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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.

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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.

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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.

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Significant effort has been directed in recent times to the use of plants to extract and detoxify nitroaromatics from polluted industrial facilities. We have explored the possibility of overcoming the phytotoxicity of the highly toxic and recalcitrant nitroderivative 2,4-dinitrotoluene (2,4-DNT) by expressing a cyanobacterial flavodoxin (Fld) in tobacco plants. We demonstrate here that transformants accumulating Fld in plastids display a remarkable increase in the ability to tolerate, take up, and transform 2,4-DNT, as compared to their wild-type siblings. We also show that Fld mediates one-electron reduction of 2,4-DNT in the presence of oxygen and especially in anaerobiosis. Moreover, Fld-loaded chloroplasts are able to convert 2,4-DNT into its aminoderivatives in the presence of light. The results suggest that expression of Fld in landscape plants could facilitate effective cleanup of sites contaminated with this class of pollutants.

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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.

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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.

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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 micromol 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.

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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.

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Ferredoxin-NADP(H) reductase (FNR) catalyses the final step of the photosynthetic electron transport in chloroplasts. Using an antisense RNA strategy to reduce expression of this flavoenzyme in transgenic tobacco plants, it has been demonstrated that FNR mediates a rate-limiting step of photosynthesis under both limiting and saturating light conditions. Here, we show that these FNR-deficient plants are abnormally prone to photo-oxidative injury. When grown under autotrophic conditions for 3 weeks, specimens with 20-40% extant reductase undergo leaf bleaching, lipid peroxidation and membrane damage. The magnitude of the effect was proportional to the light intensity and to the extent of FNR depletion, and was accompanied by morphological changes involving accumulation of aberrant plastids with defective thylakoid stacking. Damage was initially confined to chloroplast membranes, whereas Rubisco and other stromal proteins began to decline only after several weeks of autotrophic growth, paralleled by partial recovery of NADPH levels. Exposure of the transgenic plants to moderately high irradiation resulted in rapid loss of photosynthetic capacity and accumulation of singlet oxygen in leaves. The collected results suggest that the extensive photo-oxidative damage sustained by plants impaired in FNR expression was caused by singlet oxygen building up to toxic levels in these tissues, as a direct consequence of the over-reduction of the electron transport chain in FNR-deficient chloroplasts.

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