice chloroplasts from cold stress by interacting with OsTrxZ. These results provide novel insights into ways in which rice chloroplast development and chlorophyll synthesis are protected by TSV under cold stress.


Assuntos
Tiorredoxinas de Cloroplastos/metabolismo , Resposta ao Choque Frio , Oryza/fisiologia , Proteínas de Plantas/metabolismo , Clorofila/metabolismo , Cloroplastos/metabolismo , Cloroplastos/fisiologia , Cloroplastos/ultraestrutura , Clonagem Molecular , Análise Mutacional de DNA , Oryza/metabolismo , Oryza/ultraestrutura , Temperatura
20.
Biochem J ; 474(8): 1347-1360, 2017 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-28246333

RESUMO

Thiol-based redox regulation is considered to support light-responsive control of various chloroplast functions. The redox cascade via ferredoxin-thioredoxin reductase (FTR)/thioredoxin (Trx) has been recognized as a key to transmitting reducing power; however, Arabidopsis thaliana genome sequencing has revealed that as many as five Trx subtypes encoded by a total of 10 nuclear genes are targeted to chloroplasts. Because each Trx isoform seems to have a distinct target selectivity, the electron distribution from FTR to multiple Trxs is thought to be the critical branch point for determining the consequence of chloroplast redox regulation. In the present study, we aimed to comprehensively characterize the kinetics of electron transfer from FTR to 10 Trx isoforms. We prepared the recombinant FTR protein from Arabidopsis in the heterodimeric form containing the Fe-S cluster. By reconstituting the FTR/Trx system in vitro, we showed that FTR prepared here was enzymatically active and suitable for uncovering biochemical features of chloroplast redox regulation. A series of redox state determinations using the thiol-modifying reagent, 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonate, indicated that all chloroplast Trx isoforms are commonly reduced by FTR; however, significantly different efficiencies were evident. These differences were apparently correlated with the distinct midpoint redox potentials among Trxs. Even when the experiments were performed under conditions of hypothetical in vivo stoichiometry of FTR and Trxs, a similar trend in distinguishable electron transfers was observed. These data highlight an aspect of highly organized circuits in the chloroplast redox regulation network.


Assuntos
Proteínas de Arabidopsis/metabolismo , Tiorredoxinas de Cloroplastos/metabolismo , Cloroplastos/metabolismo , Transporte de Elétrons , Proteínas Ferro-Enxofre/metabolismo , Modelos Moleculares , Oxirredutases/metabolismo , Arabidopsis/enzimologia , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Biocatálise/efeitos dos fármacos , Domínio Catalítico , Tiorredoxinas de Cloroplastos/química , Tiorredoxinas de Cloroplastos/genética , Cloroplastos/enzimologia , Transporte de Elétrons/efeitos dos fármacos , Proteínas Ferro-Enxofre/química , Proteínas Ferro-Enxofre/genética , Oxirredução , Oxirredutases/química , Oxirredutases/genética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Estilbenos/farmacologia , Reagentes de Sulfidrila/farmacologia , Ácidos Sulfônicos/farmacologia
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