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Functional Replacement of Ferredoxin by a Cyanobacterial Flavodoxin in Tobacco Confers Broad-Range Stress Tolerance^[W]

^a Instituto de Biología Molecular y Celular de Rosario, Consejo Nacional de Investigaciones Científicas y Técnicas, División Biología Molecular, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, S2002LRK Rosario, Argentina
^b Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, 50009 Zaragoza, Spain
^c Institut für Pflanzengenetik und Kulturpflanzenforschung, 06466 Gatersleben, Germany

¹ To whom correspondence should be addressed. E-mail carrillo{at}ibr.gov.ar; fax 54-341-4390465.

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

 
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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

 
Ferredoxins (Fds) are ubiquitous iron–sulfur proteins involved in many different electron transfer pathways in plants, animals, and microorganisms (Knaff, 2005). As the major low-potential mobile electron carrier protein of chloroplasts, Fd plays a central role in the physiology of the plant cell, distributing the reducing equivalents generated in the photosynthetic electron transport chain (PETC) to various electron-consuming reactions of the chloroplast. Part of the Fd reduced at the level of photosystem I (PSI) delivers reducing equivalents to ferredoxin-NADP⁺ reductase (FNR) for NADP⁺ photoreduction, generating the NADPH needed for CO₂ fixation and other biosynthetic routes, but a substantial fraction engages in electron transfer to alternative plastidic enzymes involved in crucial cellular pathways. They include nitrogen and sulfur assimilation via Fd-dependent nitrite reductase and sulfite reductase as well as amino acid and fatty acid metabolism through Gln-oxoglutarate amino transferase and fatty acid desaturase. Reductive activation of key chloroplast enzymes by the thioredoxin (Trx)/ferredoxin-thioredoxin reductase (FTR) system also requires a steady supply of Fd to ensure proper function of the Calvin cycle, the malate valve, and several other metabolic and dissipative processes (Balmer et al., 2003). In addition, Fd is a key player in different routes of cyclic electron flow that operate under physiological and stress conditions to eliminate the excess of reducing power and prevent uncontrolled overreduced states in the PETC and the stroma (Kramer et al., 2004; Munekage et al., 2004). The Fd redox state also functions as a signal in the intracellular signaling pathway between chloroplast and nucleus (Knaff, 2005). The various Fd acceptors are organized in a hierarchical manner, attaining their maximal activities at different electronic pressures (Backhausen et al., 2000). Most of these Fd activities are also found in eukaryotic algae and in cyanobacteria, the closest living relatives of the ancient endosymbiont that gave origin to chloroplasts (Falk et al., 1995).

In agreement with this multiplicity of functions, Fd accumulates above the tight stoichiometry of its redox partners in the PETC (PSI, FNR) in both plants (Bohme, 1978) and algae (Inda and Peleato, 2002). The amounts of Fd in photosynthetic microorganisms fluctuate in response to different environmental stimuli, including light-dependent induction (Mazouni et al., 2003), and downregulation by iron starvation, environmental adversities, and oxidative stress episodes (Erdner et al., 1999; Mazouni et al., 2003; Singh et al., 2004). In higher plants, Fd is encoded by a small gene family whose members display tissue-specific expression and perform specialized tasks in different organs (Hanke et al., 2004). Chloroplast Fd is also induced by light, through a redox-based mechanism (Petracek et al., 1998). Repression of Fd expression by hostile environmental situations has not yet been reported for plants. However, genome-wide analysis of Arabidopsis thaliana transcription profiles indicates that steady state levels of transcripts encoding Fd are decreased, relative to control conditions, under virtually all kinds of abiotic stress (Zimmermann et al., 2004). Such an effect could deeply affect central metabolic pathways of the chloroplast and the cell and contribute to the growth penalties exhibited by plants exposed to adverse conditions. Transgenic potato (Solanum tuberosum) plants in which the contents of Fd were diminished by the expression of antisense RNA displayed lower CO₂ assimilation rates, growth arrest, and altered distribution of reducing equivalents to oxidoreductive pathways of the stroma (Holtgrefe et al., 2003).

Although each of the various stress conditions to which a plant may be subjected has distinctive characteristics and elicits its own specific responses, they share a common feature: the establishment of an oxidative situation that accounts for a substantial part of the damage inflicted upon the stressed organism. NADPH-consuming reactions of photosynthesis are early targets of inhibition by most environmental hardships. As a consequence, NADP⁺ levels decline, leading to overreduction of the PETC as its terminal acceptor becomes unavailable (Allen, 1995; Apel and Hirt, 2004). Reduced intermediates of the chain may then engage in energy and electron transfer to adventitious acceptors, most conspicuously oxygen, resulting in the generation of reactive oxygen species (ROS), such as superoxide radicals, H₂O₂, hydroxyl radicals, and carbon-based organic radicals (Allen, 1995).

