Néstor Carrillo

A productive NADP+ binding mode of ferredoxin-NADP+ reductase revealed by protein engineering and crystallographic studies.

The flavoenzyme ferredoxin–NADP+ reductase (FNR) catalyzes the production of NADPH during photosynthesis. Whereas the structures of FNRs from spinach leaf and a cyanobacterium as well as many of their homologs have been solved, none of these studies has yielded a productive geometry of the flavin–nicotinamide interaction. Here, we show that this failure occurs because nicotinamide binding to wild type FNR involves the energetically unfavorable displacement of the C-terminal Tyr side chain. We used mutants of this residue (Tyr 308) of pea FNR to obtain the structures of productive NADP+ and NADPH complexes. These structures reveal a unique NADP+ binding mode in which the nicotinamide ring is not parallel to the flavin isoalloxazine ring, but lies against it at an angle of ~30°, with the C4 atom 3 Å from the flavin N5 atom.

Generation of superoxide anion in chloroplasts of Arabidopsis thaliana during active photosynthesis: a focus on rapidly induced genes

The antioxidant defense system involves complex functional coordination of multiple components in different organelles within the plant cell. Here, we have studied the Arabidopsis thaliana early response to the generation of superoxide anion in chloroplasts during active photosynthesis. We exposed plants to methyl viologen (MV), a superoxide anion propagator in the light, and performed biochemical and expression profiling experiments using Affymetrix ATH1 GeneChip® microarrays under conditions in which photosynthesis and antioxidant enzymes were active. Data analysis identified superoxide-responsive genes that were compared with available microarray results. Examples include genes encoding proteins with unknown function, transcription factors and signal transduction components. A common GAAAAGTCAAAC motif containing the W-box consensus sequence of WRKY transcription factors, was found in the promoters of genes highly up-regulated by superoxide. Band shift assays showed that oxidative treatments enhanced the specific binding of leaf protein extracts to this motif. In addition, GUS reporter gene fused to WRKY30 promoter, which contains this binding motif, was induced by MV and H2O2. Overall, our study suggests that genes involved in signalling pathways and with unknown functions are rapidly activated by superoxide anion generated in photosynthetically active chloroplasts, as part of the early antioxidant response of Arabidopsis leaves.

Functional replacement of ferredoxin by a cyanobacterial flavodoxin in tobacco confers broad-range stress tolerance.

Chloroplast ferredoxin (Fd) plays a pivotal role in plant cell metabolism by delivering reducing equivalents to various essential oxidoreductive pathways. Fd levels decrease under adverse environmental conditions in many microorganisms, including cyanobacteria, which share a common ancestor with chloroplasts. Conversely, stress situations induce the synthesis of flavodoxin (Fld), an electron carrier flavoprotein not found in plants, which can efficiently replace Fd in most electron transfer processes. We report here that chloroplast Fd also declined in plants exposed to oxidants or stress conditions. A purified cyanobacterial Fld was able to mediate plant Fd-dependent reactions in vitro, including NADP+ and thioredoxin reduction. Tobacco (Nicotiana tabacum) plants expressing Fld in chloroplasts displayed increased tolerance to multiple sources of stress, including redox-cycling herbicides, extreme temperatures, high irradiation, water deficit, and UV radiation. Oxidant buildup and oxidative inactivation of thioredoxin-dependent plastidic enzymes were decreased in stressed plants expressing plastid-targeted Fld, suggesting that development of the tolerant phenotype relied on productive interaction of this flavoprotein with Fd-dependent oxidoreductive pathways of the host, most remarkably, thioredoxin reduction. The use of Fld provides new tools to investigate the requirements of photosynthesis in planta and to increase plant stress tolerance based on the introduction of a cyanobacterial product that is free from endogenous regulation in higher plants.

The Flavoenzyme Ferredoxin (Flavodoxin)-NADP(H) Reductase Modulates NADP(H) Homeostasis during the soxRS Response of Escherichia coli

Escherichia coli cells from strain fpr, deficient in the soxRS-induced ferredoxin (flavodoxin)-NADP(H) reductase (FPR), display abnormal sensitivity to the bactericidal effects of the superoxide-generating reagent methyl viologen (MV). Neither bacteriostatic effects nor inactivation of oxidant-sensitive hydrolyases could be detected in fpr cells exposed to MV. FPR inactivation did not affect the MV-driven soxRS response, whereas FPR overexpression led to enhanced stimulation of the regulon, with concomitant oxidation of the NADPH pool. Accumulation of a site-directed FPR mutant that uses NAD(H) instead of NADP(H) had no effect on soxRS induction and failed to protect fpr cells from MV toxicity, suggesting that FPR contributes to NADP(H) homeostasis in stressed bacteria.

