Introduction of a terminal electron sink in chloroplasts decreases leaf cell expansion associated with higher proteasome activity and lower endoreduplication.

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.

Lighting the light reactions of photosynthesis by means of redox-responsive genetically encoded biosensors for photosynthetic intermediates.

Oxygenic photosynthesis involves light and dark phases. In the light phase, photosynthetic electron transport provides reducing power and energy to support the carbon assimilation process. It also contributes signals to defensive, repair, and metabolic pathways critical for plant growth and survival. The redox state of components of the photosynthetic machinery and associated routes determines the extent and direction of plant responses to environmental and developmental stimuli, and therefore, their space- and time-resolved detection in planta becomes critical to understand and engineer plant metabolism. Until recently, studies in living systems have been hampered by the inadequacy of disruptive analytical methods. Genetically encoded indicators based on fluorescent proteins provide new opportunities to illuminate these important issues. We summarize here information about available biosensors designed to monitor the levels and redox state of various components of the light reactions, including NADP(H), glutathione, thioredoxin, and reactive oxygen species. Comparatively few probes have been used in plants, and their application to chloroplasts poses still additional challenges. We discuss advantages and limitations of biosensors based on different principles and propose rationales for the design of novel probes to estimate the NADP(H) and ferredoxin/flavodoxin redox poise, as examples of the exciting questions that could be addressed by further development of these tools. Genetically encoded fluorescent biosensors are remarkable tools to monitor the levels and/or redox state of components of the photosynthetic light reactions and accessory pathways. Reducing equivalents generated at the photosynthetic electron transport chain in the form of NADPH and reduced ferredoxin (FD) are used in central metabolism, regulation, and detoxification of reactive oxygen species (ROS). Redox components of these pathways whose levels and/or redox status have been imaged in plants using biosensors are highlighted in green (NADPH, glutathione, H2O2, thioredoxins). Analytes with available biosensors not tried in plants are shown in pink (NADP+). Finally, redox shuttles with no existing biosensors are circled in light blue. APX, ASC peroxidase; ASC, ascorbate; DHA, dehydroascorbate; DHAR, DHA reductase; FNR, FD-NADP+ reductase; FTR, FD-TRX reductase; GPX, glutathione peroxidase; GR, glutathione reductase; GSH, reduced glutathione; GSSG, oxidized glutathione; MDA, monodehydroascorbate; MDAR, MDA reductase; NTRC, NADPH-TRX reductase C; OAA, oxaloacetate; PRX, peroxiredoxin; PSI, photosystem I; PSII: photosystem II; SOD, superoxide dismutase; TRX, thioredoxin.

The chloroplast redox-responsive transcriptome of solanaceous plants reveals significant nuclear gene regulatory motifs associated to stress acclimation.

KEY MESSAGE: Transcriptomes of solanaceous plants expressing a plastid-targeted antioxidant protein were analysed to identify chloroplast redox networks modulating the expression of nuclear genes associated with stress acclimation. Plastid functions depend on the coordinated expression of nuclear genes, many of them associated to developmental and stress response pathways. Plastid-generated signals mediate this coordination via retrograde signaling, which includes sensing of chloroplast redox state and levels of reactive oxygen species (ROS), although it remains a poorly understood process. Chloroplast redox poise and ROS build-up can be modified by recombinant expression of a plastid-targeted antioxidant protein, i.e., cyanobacterial flavodoxin, with the resulting plants displaying increased tolerance to multiple environmental challenges. Here we analysed the transcriptomes of these flavodoxin-expressing plants to study the coordinated transcriptional responses of the nucleus to the chloroplast redox status and ROS levels during normal growth and stress responses (drought or biotic stress) in tobacco and potato, members of the economically important Solanaceae family. We compared their transcriptomes against those from stressed and mutant plants accumulating ROS in different subcellular compartments and found distinct ROS-related imprints modulated by flavodoxin expression and/or stress. By introducing our datasets in a large-scale interaction network, we identified transcriptional factors related to ROS and stress responses potentially involved in flavodoxin-associated signaling. Finally, we discovered identical cis elements in the promoters of many genes that respond to flavodoxin in the same direction as in wild-type plants under stress, suggesting a priming effect of flavodoxin before stress manifestation. The results provide a genome-wide picture illustrating the relevance of chloroplast redox status on biotic and abiotic stress responses and suggest new cis and trans targets to generate stress-tolerant solanaceous crops.

Photosynthesis and chloroplast redox signaling in the age of global warming: stress tolerance, acclimation, and developmental plasticity.

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.

Engineering Climate-Change-Resilient Crops: New Tools and Approaches.

