RESUMEN
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.
Asunto(s)
Cloroplastos , Nicotiana , Hojas de la Planta , Complejo de la Endopetidasa Proteasomal , Cloroplastos/metabolismo , Hojas de la Planta/metabolismo , Hojas de la Planta/crecimiento & desarrollo , Hojas de la Planta/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Nicotiana/genética , Nicotiana/metabolismo , Nicotiana/crecimiento & desarrollo , Transporte de Electrón , Fotosíntesis , Flavodoxina/metabolismo , Flavodoxina/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genéticaRESUMEN
Contemporary climate change is characterized by the increased intensity and frequency of environmental stress events such as floods, droughts, and heatwaves, which have a debilitating impact on photosynthesis and growth, compromising the production of food, feed, and biofuels for an expanding population. The need to increase crop productivity in the context of global warming has fueled attempts to improve several key plant features such as photosynthetic performance, assimilate partitioning, and tolerance to environmental stresses. Chloroplast redox metabolism, including photosynthetic electron transport and CO2 reductive assimilation, are primary targets of most stress conditions, leading to excessive excitation pressure, photodamage, and propagation of reactive oxygen species. Alterations in chloroplast redox poise, in turn, provide signals that exit the plastid and modulate plant responses to the environmental conditions. Understanding the molecular mechanisms involved in these processes could provide novel tools to increase crop yield in suboptimal environments. We describe herein various interventions into chloroplast redox networks that resulted in increased tolerance to multiple sources of environmental stress. They included manipulation of endogenous components and introduction of electron carriers from other organisms, which affected not only stress endurance but also leaf size and longevity. The resulting scenario indicates that chloroplast redox pathways have an important impact on plant growth, development, and defense that goes beyond their roles in primary metabolism. Manipulation of these processes provides additional strategies for the design of crops with improved performance under destabilized climate conditions as foreseen for the future.
Asunto(s)
Cloroplastos , Calentamiento Global , Aclimatación , Cloroplastos/metabolismo , Productos Agrícolas , Oxidación-Reducción , FotosíntesisRESUMEN
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.
Asunto(s)
Adaptación Biológica , Arabidopsis/fisiología , Cloroplastos/genética , Nicotiana/fisiología , Proteínas de Plantas/genética , Estrés Fisiológico/genética , Sequías , Regulación de la Expresión Génica de las Plantas , Estrés Oxidativo , Fenotipo , Complejo de Proteína del Fotosistema II/genética , Complejo de Proteína del Fotosistema II/metabolismo , Fenómenos Fisiológicos de las Plantas , Plantas Modificadas Genéticamente , Plastidios/genética , Tolerancia a la Sal/genéticaRESUMEN
The ability of plants to maintain photosynthesis in a dynamically changing environment is of central importance for their growth. As the photosynthetic machinery is a sensitive and early target of adverse environmental conditions as those typically found in the field, photosynthetic efficiency is not always optimal. Cyanobacteria, algae, mosses, liverworts and gymnosperms produce flavodiiron proteins (Flvs), a class of electron sinks not represented in angiosperms; these proteins act to mitigate the photoinhibition of photosystem I under high or fluctuating light. Here, genes specifying two cyanobacterial Flvs have been expressed in the chloroplasts of Arabidopsis thaliana in an attempt to improve plant growth. Co-expression of Flv1 and Flv3 enhanced the efficiency of light utilization, boosting the plant's capacity to accumulate biomass as the growth light intensity was raised. The Flv1/Flv3 transgenics displayed an increased production of ATP, an acceleration of carbohydrate metabolism and a more pronounced partitioning of sucrose into starch. The results suggest that Flvs are able to establish an efficient electron sink downstream of PSI, thereby ensuring efficient photosynthetic electron transport at moderate to high light intensities. The expression of Flvs thus acts to both protect photosynthesis and to control the ATP/NADPH ratio; together, their presence is beneficial for the plant's growth potential.
