RESUMO
Foliar development involves successive phases of cell proliferation and expansion that determine the final leaf size, and is characterized by an early burst of reactive oxygen species generated in the photosynthetic electron transport chain (PETC). Introduction of the alternative PETC acceptor flavodoxin in tobacco chloroplasts led to a reduction in leaf size associated to lower cell expansion, without affecting cell number per leaf. Proteomic analysis showed that the biogenesis of the PETC proceeded stepwise in wild-type leaves, with accumulation of light-harvesting proteins preceding that of electron transport components, which might explain the increased energy and electron transfer to oxygen and reactive oxygen species build-up at this stage. Flavodoxin expression did not affect biogenesis of the PETC but prevented hydroperoxide formation through its function as electron sink. Mature leaves from flavodoxin-expressing plants were shown to contain higher levels of transcripts encoding components of the proteasome, a key negative modulator of organ size. Proteome profiling revealed that this differential accumulation was initiated during expansion and led to increased proteasomal activity, whereas a proteasome inhibitor reverted the flavodoxin-dependent size phenotype. Cells expressing plastid-targeted flavodoxin displayed lower endoreduplication, also associated to decreased organ size. These results provide novel insights into the regulation of leaf growth by chloroplast-generated redox signals, and highlight the potential of alternative electron shuttles to investigate the link(s) between photosynthesis and plant development.
Assuntos
Cloroplastos , Nicotiana , Folhas de Planta , Complexo de Endopeptidases do Proteassoma , Cloroplastos/metabolismo , Folhas de Planta/metabolismo , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Nicotiana/genética , Nicotiana/metabolismo , Nicotiana/crescimento & desenvolvimento , Transporte de Elétrons , Fotossíntese , Flavodoxina/metabolismo , Flavodoxina/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genéticaRESUMO
Flavodoxins are electron carrier flavoproteins present in bacteria and photosynthetic microorganisms which duplicate the functional properties of iron-sulphur containing ferredoxins and replace them under adverse environmental situations that lead to ferredoxin decline. When expressed in plant chloroplasts, flavodoxin complemented ferredoxin deficiency and improved tolerance to multiple sources of biotic, abiotic and xenobiotic stress. Analysis of flavodoxin-expressing plants grown under normal conditions, in which the two carriers are present, revealed phenotypic effects unrelated to ferredoxin replacement. Flavodoxin thus provided a tool to alter the chloroplast redox poise in a customized way and to investigate its consequences on plant physiology and development. We describe herein the effects exerted by the flavoprotein on the function of the photosynthetic machinery. Pigment analysis revealed significant increases in chlorophyll a, carotenoids and chlorophyll a/b ratio in flavodoxin-expressing tobacco lines. Results suggest smaller antenna size in these plants, supported by lower relative contents of light-harvesting complex proteins. Chlorophyll a fluorescence and P700 spectroscopy measurements indicated that transgenic plants displayed higher quantum yields for both photosystems, a more oxidized plastoquinone pool under steady-state conditions and faster plastoquinone dark oxidation after a pulse of saturating light. Many of these effects resemble the phenotypes exhibited by leaves adapted to high irradiation, a most common environmental hardship faced by plants growing in the field. The results suggest that flavodoxin-expressing plants would be better prepared to cope with this adverse situation, and concur with earlier observations reporting that hundreds of stress-responsive genes were induced in the absence of stress in these lines.