Environmental adversities, particularly drought and nutrient limitation, are among the major causes of crop losses worldwide. Due to the rapid increase of the world's population, there is an urgent need to combine knowledge of plant science with innovative applications in agriculture to protect plant growth and thus enhance crop yield. In recent decades, engineering strategies have been successfully developed with the aim to improve growth and stress tolerance in plants. Most strategies applied so far have relied on transgenic approaches and/or chemical treatments. However, to cope with rapid climate change and the need to secure sustainable agriculture and biomass production, innovative approaches need to be developed to effectively meet these challenges and demands. In this review, we summarize recent and advanced strategies that involve the use of plant-related cyanobacterial proteins, macro- and micronutrient management, nutrient-coated nanoparticles, and phytopathogenic organisms, all of which offer promise as protective resources to shield plants from climate challenges and to boost stress tolerance in crops.

Expression of Flavodiiron Proteins Flv2-Flv4 in Chloroplasts of Arabidopsis and Tobacco Plants Provides Multiple Stress Tolerance.

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.

Transcriptional and Metabolic Profiling of Potato Plants Expressing a Plastid-Targeted Electron Shuttle Reveal Modulation of Genes Associated to Drought Tolerance by Chloroplast Redox Poise.

Water limitation represents the main environmental constraint affecting crop yield worldwide. Photosynthesis is a primary drought target, resulting in over-reduction of the photosynthetic electron transport chain and increased production of reactive oxygen species in plastids. Manipulation of chloroplast electron distribution by introducing alternative electron transport sinks has been shown to increase plant tolerance to multiple environmental challenges including hydric stress, suggesting that a similar strategy could be used to improve drought tolerance in crops. We show herein that the expression of the cyanobacterial electron shuttle flavodoxin in potato chloroplasts protected photosynthetic activities even at a pre-symptomatic stage of drought. Transcriptional and metabolic profiling revealed an attenuated response to the adverse condition in flavodoxin-expressing plants, correlating with their increased stress tolerance. Interestingly, 5-6% of leaf-expressed genes were affected by flavodoxin in the absence of drought, representing pathways modulated by chloroplast redox status during normal growth. About 300 of these genes potentially contribute to stress acclimation as their modulation by flavodoxin proceeds in the same direction as their drought response in wild-type plants. Tuber yield losses under chronic water limitation were mitigated in flavodoxin-expressing plants, indicating that the flavoprotein has the potential to improve major agronomic traits in potato.

Long-Day Photoperiod Enhances Jasmonic Acid-Related Plant Defense.

Agricultural crops are exposed to a range of daylengths, which act as important environmental cues for the control of developmental processes such as flowering. To explore the additional effects of daylength on plant function, we investigated the transcriptome of Arabidopsis (Arabidopsis thaliana) plants grown under short days (SD) and transferred to long days (LD). Compared with that under SD, the LD transcriptome was enriched in genes involved in jasmonic acid-dependent systemic resistance. Many of these genes exhibited impaired expression induction under LD in the phytochrome A (phyA), cryptochrome 1 (cry1), and cry2 triple photoreceptor mutant. Compared with that under SD, LD enhanced plant resistance to the necrotrophic fungus Botrytis cinerea This response was reduced in the phyA cry1 cry2 triple mutant, in the constitutive photomorphogenic1 (cop1) mutant, in the myc2 mutant, and in mutants impaired in DELLA function. Plants grown under SD had an increased nuclear abundance of COP1 and decreased DELLA abundance, the latter of which was dependent on COP1. We conclude that growth under LD enhances plant defense by reducing COP1 activity and enhancing DELLA abundance and MYC2 expression.

Manipulation of oxidative stress responses as a strategy to generate stress-tolerant crops. From damage to signaling to tolerance.

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.

Reactive oxygen species generated in chloroplasts contribute to tobacco leaf infection by the necrotrophic fungus Botrytis cinerea.

Reactive oxygen species (ROS) play fundamental roles in plant responses to pathogen infection, including modulation of cell death processes and defense-related gene expression. Cell death triggered as part of the hypersensitive response enhances resistance to biotrophic pathogens, but favors the virulence of necrotrophs. Even though the involvement of ROS in the orchestration of defense responses is well established, the relative contribution of specific subcellular ROS sources to plant resistance against microorganisms with different pathogenesis strategies is not completely known. The aim of this work was to investigate the role of chloroplastic ROS in plant defense against a typical necrotrophic fungus, Botrytis cinerea. For this purpose, we used transgenic Nicotiana tabacum (tobacco) lines expressing a plastid-targeted cyanobacterial flavodoxin (pfld lines), which accumulate lower chloroplastic ROS in response to different stresses. Tissue damage and fungal growth were significantly reduced in infected leaves of pfld plants, as compared with infected wild-type (WT) counterparts. ROS build-up triggered by Botrytis infection and associated with chloroplasts was significantly decreased (70-80%) in pfld leaves relative to the wild type. Phytoalexin accumulation and expression of pathogenesis-related genes were induced to a lower degree in pfld plants than in WT siblings. The impact of fungal infection on photosynthetic activity was also lower in pfld leaves. The results indicate that chloroplast-generated ROS play a major role in lesion development during Botrytis infection. This work demonstrates that the modulation of chloroplastic ROS levels by the expression of a heterologous antioxidant protein can provide a significant degree of protection against a canonical necrotrophic fungus.