RESUMEN
Nitric oxide (NO) is a messenger molecule widespread studied in plant physiology. Latter evidence supports the lack of a NO-producing system involving a NO synthase (NOS) activity in higher plants. However, a NOS gene from the unicellular marine alga Ostreococcus tauri (OtNOS) was characterized in recent years. OtNOS is a genuine NOS, with similar spectroscopic fingerprints to mammalian NOSs and high NO producing capacity. We are interested in investigating whether OtNOS activity alters nitrogen metabolism and nitrogen availability, thus improving growth promotion conditions in tobacco. Tobacco plants were transformed with OtNOS under the constitutive CaMV 35S promoter. Transgenic tobacco plants expressing OtNOS accumulated higher NO levels compared to siblings transformed with the empty vector, and displayed accelerated growth in different media containing sufficient nitrogen availability. Under conditions of nitrogen scarcity, the growth promoting effect of the OtNOS expression is diluted in terms of total leaf area, protein content and seed production. It is proposed that OtNOS might possess a plant growth promoting effect through facilitating N remobilization and nitrate assimilation with potential to improve crop plants performance.
RESUMEN
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.
Asunto(s)
Aclimatación/efectos de la radiación , Flavodoxina/genética , Regulación de la Expresión Génica de las Plantas/efectos de la radiación , Luz , Nicotiana/metabolismo , Fotosíntesis/efectos de la radiación , Hojas de la Planta/genética , Relación Dosis-Respuesta en la Radiación , Fenotipo , Hojas de la Planta/efectos de la radiación , Nicotiana/genética , Nicotiana/fisiología , Nicotiana/efectos de la radiaciónRESUMEN
Chloroplasts, the sites of photosynthesis in higher plants, have evolved several means to tolerate short episodes of drought stress through biosynthesis of diverse metabolites essential for plant function, but these become ineffective when the duration of the stress is prolonged. Cyanobacteria are the closest bacterial homologs of plastids with two photosystems to perform photosynthesis and to evolve oxygen as a byproduct. The presence of Flv genes encoding flavodiiron proteins has been shown to enhance stress tolerance in cyanobacteria. In an attempt to support the growth of plants exposed to drought, the Synechocystis genes Flv1 and Flv3 were expressed in barley with their products being targeted to the chloroplasts. The heterologous expression of both Flv1 and Flv3 accelerated days to heading, increased biomass, promoted the number of spikes and grains per plant, and improved the total grain weight per plant of transgenic lines exposed to drought. Improved growth correlated with enhanced availability of soluble sugars, a higher turnover of amino acids and the accumulation of lower levels of proline in the leaf. Flv1 and Flv3 maintained the energy status of the leaves in the stressed plants by converting sucrose to glucose and fructose, immediate precursors for energy production to support plant growth under drought. The results suggest that sugars and amino acids play a fundamental role in the maintenance of the energy status and metabolic activity to ensure growth and survival under stress conditions, that is, water limitation in this particular case. Engineering chloroplasts by Flv genes into the plant genome, therefore, has the potential to improve plant productivity wherever drought stress represents a significant production constraint.
RESUMEN
Leaf senescence is a developmental process critical for plant fitness, which involves genetically controlled cell death and ordered disassembly of macromolecules for reallocating nutrients to juvenile and reproductive organs. While natural leaf senescence is primarily associated with aging, it can also be induced by environmental and nutritional inputs including biotic and abiotic stresses, darkness, phytohormones and oxidants. Reactive oxygen species (ROS) are a common thread in stress-dependent cell death and also increase during leaf senescence. Involvement of chloroplast redox chemistry (including ROS propagation) in modulating cell death is well supported, with photosynthesis playing a crucial role in providing redox-based signals to this process. While chloroplast contribution to senescence received less attention, recent findings indicate that changes in the redox poise of these organelles strongly affect senescence timing and progress. In this review, the involvement of chloroplasts in leaf senescence execution is critically assessed in relation to available evidence and the role played by environmental and developmental cues such as stress and phytohormones. The collected results indicate that chloroplasts could cooperate with other redox sources (e.g., mitochondria) and signaling molecules to initiate the committed steps of leaf senescence for a best use of the recycled nutrients in plant reproduction.
