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First published online December 22, 2006; 10.1104/pp.106.090449 Plant Physiology 143:639-649 (2007) © 2007 American Society of Plant Biologists Transgenic Tobacco Plants Overexpressing Chloroplastic Ferredoxin-NADP(H) Reductase Display Normal Rates of Photosynthesis and Increased Tolerance to Oxidative Stress1Instituto de Biología Molecular y Celular de Rosario, División Biología Molecular, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, S2002LRK Rosario, Argentina (R.E.R., A.L., H.O.P., M.Z., J.F.P., V.B.T., E.M.V., N.C.); and Leibniz-Institut für Pflanzengenetik und Kulturpflanzenforschung, 06466 Gatersleben, Germany (M.P., H.T., M.-R.H.)
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.
Plant photosynthesis takes place in chloroplast-containing source tissues (mostly leaves) that subsequently export the synthesized carbohydrates to heterotrophic sink organs, such as roots or seeds, which depend strictly on the uptake of reduced carbon. The efficiency of this metabolic pathway is thought to be critical for plant growth and crop yield and is therefore a most attractive target for breeders and geneticists (Miyagawa et al., 2001  ). Accordingly, considerable effort has been devoted to the identification of limiting steps amenable to genetic improvement in both the photosynthetic electron transport chain (PETC) and the reductive pentose phosphate cycle (RPPC). These investigations gained momentum in recent years through the widespread use of antisense RNA technology to reduce the levels of individual proteins and enzymes.
Several components of the RPPC (and other enzymes of carbohydrate metabolism) are known to accumulate in excess, with their in vivo activities modulated through allosteric or covalent (i.e. thiol oxidation) modifications (Buchanan et al., 2002
On the other hand, relatively few investigations have been carried out with genotypes differing in PETC components. Transgenic plants in which the content of ferredoxin (Fd; Holtgrefe et al., 2003 To probe this contention, we constructed transgenic tobacco plants expressing a pea (Pisum sativum) FNR in chloroplasts. The foreign flavoprotein accumulated up to 6-fold over the levels of its indigenous counterpart. Contrary to our expectations, however, transformants grown at low or high irradiation exhibited phenotypes and photosynthetic activities comparable to those of their wild-type siblings. Rates of NADP+ photoreduction by isolated thylakoids correlated with FNR content only when electron donors of PSI were employed to drive the reaction. CO2 assimilation rates did not differ significantly between wild type and transformants with an approximately 4-fold increase in FNR levels. Interestingly enough, lines overexpressing the reductase showed augmented tolerance to photooxidative damage and redox-cycling oxidants.
Generation of Transgenic Tobacco Plants Expressing Pea FNR in Chloroplasts
To obtain plants with increased levels of FNR activity in chloroplasts, we cloned a full-length cDNA encoding the pea FNR precursor (Newman and Gray, 1988
Although FNR is highly specific for NADP(H) and a very poor NAD(H)-dependent oxidoreductase, it can derive electrons to a wide variety of acceptors of appropriate redox potential but very different structures, mediating the so-called diaphorase reaction (Carrillo and Ceccarelli, 2003 ). Expression of pea FNR in tobacco chloroplasts led to a concomitant increase in diaphorase activity (Fig. 2, A and B
), indicating that the introduced flavoenzyme was functional in the transgenic host. It could be recognized as an extra band of lower electrophoretic mobility when soluble extracts from transgenic plants were separated by native PAGE and stained for diaphorase activity (Fig. 2B).
Leaf homogenates of the various transformants contained no trace of FNR precursor (Fig. 1B), suggesting that the pea-derived flavoprotein was correctly imported and processed by tobacco plastids. To corroborate this subcellular location, we prepared lysates from whole leaves and isolated chloroplasts and subjected them to SDS-PAGE and immunoblotting. When identical amounts of chl were loaded, nearly all FNR was recovered in the chloroplastic fraction of both wild-type and transgenic plants (Fig. 2C), indicating that the foreign enzyme was indeed plastid targeted.