Assuntos
Aclimatação/efeitos da radiação , Flavodoxina/genética , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Luz , Nicotiana/metabolismo , Fotossíntese/efeitos da radiação , Folhas de Planta/genética , Relação Dose-Resposta à Radiação , Fenótipo , Folhas de Planta/efeitos da radiação , Nicotiana/genética , Nicotiana/fisiologia , Nicotiana/efeitos da radiaçãoRESUMO
Flavodoxins are small electron transfer proteins containing flavin mononucleotide (FMN) as a prosthetic group, which play an important role during oxidative stress or iron limitation. The aims of this study were the identification and characterization of flavodoxins in the model aromatic-degrader Paraburkholderia xenovorans LB400 and the analyses of their protective effects during oxidative stress induced by paraquat and H2O2. Two genes (BxeA0278 and BxeB0391) encoding flavodoxins (hereafter referred to as fldX for flavodoxin from P. xenovorans), were identified at the LB400 major and minor chromosome. Genomic context of the flavodoxin-encoding genes showed genes encoding membrane proteins, transporters, and proteins involved in redox processes and biosynthesis of macromolecules. A secondary structure prediction of both LB400 flavodoxins showed the characteristic flavodoxin structure of five ß-sheets intercalated with five α-helices. FldX1 contains a loop intercalated in the fifth ß-strand, which indicates that it belongs to the long-chain flavodoxins, whereas FldX2 is a short-chain flavodoxin. A phylogenetic analysis of 73 flavodoxins from 43 bacterial genera revealed eight clusters (I-VIII), while FldX1 and FldX2 grouped separately within a long-chain and a short-chain flavodoxin clades. FldX1 and FldX2 were overexpressed in P. xenovorans. Interestingly, the strain overexpressing the long-chain flavodoxin FldX1 (p2-fldX1) showed a faster growth in glucose than the control strain. The recombinant strain overexpressing the long-chain flavodoxin FldX1 (p2-fldx1) exposed to paraquat (20 mM) possessed lower susceptibility to growth inhibition on plates and higher survival in liquid medium than the control strain. The strains overexpressing the flavodoxins FldX1 and FldX2 showed higher survival during exposure to 1 mM paraquat (>95%) than the control strain (68%). Compared to the control strain, strains overexpressing FldX1 and FldX2 showed lower lipid peroxidation (>20%) after exposure to 1 mM paraquat and a lower protein carbonylation (~30%) after exposure to 1 mM H2O2 was observed. During exposure to paraquat, strain p2-fldx1 downregulated the katG4, hpf, trxB1 and ohr genes (> 2-fold), whereas strain p2-fldx2 upregulated the oxyR and ahpC1 genes (> 2-fold). In conclusion, the flavodoxins FldX1 and FldX2 of P. xenovorans LB400 conferred protection to cells exposed to the oxidizing agents paraquat and H2O2.
Assuntos
Adaptação Biológica/efeitos dos fármacos , Betaproteobacteria/efeitos dos fármacos , Betaproteobacteria/fisiologia , Flavodoxina/genética , Peróxido de Hidrogênio/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Paraquat/farmacologia , Sequência de Aminoácidos , Biologia Computacional/métodos , Flavodoxina/química , Flavodoxina/metabolismo , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Genômica/métodos , FilogeniaRESUMO
Oxidative stress and iron limitation represent the grim side of life in an oxygen-rich atmosphere. The versatile electron transfer shuttle ferredoxin, an iron-sulfur protein, is particularly sensitive to these hardships, and its downregulation under adverse conditions severely compromises survival of phototrophs. Replacement of ferredoxin by a stress-resistant isofunctional carrier, flavin-containing flavodoxin, is a widespread strategy employed by photosynthetic microorganisms to overcome environmental adversities. The flavodoxin gene was lost in the course of plant evolution, but its reintroduction in transgenic plants confers increased tolerance to environmental stress and iron starvation, raising the question as to why a genetic asset with obvious adaptive value was not kept by natural selection. Phylogenetic analyses reveal that the evolutionary history of flavodoxin is intricate, with several horizontal gene transfer events between distant organisms, including Eukarya, Bacteria, and Archaea. The flavodoxin gene is unevenly distributed in most algal lineages, with flavodoxin-containing species being overrepresented in iron-limited regions and scarce or absent in iron-rich environments. Evaluation of cyanobacterial genomic and metagenomic data yielded essentially the same results, indicating that there was little selection pressure to retain flavodoxin in iron-rich coastal/freshwater phototrophs. Our results show a highly dynamic evolution pattern of flavodoxin tightly connected to the bioavailability of iron. Evidence presented here also indicates that the high concentration of iron in coastal and freshwater habitats may have facilitated the loss of flavodoxin in the freshwater ancestor of modern plants during the transition of photosynthetic organisms from the open oceans to the firm land.
Assuntos
Evolução Molecular , Flavodoxina/genética , Genoma de Planta , Cianobactérias/genética , Meio Ambiente , Flavodoxina/classificação , Genes de Plantas , Ferro/metabolismo , Complexo de Proteína do Fotossistema II/genética , Processos Fototróficos/genética , FilogeniaRESUMO
Long-chain flavodoxins, ubiquitous electron shuttles containing flavin mononucleotide (FMN) as prosthetic group, play an important protective role against reactive oxygen species (ROS) in various microorganisms. Pseudomonas aeruginosa is an opportunistic pathogen which frequently has to face ROS toxicity in the environment as well as within the host. We identified a single ORF, hereafter referred to as fldP (for fl avo d oxin from P . aeruginosa), displaying the highest similarity in length, sequence identity and predicted secondary structure with typical long-chain flavodoxins. The gene was cloned and expressed in Escherichia coli. The recombinant product (FldP) could bind FMN and exhibited flavodoxin activity in vitro. Expression of fldP in P. aeruginosa was induced by oxidative stress conditions through an OxyR-independent mechanism, and an fldP-null mutant accumulated higher intracellular ROS levels and exhibited decreased tolerance to H2O2 toxicity compared to wild-type siblings. The mutant phenotype could be complemented by expression of a cyanobacterial flavodoxin. Overexpression of FldP in a mutT-deficient P. aeruginosa strain decreased H2O2-induced cell death and the hypermutability caused by DNA oxidative damage. FldP contributed to the survival of P. aeruginosa within cultured mammalian macrophages and in infected Drosophila melanogaster, which led in turn to accelerated death of the flies. Interestingly, the fldP gene is present in some but not all P. aeruginosa strains, constituting a component of the P. aeruginosa accessory genome. It is located in a genomic island as part of a self-regulated polycistronic operon containing a suite of stress-associated genes. The collected results indicate that the fldP gene encodes a long-chain flavodoxin, which protects the cell from oxidative stress, thereby expanding the capabilities of P. aeruginosa to thrive in hostile environments.