RESUMEN
Plants exposed to hostile environmental conditions such as drought or extreme temperatures usually undergo oxidative stress, which has long been assumed to significantly contribute to the damage suffered by the organism. Reactive oxygen species (ROS) overproduced under stress conditions were proposed to destroy membrane lipids and to inactivate proteins and photosystems, ultimately leading to cell death. Accordingly, considerable effort has been devoted, over the years, to improve stress tolerance by strengthening antioxidant and dissipative mechanisms. Although the notion that ROS cause indiscriminate damage in vivo has been progressively replaced by the alternate concept that they act as signaling molecules directing critical plant developmental and environmental responses including cell death, the induction of genes encoding antioxidant activities is commonplace under many environmental stresses, suggesting that their manipulation still offers promise. The features and consequences of ROS effects depend on the balance between various interacting pathways including ROS synthesis and scavenging, energy dissipation, conjugative reactions, and eventually reductive repair. They represent many possibilities for genetic manipulation. We report, herein, a comprehensive survey of transgenic plants in which components of the ROS-associated pathways were overexpressed, and of the stress phenotypes displayed by the corresponding transformants. Genetic engineering of different stages of ROS metabolism such as synthesis, scavenging, and reductive repair revealed a strong correlation between down-regulation of ROS levels and increased stress tolerance in plants grown under controlled conditions. Field assays are scarce, and are eagerly required to assess the possible application of this strategy to agriculture.
Asunto(s)
Adaptación Fisiológica , Productos Agrícolas/genética , Productos Agrícolas/metabolismo , Estrés Oxidativo , Estrés Fisiológico , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Especies Reactivas de Oxígeno/metabolismoRESUMEN
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.
RESUMEN
Plants grown in the field experience sharp changes in irradiation due to shading effects caused by clouds, other leaves, etc. The excess of absorbed light energy is dissipated by a number of mechanisms including cyclic electron transport, photorespiration, and Mehler-type reactions. This protection is essential for survival but decreases photosynthetic efficiency. All phototrophs except angiosperms harbor flavodiiron proteins (Flvs) which relieve the excess of excitation energy on the photosynthetic electron transport chain by reducing oxygen directly to water. Introduction of cyanobacterial Flv1/Flv3 in tobacco chloroplasts resulted in transgenic plants that showed similar photosynthetic performance under steady-state illumination, but displayed faster recovery of various photosynthetic parameters, including electron transport and non-photochemical quenching during dark-light transitions. They also kept the electron transport chain in a more oxidized state and enhanced the proton motive force of dark-adapted leaves. The results indicate that, by acting as electron sinks during light transitions, Flvs contribute to increase photosynthesis protection and efficiency under changing environmental conditions as those found by plants in the field.