Oxidative Stress Causes Ferredoxin-NADP+ Reductase Solubilization from the Thylakoid Membranes in Methyl Viologen-Treated Plants

The flavoenzyme ferredoxin-NADP+ reductase (FNR) is a member of the cellular defense barrier against oxidative damage in Escherichia coli. We evaluated the responses of chloroplast FNR to methyl viologen, a superoxide radical propagator, in wheat (Triticum aestivum L.) plants and chloroplasts. Treatments with the herbicide showed little effect on the levels of FNR protein or transcripts, indicating that expression of this reductase is not up-regulated by oxidants in plants. Viologens and peroxides caused solubilization of active FNR from the thylakoids into the stroma, converting the enzyme from a membrane-bound NADPH producer to a soluble NADPH consumer. This response appeared specific for FNR, since other thylakoid proteins were unaffected by the treatments. The reductase-binding protein was released together with FNR, suggesting that it might be the target of oxidative modification. Stromal accumulation of a functional NADPH reductase in response to oxidative stress is formally analogous to the induction of FNR synthesis observed in E. coli under similar conditions. FNR solubilization may be playing a crucial role in maintaining the NADPH/NADP+ homeostasis of the stressed plastid. The unchecked accumulation of NADPH might otherwise increase the risks of oxidative damage through a rise in the Mehler reaction rates and/or the production of hydroxyl radicals.

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.

Transgenic Tobacco Plants Overexpressing Chloroplastic Ferredoxin-NADP(H) Reductase Display Normal Rates of Photosynthesis and Increased Tolerance to Oxidative Stress

Ferredoxin-NADP(H) reductase (FNR) catalyzes the last step of photosynthetic electron transport in chloroplasts, driving electrons from reduced ferredoxin to NADP+. This reaction is rate limiting for photosynthesis under a wide range of illumination conditions, as revealed by analysis of plants transformed with an antisense version of the FNR gene. To investigate whether accumulation of this flavoprotein over wild-type levels could improve photosynthetic efficiency and growth, we generated transgenic tobacco (Nicotiana tabacum) plants expressing a pea (Pisum sativum) FNR targeted to chloroplasts. The alien product distributed between the thylakoid membranes and the chloroplast stroma. Transformants grown at 150 or 700 μmol quanta m−2 s−1 displayed wild-type phenotypes regardless of FNR content. Thylakoids isolated from plants with a 5-fold FNR increase over the wild type displayed only moderate stimulation (approximately 20%) in the rates of electron transport from water to NADP+. In contrast, when donors of photosystem I were used to drive NADP+ photoreduction, the activity was 3- to 4-fold higher than the wild-type controls. Plants expressing various levels of FNR (from 1- to 3.6-fold over the wild type) failed to show significant differences in CO2 assimilation rates when assayed over a range of light intensities and CO2 concentrations. Transgenic lines exhibited enhanced tolerance to photooxidative damage and redox-cycling herbicides that propagate reactive oxygen species. The results suggest that photosynthetic electron transport has several rate-limiting steps, with FNR catalyzing just one of them.

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.

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.

The soxRS response of Escherichia coli can be induced in the absence of oxidative stress and oxygen by modulation of NADPH content

The soxRS regulon protects Escherichia coli cells against superoxide and nitric oxide. Oxidation of the SoxR sensor, a [2Fe-2S]-containing transcriptional regulator, triggers the response, but the nature of the cellular signal sensed by SoxR is still a matter of debate. In vivo, the sensor is maintained in a reduced, inactive state by the activities of SoxR reductases, which employ NADPH as an electron donor. The hypothesis that NADPH levels affect deployment of the soxRS response was tested by transforming E. coli cells with genes encoding enzymes and proteins that lead to either build-up or depletion of the cellular NADPH pool. Introduction of NADP(+)-reducing enzymes, such as wheat non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase or E. coli malic enzyme, led to NADPH accumulation, inhibition of the soxRS regulon and enhanced sensitivity to the superoxide propagator methyl viologen (MV). Conversely, expression of pea ferredoxin (Fd), a redox shuttle that can oxidize NADPH via ferredoxin-NADP(H) reductase, resulted in execution of the soxRS response in the absence of oxidative stress, and in higher tolerance to MV. Processes that caused NADPH decline, including oxidative stress and Fd activity, correlated with an increase in total (NADP(+)+NADPH) stocks. SoxS expression can be induced by Fd expression or by MV in anaerobiosis, under conditions in which NADPH is oxidized but no superoxide can be formed. The results indicate that activation of the soxRS regulon in E. coli cells exposed to superoxide-propagating compounds can be triggered by depletion of the NADPH stock rather than accumulation of superoxide itself. They also suggest that bacteria need to finely regulate homeostasis of the NADP(H) pool to enable proper deployment of this defensive response.