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.

Organ-specific COP1 control of BES1 stability adjusts plant growth patterns under shade or warmth.

Under adverse conditions such as shade or elevated temperatures, cotyledon expansion is reduced and hypocotyl growth is promoted to optimize plant architecture. The mechanisms underlying the repression of cotyledon cell expansion remain unknown. Here, we report that the nuclear abundance of the BES1 transcription factor decreased in the cotyledons and increased in the hypocotyl in Arabidopsis thaliana under shade or warmth. Brassinosteroid levels did not follow the same trend. PIF4 and COP1 increased their nuclear abundance in both organs under shade or warmth. PIF4 directly bound the BES1 promoter to enhance its activity but indirectly reduced BES1 expression. COP1 physically interacted with the BES1 protein, promoting its proteasome degradation in the cotyledons. COP1 had the opposite effect in the hypocotyl, demonstrating organ-specific regulatory networks. Our work indicates that shade or warmth reduces BES1 activity by transcriptional and post-translational regulation to inhibit cotyledon cell expansion.

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.

Shade avoidance responses become more aggressive in warm environments.

When exposed to neighbour cues, competitive plants increase stem growth to reduce the degree of current or future shade. The aim of this work is to investigate the impact of weather conditions on the magnitude of shade avoidance responses in Arabidopsis thaliana. We first generated a growth rate database under controlled conditions and elaborated a model that predicts daytime hypocotyl growth as a function of the activity of the main photosensory receptors (phytochromes A and B, cryptochromes 1 and 2) in combination with light and temperature inputs. We then incorporated the action of thermal amplitude to account for its effect on selected genotypes, which correlates with the dynamics of the growth-promoting transcription factor PHYTOCHROME-INTERACTING FACTOR 4. The model predicted growth rate in the field with reasonable accuracy. Thus, we used the model in combination with a worldwide data set of current and future whether conditions. The analysis predicted enhanced shade avoidance responses as a result of higher temperatures due to the geographical location or global warming. Irradiance and thermal amplitude had no effects. These trends were also observed for our local growth rate measurements. We conclude that, if water and nutrients do not become limiting, warm environments enhance the shade avoidance response.

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.

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.

Multiple links between shade avoidance and auxin networks.

Auxin has emerged as a key player in the adjustment of plant morphology to the challenge imposed by variable environmental conditions. Shade-avoidance responses, including the promotion of stem and petiole growth, leaf hyponasty, and the inhibition of branching, involve an intimate connection between light and auxin signalling. Low activity of photo-sensory receptors caused by the presence of neighbouring vegetation enhances the activity of PHYTOCHROME INTERACTING FACTORs (PIFs), which directly promote the expression of genes involved in auxin biosynthesis, conjugation, transport, perception, and signalling. In seedlings, neighbour signals increase auxin levels in the foliage, which then moves to the stem, where it reaches epidermal tissues to promote growth. However, this model only partially accounts for shade-avoidance responses (which may also occur in the absence of increased auxin levels), and understanding the whole picture will require further insight into the functional significance of the multiple links between shade and auxin networks.

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.

Novel perspectives for the engineering of abiotic stress tolerance in plants.

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.

Bacterial signaling systems as platforms for rational design of new generations of biosensors.

Bacterial signal-responsive regulatory circuits have been employed as platform to design and construct whole-cell bacterial biosensors for reporting toxicity. A new generation of biosensors with improved performance and a wide application range has emerged after the application of synthetic biology concepts to biosensor design. Site-directed mutagenesis, directed evolution and domain swapping were applied to upgrade signal detection or to create novel sensor modules. Rewiring of the genetic circuits allows improving the determinations and reduces the heterogeneity of the response between individual reporter cells. Moreover, the assembly of natural or engineered modules to biosensor platforms provides innovative outputs, expanding the range of application of these devises, from monitoring toxics and bioremediation to killing targeted cells.

ROS signaling in the hypersensitive response: when, where and what for?