Asunto(s)
Proteínas Bacterianas/genética , Cloroplastos/metabolismo , Nicotiana/fisiología , Fotosíntesis/fisiología , Synechocystis/genética , Antimicina A/farmacología , Proteínas Bacterianas/metabolismo , Cloroplastos/genética , Transporte de Electrón , Proteínas del Complejo de Cadena de Transporte de Electrón/metabolismo , Plantas Modificadas Genéticamente , Nicotiana/efectos de los fármacos , Nicotiana/genéticaRESUMEN
Crop yield reduction due to salinity is a growing agronomical concern in many regions. Increased production of reactive oxygen species (ROS) in plant cells accompanies many abiotic stresses including salinity, acting as toxic and signaling molecules during plant stress responses. While ROS are generated in various cellular compartments, chloroplasts represent a main source in the light, and plastid ROS synthesis and/or elimination have been manipulated to improve stress tolerance. Transgenic tobacco plants expressing a plastid-targeted cyanobacterial flavodoxin, a flavoprotein that prevents ROS accumulation specifically in chloroplasts, displayed increased tolerance to many environmental stresses, including drought, excess irradiation, extreme temperatures and iron starvation. Surprisingly, flavodoxin expression failed to protect transgenic plants against NaCl toxicity. However, when high salt was directly applied to leaf discs, flavodoxin did increase tolerance, as reflected by preservation of chlorophylls, carotenoids and photosynthetic activities. Flavodoxin decreased salt-dependent ROS accumulation in leaf tissue from discs and whole plants, but this decline did not improve tolerance at the whole plant level. NaCl accumulation in roots, as well as increased osmotic pressure and salt-induced root damage, were not prevented by flavodoxin expression. The results indicate that ROS formed in chloroplasts have a marginal effect on plant responses during salt stress, and that sensitive targets are present in roots which are not protected by flavodoxin.
Asunto(s)
Cloroplastos/metabolismo , Nicotiana/crecimiento & desarrollo , Hojas de la Planta/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Cloruro de Sodio/toxicidad , Estrés Fisiológico/efectos de los fármacos , Adaptación Fisiológica/efectos de los fármacos , Cloroplastos/efectos de los fármacos , Flavodoxina/metabolismo , Iones , Peróxidos Lipídicos/metabolismo , Ósmosis/efectos de los fármacos , Hojas de la Planta/efectos de los fármacos , Raíces de Plantas/efectos de los fármacos , Raíces de Plantas/metabolismo , Plantas Modificadas Genéticamente , Plastidios/efectos de los fármacos , Plastidios/metabolismo , Salinidad , Nicotiana/efectos de los fármacosRESUMEN
Nitric oxide (NO) is a signaling molecule with diverse biological functions in plants. NO plays a crucial role in growth and development, from germination to senescence, and is also involved in plant responses to biotic and abiotic stresses. In animals, NO is synthesized by well-described nitric oxide synthase (NOS) enzymes. NOS activity has also been detected in higher plants, but no gene encoding an NOS protein, or the enzymes required for synthesis of tetrahydrobiopterin, an essential cofactor of mammalian NOS activity, have been identified so far. Recently, an NOS gene from the unicellular marine alga Ostreococcus tauri (OtNOS) has been discovered and characterized. Arabidopsis thaliana plants were transformed with OtNOS under the control of the inducible short promoter fragment (SPF) of the sunflower (Helianthus annuus) Hahb-4 gene, which responds to abiotic stresses and abscisic acid. Transgenic plants expressing OtNOS accumulated higher NO concentrations compared with siblings transformed with the empty vector, and displayed enhanced salt, drought and oxidative stress tolerance. Moreover, transgenic OtNOS lines exhibited increased stomatal development compared with plants transformed with the empty vector. Both in vitro and in vivo experiments indicate that OtNOS, unlike mammalian NOS, efficiently uses tetrahydrofolate as a cofactor in Arabidopsis plants. The modulation of NO production to alleviate abiotic stress disturbances in higher plants highlights the potential of genetic manipulation to influence NO metabolism as a tool to improve plant fitness under adverse growth conditions.