Photosynthetic microorganisms have adapted to the undesirable consequences of Fd decline under stress by inducing the expression of isofunctional electron carriers termed flavodoxins (Flds), small soluble proteins (19 kD) containing one molecule of noncovalently bound flavin mononucleotide as a prosthetic group (Falk et al., 1995). Cyanobacterial Fld has been shown to efficiently replace Fd in NADP⁺ photoreduction, nitrogen fixation, sulfite reduction, and the phosphoroclastic reaction (Falk et al., 1995). Unlike FNR and Fd, which are widely distributed among plastids, mitochondria, and bacteria, Flds are restricted to prokaryotes and some eukaryotic algae (Erdner et al., 1999). In cyanobacteria and enterobacteria, Fld levels increase severalfold on exposure of the cells to the redox-cycling herbicide methyl viologen (MV) and other superoxide-propagating compounds (Zheng et al., 1999; Yousef et al., 2003; Singh et al., 2004), and overexpression of the flavoprotein in Escherichia coli leads to augmented tolerance toward various sources of oxidative stress (Zheng et al., 1999).

Somewhere in the evolutionary path that led to the appearance of vascular plants, the Fld gene disappeared from the plant genome (Arabidopsis Genome Initiative, 2000) and the considerable advantages derived from its expression and function were lost or became dispensable. Interestingly, at least some of the plant enzymes whose bacterial ancestors used Fld as a normal or occasional substrate have retained the ability to efficiently interact with this electron carrier in vitro, with chloroplast FNR being a conspicuous example (Nogués et al., 2004). The aim of this research, therefore, was twofold: to investigate the effects of adverse environments on the contents of chloroplast Fd, and to determine whether bacterial Fld could also exert its protective functions in planta. We show here that Fd levels declined in tobacco (Nicotiana tabacum) plants exposed to oxidants or to adverse environmental conditions and that the expression of Fld in chloroplasts, but not in the cytosol, compensated for this loss and resulted in transgenic lines displaying increased tolerance to oxidative stress and to a wide range of environmental challenges. We also demonstrate that Fld is reduced in vitro by the chloroplast PETC and can provide reducing equivalents for NADP⁺ photoreduction and Trx reduction via FTR. These activities decisively contribute to the increased stress tolerance of Fld-expressing plants by preserving the active state of redox-sensitive photosynthetic and scavenging enzymes under harmful conditions.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

 
Levels of Chloroplast Fd Decline in Stressed Tobacco Plants
We first tested the effects of several adverse conditions on the leaf contents of Fd using immunoblot analysis. Exposure of 2-month-old wild-type tobacco plants (cv Petit Havana) grown in soil to MV led to a significant decrease in Fd levels (Figure 1A , wild-type lanes). Depletion of the iron–sulfur protein was also evident when plants were illuminated at low temperature or subjected to water deprivation (Figure 1A, wild-type lanes). These lower levels were not a reflection of general protein breakdown or aggregation caused by the stress situations. When leaf extracts corresponding to the same amounts of soluble protein were resolved by SDS-PAGE, similar protein profiles were obtained for all treatments and the fraction of aggregates was negligible (Figure 1C, wild-type lanes), indicating that Fd declined with respect to the bulk of stromal protein in the stressed plants.

View larger version (50K): [in this window] [in a new window]   Figure 1. Fd Declines in Plants Subjected to Environmental Stress. (A) and (B) Two-month-old plants grown in soil were exposed to 30 µM MV (sprayed on the aerial parts), low temperature (LT), or water deficit (WD). Growth chamber (Co) and stress conditions are detailed in Methods. Extracts were prepared from the youngest fully expanded leaves (node 8) by the freeze-clamp procedure and analyzed by SDS-PAGE and immunoblotting with Fd (A) or Fld (B) antiserum. (C) Extracts were resolved as described above but stained for protein with Coomassie blue. Lysates corresponding to 15 µg of soluble protein were loaded onto each lane. The positions of Fd and Fld are indicated by arrowheads.