Plant FNR is regarded as a thylakoid-bound protein (Vallejos et al., 1984
Transformants of the T2 generation were used for phenotypic analysis. They grew as their nontransformed siblings when cultivated in a growth chamber at moderate light intensities (150 µmol quanta m–2 s–1) and contained similar amounts of leaf chl and carotenoids per unit leaf area (Table I
). Maximal CO2 assimilation rates at saturating light intensity and ambient CO2 concentrations were 19 to 20 µmol CO2 cm–2 s–1 for all lines and were even slightly lower (approximately 15% relative to the wild type) in plants overexpressing FNR when assayed at saturating CO2 and two different light intensities (Table I). Photosynthetic parameters reflecting photon capture and integrity of PSII (Fv/Fm), electron transport (
To rule out the possibility that the lack of increase in photosynthesis and biomass accumulation was caused by remodeling of the photosynthetic apparatus during growth under relatively low light intensities, plants were also cultured at 700 µmol quanta m–2 s–1 with the same photoperiod and a daytime temperature of 29°C. Once again, the various transgenic and wild-type plants failed to show significant differences in fresh or dry weight, chl and carotenoid content, CO2 assimilation rates, or the Fv/Fm,  PSII, and NPQ values (Table I).
Plants from all lines were, on average, 35% shorter and contained approximately 50% less chl per unit leaf area when compared to siblings grown at 150 µmol quanta m–2 s–1 (Table I). This behavior has been regularly observed in plants cultured under similar conditions (Flachmann, 1997 To evaluate this possibility, we measured photosynthetic electron transport by thylakoids isolated from wild-type and pFNR-3 plants that contained FNR, on average, 4.6-fold over wild-type levels. Water-driven NADP+ photoreduction rates were 0.57 ± 0.11 and 0.67 ± 0.12 µmol NADPH mg–1 chl min–1 (P < 0.05) for wild-type and pFNR-3 plastids, respectively, corresponding to an increase of only 20% (Fig. 3A ). When the ascorbate/N,N,N',N'-tetramethyl-p-phenylendiamine (TMPD) pair was employed as a PSI electron donor in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU), an inhibitor that prevents electron transfer from PSII, pFNR-3 chloroplasts displayed approximately 3-fold more activity (2.36 ± 0.31 µmol NADPH mg–1 chl min–1) than wild-type plastids (0.74 ± 0.15 µmol NADPH mg–1 chl min–1; Fig. 3B). In contrast, light-driven reduction of methyl viologen (MV) proceeded at similar rates in all lines, with specific activity of 2.05 ± 0.25 and 1.93 ± 0.31 µmol O2 mg–1 chl min–1 for wild type and pFNR-3 thylakoids, respectively.
Plants Accumulating FNR Display Increased Tolerance to Photooxidative Damage and Redox-Cycling Herbicides
FNR-deficient plants were highly sensitive to photoinactivation, undergoing leaf bleaching, lipid peroxidation, and membrane damage even under moderate irradiation (Palatnik et al., 2003
After photooxidative treatment resulting from a combination of relatively high light intensity (700 µmol quanta m–2 s–1) and low temperature (8°C), the maximal quantum efficiency of PSII photochemistry (Fv/Fm) was reduced to a lower value in wild-type plants in comparison to plants overexpressing FNR (Fig. 4C). Decreases in Fv/Fm can be due to photodamage to PSII reaction centers as well as to the development of slowly relaxing NPQ (Baker and Rosenqvist, 2004 ). No differences were observed in the deactivation kinetics of the energy-dependent NPQ values between wild-type and transgenics plants (data not shown). Thus, the higher Fv/Fm values measured after photooxidative treatment of pFNR-3 plants suggest that light-driven damage to PSII was ameliorated in transformants relative to wild-type siblings.
Oxidative damage can also be inflicted by subjecting plants to the toxic effects of MV, an herbicide that propagates superoxide anion radicals through a redox-cycling reaction of the reduced form with molecular oxygen (Babbs et al., 1989
FNR catalyzes a key step of photosynthetic electron transport, collecting one electron from each of two molecules of reduced Fd in its flavin cofactor to convey a hydride group to NADP+ in a single elementary reaction (Bruns and Karplus, 1995 ; Carrillo and Ceccarelli, 2003). Whereas many functional properties of FNR have been thoroughly characterized (Carrillo and Ceccarelli, 2003; Ceccarelli et al., 2004), until recently, little was known on the actual contribution of this flavoenzyme to electron transport and photosynthesis in planta. The combined use of FNR antisense (Hajirezaei et al., 2002) and overexpressing transgenic plants (this work) revealed somehow paradoxical behavior. Net CO2 fixation rose steeply with FNR content in antisense plants until wild-type levels were reached, but further accumulation failed to increase photosynthetic rates over wild-type values (Fig. 6
). The lack of effect could be due, in part, to mistargeting of the introduced protein to the chloroplast stroma instead of the thylakoids (Fig. 2D), taking into account that the ability of the soluble form of FNR to engage in photosynthetic electron transport is very limited (Forti and Bracale, 1984; Nakatani and Shin, 1992). However, a considerable fraction of the transgenic product was rescued in a membrane-bound form (Fig. 2D) and was shown to display full activity in the ferricyanide reduction assay (Fig. 2A).
