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
With the notable exception of angiosperms, all phototrophs contain different sets of flavodiiron proteins that help to relieve the excess of excitation energy on the photosynthetic electron transport chain during adverse environmental conditions, presumably by reducing oxygen directly to water. Among them, the Flv2-Flv4 dimer is only found in ß-cyanobacteria and induced by high light, supporting a role in stress protection. The possibility of a similar protective function in plants was assayed by expressing Synechocystis Flv2-Flv4 in chloroplasts of tobacco and Arabidopsis. Flv-expressing plants exhibited increased tolerance toward high irradiation, salinity, oxidants, and drought. Stress tolerance was reflected by better growth, preservation of photosynthetic activity, and membrane integrity. Metabolic profiling under drought showed enhanced accumulation of soluble sugars and amino acids in transgenic Arabidopsis and a remarkable shift of sucrose into starch, in line with metabolic responses of drought-tolerant genotypes. Our results indicate that the Flv2-Flv4 complex retains its stress protection activities when expressed in chloroplasts of angiosperm species by acting as an additional electron sink. The flv2-flv4 genes constitute a novel biotechnological tool to generate plants with increased tolerance to agronomically relevant stress conditions that represent a significant productivity constraint.
Assuntos
Adaptação Biológica , Arabidopsis/fisiologia , Cloroplastos/genética , Nicotiana/fisiologia , Proteínas de Plantas/genética , Estresse Fisiológico/genética , Secas , Regulação da Expressão Gênica de Plantas , Estresse Oxidativo , Fenótipo , Complexo de Proteína do Fotossistema II/genética , Complexo de Proteína do Fotossistema II/metabolismo , Fenômenos Fisiológicos Vegetais , Plantas Geneticamente Modificadas , Plastídeos/genética , Tolerância ao Sal/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
Leaf senescence is a concerted physiological process involving controlled degradation of cellular structures and reallocation of breakdown products to other plant organs. It is accompanied by increased production of reactive oxygen species (ROS) that are proposed to signal cell death, although both the origin and the precise role of ROS in the execution of this developmental program are still poorly understood. To investigate the contribution of chloroplast-associated ROS to natural leaf senescence, we used tobacco plants expressing a plastid-targeted flavodoxin, an electron shuttle flavoprotein present in prokaryotes and algae. When expressed in plants, flavodoxin specifically prevents ROS formation in chloroplasts during stress situations. Senescence symptoms were significantly mitigated in these transformants, with decreased accumulation of chloroplastic ROS and differential preservation of chlorophylls, carotenoids, protein contents, cell and chloroplast structures, membrane integrity and cell viability. Flavodoxin also improved maintenance of chlorophyll-protein complexes, photosynthetic electron flow, CO2 assimilation, central metabolic routes and levels of bioactive cytokinins and auxins in aging leaves. Delayed induction of senescence-associated genes indicates that the entire genetic program of senescence was affected by flavodoxin. The results suggest that ROS generated in chloroplasts are involved in the regulation of natural leaf senescence.
RESUMO
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
Attempted infection of plants by pathogens elicits a complex defensive response. In many non-host and incompatible host interactions it includes the induction of defence-associated genes and a form of localized cell death (LCD), purportedly designed to restrict pathogen advance, collectively known as the hypersensitive response (HR). It is preceded by an oxidative burst, generating reactive oxygen species (ROS) that are proposed to cue subsequent deployment of the HR, although neither the origin nor the precise role played by ROS in the execution of this response are completely understood. We used tobacco plants expressing cyanobacterial flavodoxin to address these questions. Flavodoxin is an electron shuttle present in prokaryotes and algae that, when expressed in chloroplasts, specifically prevents ROS formation in plastids during abiotic stress episodes. Infiltration of tobacco wild-type leaves with high titres of Xanthomonas campestris pv. vesicatoria (Xcv), a non-host pathogen, resulted in ROS accumulation in chloroplasts, followed by the appearance of localized lesions typical of the HR. In contrast, chloroplast ROS build-up and LCD were significantly reduced in Xcv-inoculated plants expressing plastid-targeted flavodoxin. Metabolic routes normally inhibited by pathogens were protected in the transformants, whereas other aspects of the HR, including the induction of defence-associated genes and synthesis of salicylic and jasmonic acid, proceeded as in inoculated wild-type plants. Therefore, ROS generated in chloroplasts during this non-host interaction are essential for the progress of LCD, but do not contribute to the induction of pathogenesis-related genes or other signalling components of the response.
Assuntos
Morte Celular , Cloroplastos/metabolismo , Nicotiana/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Xanthomonas campestris/fisiologia , Ciclopentanos/metabolismo , Flavodoxina/metabolismo , Regulação da Expressão Gênica de Plantas , Oxilipinas/metabolismo , Doenças das Plantas , Folhas de Planta/genética , Folhas de Planta/metabolismo , Folhas de Planta/microbiologia , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Plantas Geneticamente Modificadas/microbiologia , RNA de Plantas/genética , Ácido Salicílico/metabolismo , Nicotiana/genética , Nicotiana/microbiologiaRESUMO
Chloroplast ferredoxin (Fd) plays a pivotal role in plant cell metabolism by delivering reducing equivalents to various essential oxidoreductive pathways. Fd levels decrease under adverse environmental conditions in many microorganisms, including cyanobacteria, which share a common ancestor with chloroplasts. Conversely, stress situations induce the synthesis of flavodoxin (Fld), an electron carrier flavoprotein not found in plants, which can efficiently replace Fd in most electron transfer processes. We report here that chloroplast Fd also declined in plants exposed to oxidants or stress conditions. A purified cyanobacterial Fld was able to mediate plant Fd-dependent reactions in vitro, including NADP+ and thioredoxin reduction. Tobacco (Nicotiana tabacum) plants expressing Fld in chloroplasts displayed increased tolerance to multiple sources of stress, including redox-cycling herbicides, extreme temperatures, high irradiation, water deficit, and UV radiation. Oxidant buildup and oxidative inactivation of thioredoxin-dependent plastidic enzymes were decreased in stressed plants expressing plastid-targeted Fld, suggesting that development of the tolerant phenotype relied on productive interaction of this flavoprotein with Fd-dependent oxidoreductive pathways of the host, most remarkably, thioredoxin reduction. The use of Fld provides new tools to investigate the requirements of photosynthesis in planta and to increase plant stress tolerance based on the introduction of a cyanobacterial product that is free from endogenous regulation in higher plants.