Assuntos
Flavodoxina/genética , Interações Hospedeiro-Parasita/genética , Estresse Oxidativo , Pseudomonas aeruginosa/genética , Clonagem Molecular , Flavodoxina/metabolismo , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Pseudomonas aeruginosa/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Ferredoxins are iron-sulfur proteins involved in various one-electron transfer pathways. Ferredoxin levels decrease under adverse environmental conditions in photosynthetic organisms. In cyanobacteria, this decline is compensated by induction of flavodoxin, an isofunctional flavoprotein. Flavodoxin is not present in higher plants, but transgenic Nicotiana tabacum lines accumulating Anabaena flavodoxin in plastids display increased tolerance to different sources of environmental stress. As the degree of tolerance correlated with flavodoxin dosage in plastids of nuclear-transformed transgenic tobacco, we prepared plants expressing even higher levels of flavodoxin by direct plastid transformation. A suite of nuclear- and chloroplast-transformed lines expressing a wide range of flavodoxin levels, from 0.3 to 10.8 µmol m(-2), did not exhibit any detectable growth phenotype relative to the wild type. In the absence of stress, the contents of both chlorophyll a and carotenoids, as well as the photosynthetic performance (photosystem II maximum efficiency, photosystem II operating efficiency, electron transport rates and carbon assimilation rates), displayed a moderate increase with flavodoxin concentrations up to 1.3-2.6 µmol flavodoxin m(-2), and then declined to wild-type levels. Stress tolerance, as estimated by the damage inflicted on exposure to the pro-oxidant methyl viologen, also exhibited a bell-shaped response, with a significant, dose-dependent increase in tolerance followed by a drop in the high-expressing lines. The results indicate that optimal photosynthetic performance and stress tolerance were observed at flavodoxin levels comparable to those of endogenous ferredoxin. Further increases in flavodoxin content become detrimental to plant fitness.
Assuntos
Flavodoxina/genética , Nicotiana/genética , Fotossíntese/fisiologia , Estresse Fisiológico/genética , Anabaena/genética , Carotenoides/metabolismo , Clorofila/metabolismo , Clorofila A , Cloroplastos/genética , Relação Dose-Resposta a Droga , Flavodoxina/metabolismo , Flavodoxina/farmacologia , Regulação da Expressão Gênica , Estresse Oxidativo/genética , Paraquat/farmacologia , Complexo de Proteína do Fotossistema II/metabolismo , Plantas Geneticamente Modificadas/fisiologia , Plastídeos/genética , Nicotiana/efeitos dos fármacos , Nicotiana/crescimento & desenvolvimento , Nicotiana/fisiologiaRESUMO
Ferredoxins are the main electron shuttles in chloroplasts, accepting electrons from photosystem I and delivering them to essential oxido-reductive pathways in the stroma. Ferredoxin levels decrease under adverse environmental conditions in both plants and photosynthetic micro-organisms. In cyanobacteria and some algae, this decrease is compensated for by induction of flavodoxin, an isofunctional flavoprotein that can replace ferredoxin in many reactions. Flavodoxin is not present in plants, but tobacco lines expressing a plastid-targeted cyanobacterial flavodoxin developed increased tolerance to environmental stress. Chloroplast-located flavodoxin interacts productively with endogenous ferredoxin-dependent pathways, suggesting that its protective role results from replacement of stress-labile ferredoxin. We tested this hypothesis by using RNA antisense and interference techniques to decrease ferredoxin levels in transgenic tobacco. Ferredoxin-deficient lines showed growth arrest, leaf chlorosis and decreased CO(2) assimilation. Chlorophyll fluorescence measurements indicated impaired photochemistry, over-reduction of the photosynthetic electron transport chain and enhanced non-photochemical quenching. Expression of flavodoxin from the nuclear or plastid genome restored growth, pigment contents and photosynthetic capacity, and relieved the electron pressure on the electron transport chain. Tolerance to oxidative stress also recovered. In the absence of flavodoxin, ferredoxin could not be decreased below 45% of physiological content without fatally compromising plant survival, but in its presence, lines with only 12% remaining ferredoxin could grow autotrophically, with almost wild-type phenotypes. The results indicate that the stress tolerance conferred by flavodoxin expression in plants stems largely from functional complementation of endogenous ferredoxin by the cyanobacterial flavoprotein.