The failure of FNR to boost carbon assimilation when expressed beyond wild-type levels might reflect the existence of tight coordination in the relative proportions and reaction rates at which components of the PETC operate, resulting in more than one rate-limiting step. Depletion of some chain intermediates could lead to impairment of photosynthesis, but, as their levels build up (e.g. in transgenic plants), other components become immediately limiting so that the overall output of the process is hardly changed. Therefore, even when functional FNR molecules were incorporated into the thylakoid membranes in the transformants, NADP+ photoreduction could still be restrained if the provision of reduced Fd is already limited by a previous step. This contention gained support from the observation that thylakoids isolated from plants overexpressing FNR displayed at least 3-fold higher NADP+ photoreduction activity with respect to wild-type membranes when electron transfer from water to NADP+ was blocked at the reducing side of PSII, and PSI electron donors were provided, therefore bypassing electron transfer processes from water to plastocyanin (Fig. 3A). These data suggest that at least an additional rate-limiting step does exist either between photosystems or at the oxidizing side of PSII.
Electron transport in thylakoids involves the function of four membrane-bound components (the PSII/oxygen evolution, cytochrome b6f and PSI complexes, and FNR) linked by three electron transfer processes mediated by the diffusible components plastoquinone, plastocyanin, and Fd (Haehnel, 1984
In agreement with the kinetic determinations, plants expressing an antisense version of the gene encoding the Rieske FeS protein exhibited phenotypes that shared many common features with those of FNR-deficient lines (Price et al., 1998
It is not evident why overexpression of FNR should give rise to increased tolerance to oxidative stress. Interestingly, homologs of this reductase have been shown to participate in concerted antioxidant responses in many different heterotrophic organisms, including bacteria (Bianchi et al., 1995 NADPH-Fd reductase activity of FNR could also contribute to protection by eliminating the excess of reducing equivalents from the PETC. In plants, photooxidative damage stems from the inability of the PETC to handle the surplus of electrons produced by high irradiation or other environmental hardships that inhibit NADPH-consuming reactions of chloroplasts, especially those belonging to the RPPC. These situations often lead to NADPH accumulation, NADP+ shortage, overreduction of the PETC, and adventitious electron and energy transfer to oxygen, resulting in propagation of reactive oxygen species. The introduced FNR may be relieving the reductive burden of the PETC through conversion of NADPH into NADP+ (namely, by regenerating the final acceptor of linear electron flow). We therefore propose that the NADPH-Fd reductase activity of soluble FNR accumulated in the stroma is responsible for the enhanced tolerance of transgenic plants. Research is currently under way to prove this hypothesis. Preliminary observations indicate that introduction into tobacco chloroplasts of a cyanobacterial FNR that is unable to bind to thylakoids (and consequently to productively engage in the PETC), leads to plants with increased stress tolerance without affecting photosynthesis or growth (A. Lodeyro, M. Giró, E.M. Valle, and N. Carrillo, unpublished data).
Construction of Binary Vectors and Transformation of Agrobacterium tumefaciens and Tobacco
A DNA fragment encoding the pea (Pisum sativum) FNR precursor was obtained by PCR amplification of a previously isolated gene (Newman and Gray, 1988
Plants were grown on soil in a growth chamber at 80% humidity and a 14-h light/10-h dark photoperiod. Two different light intensities were employed: 150 µmol quanta m–2 s–1 and 700 µmol quanta m–2 s–1, with daytime temperatures of 22°C and 29°C, respectively. The temperature during the dark periods was fixed to 20°C in both cases. Plants were watered twice a week with nutrient medium (Geiger et al., 1999 To evaluate the effects of excess light, discs (1.2 cm in diameter) were punched from the fourth fully expanded leaf of 6-week-old plants, floated on distilled water with the abaxial side down, and irradiated at 2,000 µmol quanta m–2 s–1 and 25°C for 17 h, or at 700 µmol quanta m–2 s–1 and 8°C for the indicated times. Leaf discs treated with MV were placed on solutions containing various concentrations of the herbicide, vacuum infiltrated, and illuminated at 500 µmol quanta m–2 s–1 and 25°C during 12 h. Electrolyte leakage was measured as the increase in conductivity of the medium, using a Horiba B-173 conductivity meter. To estimate the total ion content of the tissue, discs were autoclaved after the experiment and the conductivity of the resulting solution was determined as above. The results are expressed as the fraction of total electrolytes released after photooxidative treatment. For treatments of whole plants, 4-week-old specimens were sprayed with 100 µM MV in 0.05% (v/v) Tween 20 and incubated in a growth chamber at 80% humidity and a 14-h light (25°C)/10-h dark (22°C) photoperiod under 150 µmol quanta m–2 s–1.