Asunto(s)
Arabidopsis/fisiología , Chlorophyta/genética , Óxido Nítrico Sintasa/genética , Estomas de Plantas/crecimiento & desarrollo , Estrés Fisiológico/genética , Ácido Abscísico/metabolismo , Ácido Abscísico/farmacología , Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Germinación/genética , Helianthus/genética , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raíces de Plantas/efectos de los fármacos , Raíces de Plantas/genética , Raíces de Plantas/crecimiento & desarrollo , Brotes de la Planta/efectos de los fármacos , Brotes de la Planta/genética , Brotes de la Planta/crecimiento & desarrollo , Estomas de Plantas/genética , Plantas Modificadas Genéticamente , Regiones Promotoras Genéticas , Cloruro de Sodio/farmacología , Tetrahidrofolatos/metabolismoRESUMEN
Ferredoxins are electron shuttles harbouring iron-sulfur clusters that connect multiple oxido-reductive pathways in organisms displaying different lifestyles. Some prokaryotes and algae express an isofunctional electron carrier, flavodoxin, which contains flavin mononucleotide as cofactor. Both proteins evolved in the anaerobic environment preceding the appearance of oxygenic photosynthesis. The advent of an oxygen-rich atmosphere proved detrimental to ferredoxin owing to iron limitation and oxidative damage to the iron-sulfur cluster, and many microorganisms induced flavodoxin expression to replace ferredoxin under stress conditions. Paradoxically, ferredoxin was maintained throughout the tree of life, whereas flavodoxin is absent from plants and animals. Of note is that flavodoxin expression in transgenic plants results in increased tolerance to multiple stresses and iron deficit, through mechanisms similar to those operating in microorganisms. Then, the question remains open as to why a trait that still confers plants such obvious adaptive benefits was not retained. We compare herein the properties of ferredoxin and flavodoxin, and their contrasting modes of expression in response to different environmental stimuli. Phylogenetic analyses suggest that the flavodoxin gene was already absent in the algal lineages immediately preceding land plants. Geographical distribution of phototrophs shows a bias against flavodoxin-containing organisms in iron-rich coastal/freshwater habitats. Based on these observations, we propose that plants evolved from freshwater macroalgae that already lacked flavodoxin because they thrived in an iron-rich habitat with no need to back up ferredoxin functions and therefore no selective pressure to keep the flavodoxin gene. Conversely, ferredoxin retention in the plant lineage is probably related to its higher efficiency as an electron carrier, compared with flavodoxin. Several lines of evidence supporting these contentions are presented and discussed.
Asunto(s)
Flavodoxina/metabolismo , Evolución Biológica , Transporte de Electrón , Ferredoxinas/metabolismo , Hierro/metabolismo , FilogeniaRESUMEN
Adverse environmental conditions pose serious limitations to agricultural production. Classical biotechnological approaches towards increasing abiotic stress tolerance focus on boosting plant endogenous defence mechanisms. However, overexpression of regulatory elements or effectors is usually accompanied by growth handicap and yield penalties due to crosstalk between developmental and stress-response networks. Herein we offer an overview on novel strategies with the potential to overcome these limitations based on the engineering of regulatory systems involved in the fine-tuning of the plant response to environmental hardships, including post-translational modifications, small RNAs, epigenetic control of gene expression and hormonal networks. The development and application of plant synthetic biology tools and approaches will add new functionalities and perspectives to genetic engineering programs for enhancing abiotic stress tolerance.
Asunto(s)
Biotecnología/métodos , Ingeniería Genética , Plantas/genética , Plantas/metabolismo , Estrés Fisiológico/genética , Antioxidantes/metabolismo , Carbohidratos , Epigénesis Genética , Homeostasis , MicroARNs/genética , MicroARNs/metabolismo , Chaperonas Moleculares/metabolismo , Concentración Osmolar , Fosforilación , Desarrollo de la Planta , Reguladores del Crecimiento de las Plantas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Sumoilación , Biología Sintética , UbiquitinaciónRESUMEN
Ferredoxins are electron shuttles harboring iron-sulfur clusters which participate in oxido-reductive pathways in organisms displaying very different lifestyles. Ferredoxin levels decline in plants and cyanobacteria exposed to environmental stress and iron starvation. Flavodoxin is an isofunctional flavoprotein present in cyanobacteria and algae (not plants) which is induced and replaces ferredoxin under stress. Expression of a chloroplast-targeted flavodoxin in plants confers tolerance to multiple stresses and iron deficit. We discuss herein the bases for functional equivalence between the two proteins, the reasons for ferredoxin conservation despite its susceptibility to aerobic stress and for the loss of flavodoxin as an adaptive trait in higher eukaryotes. We also propose a mechanism to explain the tolerance conferred by flavodoxin when expressed in plants.