Faster photosynthetic induction in tobacco by expressing cyanobacterial flavodiiron proteins in chloroplasts.

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.

Evolution of the acceptor side of photosystem I: ferredoxin, flavodoxin, and ferredoxin-NADP+ oxidoreductase.

The development of oxygenic photosynthesis by primordial cyanobacteria ~2.7 billion years ago led to major changes in the components and organization of photosynthetic electron transport to cope with the challenges of an oxygen-enriched atmosphere. We review herein, following the seminal contributions as reported by Jaganathan et al. (Functional genomics and evolution of photosynthetic systems, vol 33, advances in photosynthesis and respiration, Springer, Dordrecht, 2012), how these changes affected carriers and enzymes at the acceptor side of photosystem I (PSI): the electron shuttle ferredoxin (Fd), its isofunctional counterpart flavodoxin (Fld), their redox partner ferredoxin-NADP+ reductase (FNR), and the primary PSI acceptors F x and F A/F B. Protection of the [4Fe-4S] centers of these proteins from oxidative damage was achieved by strengthening binding between the F A/F B polypeptide and the reaction center core containing F x, therefore impairing O2 access to the clusters. Immobilization of F A/F B in the PSI complex led in turn to the recruitment of new soluble electron shuttles. This function was fulfilled by oxygen-insensitive [2Fe-2S] Fd, in which the reactive sulfide atoms of the cluster are shielded from solvent by the polypeptide backbone, and in some algae and cyanobacteria by Fld, which employs a flavin as prosthetic group and is tolerant to oxidants and iron limitation. Tight membrane binding of FNR allowed solid-state electron transfer from PSI bridged by Fd/Fld. Fine tuning of FNR catalytic mechanism led to formidable increases in turnover rates compared with FNRs acting in heterotrophic pathways, favoring Fd/Fld reduction instead of oxygen reduction.

A long-chain flavodoxin protects Pseudomonas aeruginosa from oxidative stress and host bacterial clearance.

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.

Expression of the tetrahydrofolate-dependent nitric oxide synthase from the green alga Ostreococcus tauri increases tolerance to abiotic stresses and influences stomatal development in Arabidopsis.

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.

The long goodbye: the rise and fall of flavodoxin during plant evolution.

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.

Identification of new microRNA-regulated genes by conserved targeting in plant species.

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.

The extremophilic Andean isolate Acinetobacter sp. Ver3 expresses two ferredoxin-NADP+ reductase isoforms with different catalytic properties.

Ferredoxin/flavodoxin-NADPH reductases (FPRs) catalyze the reversible electron transfer between NADPH and ferredoxin/flavodoxin. The Acinetobacter sp. Ver3 isolated from high-altitude Andean lakes contains two isoenzymes, FPR1ver3 and FPR2ver3. Absorption spectra of these FPRs revealed typical features of flavoproteins, consistent with the use of FAD as a prosthetic group. Spectral differences indicate distinct electronic arrangements for the flavin in each enzyme. Steady-state kinetic measurements show that the enzymes display catalytic efficiencies in the order of 1-6 µm-1·s-1, although FPR1ver3 exhibited higher kcat values compared to FPR2ver3. When flavodoxinver3 was used as a substrate, both reductases exhibited dissimilar behavior. Moreover, only FPR1ver3 is induced by oxidative stimuli, indicating that the polyextremophile Ver3 has evolved diverse strategies to cope with oxidative environments.

NERNST: a genetically-encoded ratiometric non-destructive sensing tool to estimate NADP(H) redox status in bacterial, plant and animal systems.

NADP(H) is a central metabolic hub providing reducing equivalents to multiple biosynthetic, regulatory and antioxidative pathways in all living organisms. While biosensors are available to determine NADP+ or NADPH levels in vivo, no probe exists to estimate the NADP(H) redox status, a determinant of the cell energy availability. We describe herein the design and characterization of a genetically-encoded ratiometric biosensor, termed NERNST, able to interact with NADP(H) and estimate ENADP(H). NERNST consists of a redox-sensitive green fluorescent protein (roGFP2) fused to an NADPH-thioredoxin reductase C module which selectively monitors NADP(H) redox states via oxido-reduction of the roGFP2 moiety. NERNST is functional in bacterial, plant and animal cells, and organelles such as chloroplasts and mitochondria. Using NERNST, we monitor NADP(H) dynamics during bacterial growth, environmental stresses in plants, metabolic challenges to mammalian cells, and wounding in zebrafish. NERNST estimates the NADP(H) redox poise in living organisms, with various potential applications in biochemical, biotechnological and biomedical research.

Cyanobacterial flavodoxin complements ferredoxin deficiency in knocked-down transgenic tobacco plants.

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.