Assuntos
Ferredoxinas/metabolismo , Flavodoxina/genética , Flavodoxina/metabolismo , Nicotiana/genética , Nicotiana/metabolismo , Anabaena/genética , Anabaena/metabolismo , Sequência de Bases , DNA de Plantas/genética , Ferredoxinas/deficiência , Ferredoxinas/genética , Técnicas de Silenciamento de Genes , Teste de Complementação Genética , Microscopia Eletrônica de Transmissão , Folhas de Planta/metabolismo , Folhas de Planta/ultraestrutura , Plantas Geneticamente Modificadas , Interferência de RNA , RNA Antissenso/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Estresse Fisiológico , Nicotiana/ultraestruturaRESUMO
Environmental stresses - especially drought and salinity - and iron limitation are the primary causes of crop yield losses. Therefore, improvement of plant stress tolerance has paramount relevance for agriculture, and vigorous efforts are underway to design stress-tolerant crops. Three aspects of this ongoing research are reviewed here. First, attempts have been made to strengthen endogenous plant defences, which are characterised by intertwined, hierarchical gene networks involved in stress perception, signalling, regulation and expression of effector proteins, enzymes and metabolites. The multigenic nature of this response requires detailed knowledge of the many actors and interactions involved in order to identify proper intervention points, followed by significant engineering of the prospective genes to prevent undesired side-effects. A second important aspect refers to the effect of concurrent stresses as plants normally meet in the field (e.g., heat and drought). Recent findings indicate that plant responses to combined environmental hardships are somehow unique and cannot be predicted from the addition of the individual stresses, underscoring the importance of programming research within this conceptual framework. Finally, the photosynthetic microorganisms from which plants evolved (i.e., algae and cyanobacteria) deploy a totally different strategy to acquire stress tolerance, based on the substitution of stress-vulnerable targets by resistant isofunctional proteins that could take over the lost functions under adverse conditions. Reintroduction of these ancient traits in model and crop plants has resulted in increased tolerance to environmental hardships and iron starvation, opening a new field of opportunities to increase the endurance of crops growing under suboptimal conditions.
Assuntos
Aclimatação/genética , Regulação da Expressão Gênica de Plantas , Engenharia Genética/tendências , Plantas Geneticamente Modificadas/genética , Cianobactérias/metabolismo , Secas , Meio Ambiente , Flavodoxina/genética , Ferro/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Salinidade , Estresse Fisiológico , Nicotiana/genética , Nicotiana/metabolismoRESUMO
Environmental stresses and iron limitation are the primary causes of crop losses worldwide. Engineering strategies aimed at gaining stress tolerance have focused on overexpression of endogenous genes belonging to molecular networks for stress perception or responses. Based on the typical response of photosynthetic microorganisms to stress, an alternative approach has been recently applied with considerable success. Ferredoxin, a stress-sensitive target, was replaced in tobacco chloroplasts by an isofunctional protein, a cyanobacterial flavodoxin, which is absent in plants. Resulting transgenic lines showed wide-range tolerance to drought, chilling, oxidants, heat and iron starvation. The survival of plants under such adverse conditions would be an enormous agricultural advantage and makes this novel strategy a potentially powerful biotechnological tool for the generation of multiple-tolerant crops in the near future.
Assuntos
Cloroplastos/metabolismo , Produtos Agrícolas/genética , Flavodoxina/metabolismo , Nicotiana/genética , Fotossíntese/fisiologia , Aclimatação/genética , Aclimatação/fisiologia , Cloroplastos/genética , Produtos Agrícolas/fisiologia , Secas , Flavodoxina/genética , Estresse Oxidativo , Plantas Geneticamente ModificadasRESUMO
Significant effort has been directed in recent times to the use of plants to extract and detoxify nitroaromatics from polluted industrial facilities. We have explored the possibility of overcoming the phytotoxicity of the highly toxic and recalcitrant nitroderivative 2,4-dinitrotoluene (2,4-DNT) by expressing a cyanobacterial flavodoxin (Fld) in tobacco plants. We demonstrate here that transformants accumulating Fld in plastids display a remarkable increase in the ability to tolerate, take up, and transform 2,4-DNT, as compared to their wild-type siblings. We also show that Fld mediates one-electron reduction of 2,4-DNT in the presence of oxygen and especially in anaerobiosis. Moreover, Fld-loaded chloroplasts are able to convert 2,4-DNT into its aminoderivatives in the presence of light. The results suggest that expression of Fld in landscape plants could facilitate effective cleanup of sites contaminated with this class of pollutants.