Net CO2 uptake rates (ACO2) and intercellular CO2 concentrations (Ci) were determined according to Hajirezaei et al. (2002)
Chl fluorescence was measured using a PAM-2000 portable fluorometer (Walz). Fv and Fm parameters were determined after keeping plants for 60 to 90 min in the dark. Later, plants were exposed first to red light (655 nm, 150 µmol quanta m–2 s–1) for 10 min and finally to actinic light (1,000 µmol quanta m–2 s–1) for another 10 min. Light-adapted parameters (F'v and F'm) were measured at the end of each particular light period. Photosynthetic parameters (Fv/Fm, After photooxidative treatments, the kinetics of relaxation of light-induced NPQ was determined by measuring Fv/Fm every tenth minute during 120 min in wild-type and transgenics plants kept in the dark at 25°C. In all cases, fast kinetics of NPQ relaxation were observed, reaching a steady value of Fv/Fm after 20 min in the dark.
Chloroplasts were isolated according to Quick et al. (1995)
For the identification of enzymes displaying NADPH-dependent diaphorase activity, leaf extracts corresponding to 20 µg of soluble protein were resolved by nondenaturing PAGE on 12% polyacrylamide gels. After electrophoresis, the gel was stained by incubation in 50 mM Tris-HCl, pH 8.5, 0.3 mM NADP+, 3 mM Glc-6-P, 1 unit mL–1 Glc-6-P dehydrogenase, and 1 mg mL–1 nitroblue tetrazolium until the appearance of the purple formazan bands.
Total FNR activity was also determined in whole broken chloroplasts, stroma, and thylakoids by measuring ferricyanide reduction in a medium containing 50 mM HEPES-KOH, pH 8.0, 5 mM MgCl2, 0.3 mM NADP+, 3 mM Glc-6-P, 1 unit mL–1 Glc-6-P dehydrogenase, 1 mM potassium ferricyanide, and samples corresponding to 0.3 µg chl mL–1 (as determined in the total chloroplast fraction). Ferricyanide reduction was monitored by the decrease in absorption at 420 nm, using
NADP+ photoreduction was measured, using water as electron donor, in 3 mL of 50 mM HEPES-K, pH 8.0, 5 mM MgCl2, 330 mM sorbitol, 0.5 mM NADP+, and thylakoids corresponding to 20 µg chl and 20 µM Fd. Illumination (2,400 µmol quanta m–2 s–1) was provided by a projector lamp and the amount of NADPH formed was estimated by measuring the increase in absorption at 340 nm (
Extracts were prepared by grinding the fourth fully expanded leaves of control and transgenic plants in liquid nitrogen. The resulting powder was resuspended in 50 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 1 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride. Proteins were determined by the method of Petersen (1977)
Chls and carotenoids were determined spectrophotometrically after extraction with 80% (v/v) aqueous acetone for leaf homogenates or 96% (v/v) ethanol for leaf discs (Lichtenthaler, 1987 Received September 28, 2006; accepted December 13, 2006; published December 22, 2006.
1 This work was supported by the National Agency for the Promotion of Science and Technology (PICT'99 grant no. 01–5105 and PICT'03 grant no. 01–14684) and Fundación Antorchas, Argentina. 
2 Present address: Department of Plant Physiology and Biochemistry/W5, University of Bielefeld, 33501 Bielefeld, Germany. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Néstor Carrillo (carrillo{at}ibr.gov.ar). www.plantphysiol.org/cgi/doi/10.1104/pp.106.090449 * Corresponding author; e-mail carrillo{at}ibr.gov.ar; fax 54–341–4390465.
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ABSTRACT
RESULTS
420 = 1 mM–1 cm–1. One activity unit is defined as the amount of enzyme that catalyzes the reduction of 1 µmol of ferricyanide/min under the conditions described above. 