Plants generally react to the attack of non-host and incompatible host microorganisms by inducing pathogenesis-related (PR) genes and localised cell death (LCD) at the site of infection, a process collectively known as the hypersensitive response (HR). Reactive oxygen species (ROS) are generated in various sub-cellular compartments shortly after pathogen recognition, and proposed to cue subsequent orchestration of the HR. Although apoplast-associated ROS production by plasma membrane NADPH oxidases have been most thoroughly studied, recent observations suggest that ROS are generated in chloroplasts earlier in the response and play a key role in execution of LCD. A model is presented in which the initial outcome of successful pathogen detection is ROS accumulation in plastids, likely mediated by mitogen-activated protein kinases and caused by dysfunction of the photosynthetic electron transport chain. ROS signaling is proposed to spread from plastids to the apoplast, through the activation of NADPH oxidases, and from there to adjacent cells, leading to suicidal death in the region of attempted infection.

Engineering the future. Development of transgenic plants with enhanced tolerance to adverse environments.

Environmental stresses - especially drought and salinity - and iron limitation are the primary causes of crop yield losses. Therefore, improvement of plant stress tolerance has paramount relevance for agriculture, and vigorous efforts are underway to design stress-tolerant crops. Three aspects of this ongoing research are reviewed here. First, attempts have been made to strengthen endogenous plant defences, which are characterised by intertwined, hierarchical gene networks involved in stress perception, signalling, regulation and expression of effector proteins, enzymes and metabolites. The multigenic nature of this response requires detailed knowledge of the many actors and interactions involved in order to identify proper intervention points, followed by significant engineering of the prospective genes to prevent undesired side-effects. A second important aspect refers to the effect of concurrent stresses as plants normally meet in the field (e.g., heat and drought). Recent findings indicate that plant responses to combined environmental hardships are somehow unique and cannot be predicted from the addition of the individual stresses, underscoring the importance of programming research within this conceptual framework. Finally, the photosynthetic microorganisms from which plants evolved (i.e., algae and cyanobacteria) deploy a totally different strategy to acquire stress tolerance, based on the substitution of stress-vulnerable targets by resistant isofunctional proteins that could take over the lost functions under adverse conditions. Reintroduction of these ancient traits in model and crop plants has resulted in increased tolerance to environmental hardships and iron starvation, opening a new field of opportunities to increase the endurance of crops growing under suboptimal conditions.

Chloroplast-generated reactive oxygen species play a major role in localized cell death during the non-host interaction between tobacco and Xanthomonas campestris pv. vesicatoria.

Attempted infection of plants by pathogens elicits a complex defensive response. In many non-host and incompatible host interactions it includes the induction of defence-associated genes and a form of localized cell death (LCD), purportedly designed to restrict pathogen advance, collectively known as the hypersensitive response (HR). It is preceded by an oxidative burst, generating reactive oxygen species (ROS) that are proposed to cue subsequent deployment of the HR, although neither the origin nor the precise role played by ROS in the execution of this response are completely understood. We used tobacco plants expressing cyanobacterial flavodoxin to address these questions. Flavodoxin is an electron shuttle present in prokaryotes and algae that, when expressed in chloroplasts, specifically prevents ROS formation in plastids during abiotic stress episodes. Infiltration of tobacco wild-type leaves with high titres of Xanthomonas campestris pv. vesicatoria (Xcv), a non-host pathogen, resulted in ROS accumulation in chloroplasts, followed by the appearance of localized lesions typical of the HR. In contrast, chloroplast ROS build-up and LCD were significantly reduced in Xcv-inoculated plants expressing plastid-targeted flavodoxin. Metabolic routes normally inhibited by pathogens were protected in the transformants, whereas other aspects of the HR, including the induction of defence-associated genes and synthesis of salicylic and jasmonic acid, proceeded as in inoculated wild-type plants. Therefore, ROS generated in chloroplasts during this non-host interaction are essential for the progress of LCD, but do not contribute to the induction of pathogenesis-related genes or other signalling components of the response.

