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.

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.

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.

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.

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.

Photosynthetic characterization of flavodoxin-expressing tobacco plants reveals a high light acclimation-like phenotype.

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.

Nitrogen Depletion Blocks Growth Stimulation Driven by the Expression of Nitric Oxide Synthase in Tobacco.

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.

Expression of a Chloroplast-Targeted Cyanobacterial Flavodoxin in Tomato Plants Increases Harvest Index by Altering Plant Size and Productivity.

Tomato is the most important horticultural crop worldwide. Domestication has led to the selection of highly fruited genotypes, and the harvest index (HI), defined as the ratio of fruit yield over total plant biomass, is usually employed as a biomarker of agronomic value. Improvement of HI might then result from increased fruit production and/or lower vegetative growth. Reduction in vegetative biomass has been accomplished in various plant species by expression of flavodoxin, an electron shuttle flavoprotein that interacts with redox-based pathways of chloroplasts including photosynthesis. However, the effect of this genetic intervention on the development of reproductive organs has not been investigated. We show herein that expression of a plastid-targeted cyanobacterial flavodoxin in tomato resulted in significant reduction of plant size affecting stems, leaves, and fruit. Decreased size correlated with smaller cells and was accompanied by higher pigment contents and photosynthetic activities per leaf cross-section. Flavodoxin accumulated in green fruit but declined with ripening. Significant increases in HI were observed in flavodoxin-expressing lines due to the production of higher fruit number per plant in smaller plants. Therefore, overall yields can be enhanced by increasing plant density in the field. Metabolic profiling of ripe red fruit showed that levels of sugars, organic acids, and amino acids were similar or higher in transgenic plants, indicating that there was no trade-off between increased HI and fruit metabolite contents in flavodoxin-expressing plants. Taken together, our results show that flavodoxin has the potential to improve major agronomic traits when introduced in tomato.

Leaf Senescence: The Chloroplast Connection Comes of Age.

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.

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.

Expression of a Plastid-Targeted Flavodoxin Decreases Chloroplast Reactive Oxygen Species Accumulation and Delays Senescence in Aging Tobacco Leaves.

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.

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.

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.

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.

Chloroplast Redox Status Modulates Genome-Wide Plant Responses during the Non-host Interaction of Tobacco with the Hemibiotrophic Bacterium Xanthomonas campestris pv. vesicatoria.

Non-host resistance is the most ample and durable form of plant resistance against pathogen infection. It includes induction of defense-associated genes, massive metabolic reprogramming, and in many instances, a form of localized cell death (LCD) at the site of infection, purportedly designed to limit the spread of biotrophic and hemibiotrophic microorganisms. Reactive oxygen species (ROS) have been proposed to act as signals for LCD orchestration. They are produced in various cellular compartments including chloroplasts, mitochondria and apoplast. We have previously reported that down-regulation of ROS build-up in chloroplasts by expression of a plastid-targeted flavodoxin (Fld) suppressed LCD in tobacco leaves inoculated with the non-host bacterium Xanthomonas campestris pv. vesicatoria (Xcv), while other defensive responses were unaffected, suggesting that chloroplast ROS and/or redox status play a major role in the progress of LCD. To better understand these effects, we compare here the transcriptomic alterations caused by Xcv inoculation on leaves of Fld-expressing tobacco plants and their wild-type siblings. About 29% of leaf-expressed genes were affected by Xcv and/or Fld. Surprisingly, 5.8% of them (1,111 genes) were regulated by Fld in the absence of infection, presumably representing pathways responsive to chloroplast ROS production and/or redox status during normal growth conditions. While the majority (∼75%) of pathogen-responsive genes were not affected by Fld, many Xcv responses were exacerbated, attenuated, or regulated in opposite direction by expression of this protein. Particularly interesting was a group of 384 genes displaying Xcv responses that were already triggered by Fld in the absence of infection, suggesting that the transgenic plants had a larger and more diversified suite of constitutive defenses against the attacking microorganism compared to the wild type. Fld modulated many genes involved in pathogenesis, signal transduction, transcriptional regulation and hormone-based pathways. Remarkable interactions with proteasomal protein degradation were observed. The results provide the first genome-wide, comprehensive picture illustrating the relevance of chloroplast redox status in biotic stress responses.

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.