Asunto(s)
Adaptación Fisiológica , Evolución Biológica , Biotecnología , Flavodoxina/metabolismo , Fotosíntesis , Plantas Modificadas GenéticamenteRESUMEN
MicroRNAs (miRNAs) are major regulators of gene expression in multicellular organisms. They recognize their targets by sequence complementarity and guide them to cleavage or translational arrest. It is generally accepted that plant miRNAs have extensive complementarity to their targets and their prediction usually relies on the use of empirical parameters deduced from known miRNA-target interactions. Here, we developed a strategy to identify miRNA targets which is mainly based on the conservation of the potential regulation in different species. We applied the approach to expressed sequence tags datasets from angiosperms. Using this strategy, we predicted many new interactions and experimentally validated previously unknown miRNA targets in Arabidopsis thaliana. Newly identified targets that are broadly conserved include auxin regulators, transcription factors and transporters. Some of them might participate in the same pathways as the targets known before, suggesting that some miRNAs might control different aspects of a biological process. Furthermore, this approach can be used to identify targets present in a specific group of species, and, as a proof of principle, we analyzed Solanaceae-specific targets. The presented strategy can be used alone or in combination with other approaches to find miRNA targets in plants.
Asunto(s)
Regulación de la Expresión Génica de las Plantas , MicroARNs/química , ARN de Planta/química , Arabidopsis/genética , Secuencia de Bases , Secuencia Conservada , Etiquetas de Secuencia Expresada , MicroARNs/metabolismo , ARN Mensajero/química , ARN Mensajero/metabolismo , ARN de Planta/metabolismo , Alineación de Secuencia , SolanaceaeRESUMEN
Oxidative stress in plants causes ferredoxin down-regulation and NADP(+) shortage, over-reduction of the photosynthetic electron transport chain, electron leakage to oxygen and generation of reactive oxygen species (ROS). Expression of cyanobacterial flavodoxin in tobacco chloroplasts compensates for ferredoxin decline and restores electron delivery to productive routes, resulting in enhanced stress tolerance. We have designed an in vivo system to optimize flavodoxin reduction and NADP(+) regeneration under stress using a version of cyanobacterial ferredoxin-NADP(+) reductase without the thylakoid-binding domain. Co-expression of the two soluble flavoproteins in the chloroplast stroma resulted in lines displaying maximal tolerance to redox-cycling oxidants, lower damage and decreased ROS accumulation. The results underscore the importance of chloroplast redox homeostasis in plants exposed to adverse conditions, and provide a tool to improve crop tolerance toward environmental hardships.
RESUMEN
Sulfate is a partial inhibitor at low and a non-essential activator at high [ATP] of the ATPase activity of F(1). Therefore, a catalytically-competent ternary F(1) x ATP x sulfate complex can be formed. In addition, the ANS fluorescence enhancement driven by ATP hydrolysis in submitochondrial particles is also stimulated by sulfate, clearly showing that the ATP hydrolysis in its presence is coupled to H(+) translocation. However, sulfate is a strong linear inhibitor of the mitochondrial ATP synthesis. The inhibition was competitive (K (i) = 0.46 mM) with respect to Pi and mixed (K (i) = 0.60 and K'(i) = 5.6 mM) towards ADP. Since it is likely that sulfate exerts its effects by binding at the Pi binding subdomain of the catalytic site, we suggest that the catalytic site involved in the H(+) translocation driven by ATP hydrolysis has a more open conformation than the half-closed one (beta(HC)), which is an intermediate in ATP synthesis. Accordingly, ATP hydrolysis is not necessarily the exact reversal of ATP synthesis.