Combating stress with flavodoxin: a promising route for crop improvement.

Environmental stresses and iron limitation are the primary causes of crop losses worldwide. Engineering strategies aimed at gaining stress tolerance have focused on overexpression of endogenous genes belonging to molecular networks for stress perception or responses. Based on the typical response of photosynthetic microorganisms to stress, an alternative approach has been recently applied with considerable success. Ferredoxin, a stress-sensitive target, was replaced in tobacco chloroplasts by an isofunctional protein, a cyanobacterial flavodoxin, which is absent in plants. Resulting transgenic lines showed wide-range tolerance to drought, chilling, oxidants, heat and iron starvation. The survival of plants under such adverse conditions would be an enormous agricultural advantage and makes this novel strategy a potentially powerful biotechnological tool for the generation of multiple-tolerant crops in the near future.

Enhanced plant tolerance to iron starvation by functional substitution of chloroplast ferredoxin with a bacterial flavodoxin.

Iron limitation affects one-third of the cultivable land on Earth and represents a major concern for agriculture. It causes decline of many photosynthetic components, including the Fe-S protein ferredoxin (Fd), involved in essential oxidoreductive pathways of chloroplasts. In cyanobacteria and some algae, Fd down-regulation under Fe deficit is compensated by induction of an isofunctional electron carrier, flavodoxin (Fld), a flavin mononucleotide-containing protein not found in plants. Transgenic tobacco lines expressing a cyanobacterial Fld in chloroplasts were able to grow in Fe-deficient media that severely compromised survival of WT plants. Fld expression did not improve Fe uptake or mobilization, and stressed transformants elicited a normal deficit response, including induction of ferric-chelate reductase and metal transporters. However, the presence of Fld did prevent decrease of several photosynthetic proteins (but not Fd) and partially protected photosynthesis from inactivation. It also preserved the activation state of enzymes depending on the Fd-thioredoxin pathway, which correlated with higher levels of intermediates of carbohydrate metabolism and the Calvin cycle, as well as increased contents of sucrose, glutamate, and other amino acids. These metabolic routes depend, directly or indirectly, on the provision of reduced Fd. The results indicate that Fld could compensate Fd decline during episodes of Fe deficiency by productively interacting with Fd-dependent pathways of the host, providing fresh genetic resources for the design of plants able to survive in Fe-poor lands.

Stress-inducible flavodoxin from photosynthetic microorganisms. The mystery of flavodoxin loss from the plant genome.

Flavodoxins (Flds) are mobile electron carriers containing flavin mononucleotide as the prosthetic group. They are isofunctional with the ubiquitous electron shuttle ferredoxin (Fd), mediating essentially the same redox processes among a promiscuous lot of donors and acceptors. While Fds are distributed throughout all kingdoms from prokaryotes to animals, Flds are only found in some bacteria and oceanic algae, in which they are induced to replace Fd functions under conditions of iron starvation and environmental stress that cause Fd decline. They thus play a key adaptive role in photosynthetic microorganisms, allowing survival and reproduction under adverse situations. The Fld gene disappeared from the plant genome somewhere between the green algal ancestor and the first terrestrial plants, and the advantages of this adaptive resource were irreversibly lost. However, reintroduction of a cyanobacterial Fld gene in the chloroplasts of transgenic tobacco resulted in remarkably enhanced tolerance to iron starvation and abiotic stress, indicating that the compensatory functions of Fld were still valuable in higher plants. A hypothesis is formulated to explain why Fld, in spite of its proven advantage, was lost from the plant genetic pool. The contention is based on two tenets: (i) iron availability was the major imperative for Fld conservation and adaptive value, and (ii) photosynthetic eukaryotes followed a succession of ecological adaptations, from the open oceans to coastal regions, and from there to the firm land, facing very different scenarios with respect to iron abundance and accessibility.