Trx reduction can be determined by measuring the reductive regeneration of peroxidases of the 2-Cys peroxiredoxin (Prx) family (Collin et al., 2003). To determine whether Fld could replace Fd as the electron donor for Prx reduction, we designed a reconstituted system in which the flavoprotein was reduced in vitro via NADPH and FNR in a medium containing purified FTR, Trx f, and Prx. The oxidized and reduced forms of Prx can be resolved by noreducing SDS-PAGE, as oxidized Prx retains an intersubunit disulfide bond and migrates as a dimer. Prx was progressively converted into the reduced form at increasing Fld concentrations (Figure 2A ), with 50% reduction (S_0.5) achieved at 5 µM Fld (Figure 2B). Under similar conditions, plant Fd was approximately twofold more efficient (Figure 2A). Two other chloroplast Trxs, Trx m and Trx x, could also mediate this reaction, with Trx x being the most effective (Figure 2C). Fld was able to sustain the Trx-dependent peroxidase activity of Prx with the three Trxs (Figure 2D). Once again, the highest activities were obtained with Trx x (Figure 2D).

View larger version (24K): [in this window] [in a new window]   Figure 2. Fld Can Donate Electrons for Trx Reduction Mediated by Fd-Trx Reductase. (A) In vitro reduction of purified 2-Cys Prx from Arabidopsis by a reconstituted electron transport chain (depicted below the gel) containing 10 µM Trx f. The amounts of Fld or Fd added to the reaction media are indicated above each lane. After incubation for 15 min, samples were derivatized with 10 mM N-ethylmaleimide, separated by nonreducing SDS-PAGE, and analyzed by immunoblotting with Prx antiserum. The positions of dimeric (Dim) and monomeric (Mr; fully reduced) Prx are shown by arrowheads. (B) Fractions of monomer as a function of Fld concentration in the reconstituted medium. The experimental values were fit to the equation of a hyperbolic curve, with 50% reduction for Fld of 4.6 µM. (C) Reduction of Prx by different Trxs. Experimental conditions were as described for (A). Reaction media contained 10 µM Fld and were supplemented with no Trx (Co) or with 5 to 10 µM Trx f, m, or x, as indicated above each lane. (D) H2O2 reduction by the Fld-driven reconstituted system. Reaction media contained 10 µM Trx f (closed circles), Trx m (closed triangles), or Trx x (closed squares). At the times indicated, the amounts of H2O2 remaining in the reaction media were determined by reaction with xylenol orange. Controls were assayed in the absence of Trx, Prx, or Fld (open symbols).

Immunodetection experiments were used to confirm Fld intracellular sorting in the various transformed lines. Fld was recovered from purified chloroplasts of pfld plants (Figures 3A , 3C, and 3D), whereas plastids isolated from specimens transformed with the cfld construct contained no detectable traces of the foreign flavoprotein (Figure 3D). In pfld specimens, Fld distribution between total leaf and chloroplast extracts matched that of two plastid markers, Fd and the large subunit (LSU) of ribulose-1,5-bis-phosphate carboxylase/oxygenase (Rubisco), and differed from those exhibited by two cytosolic enzymes: pyrophosphate fructose 6-phosphate phosphotransferase (PFP) and NAD(H)-dependent malate dehydrogenase (NAD-MDH) (Figure 3A). Samples were analyzed by SDS-PAGE and immunoblotting on the basis of equal chlorophyll loading, rendering similar amounts of the plastidic proteins per chlorophyll in leaf and chloroplast extracts (Figure 3A), as expected for this type of component. Correct Fld sorting to the chloroplast and cytosol in pfld and cfld lines, respectively, was also revealed by immunolocalization in cross sections of young fully expanded leaves. Significant immunogold label was detected within the chloroplasts in pfld plants and in the cytosol of cfld cells (Figure 3B).

View larger version (94K): [in this window] [in a new window]   Figure 3. Subcellular Distribution of Fld in Transgenic Tobacco Plants. (A) Fld is associated with the chloroplast fraction of pfld5-8 plants. Total chloroplast (Cl) and leaf (Le) extracts corresponding to 5 µg of chlorophyll were separated by SDS-PAGE on 15% acryalmide gels, blotted, and challenged with antisera specific for Fld, Fd, Rubisco LSU, NAD-MDH, or PFP. (B) Immunolocalization of Fld in mesophyll cells of leaves from wild-type, cfld1-4, and pfld5-8 plants. Significant immunogold labeling of Fld could be detected only in chloroplasts of pfld5-8 and in cytosol of cfld1-4 leaves. Cw, cell wall; M, mitochondria; S, starch. Bars = 0.2 µm. (C) Fld is internalized by tobacco chloroplasts in vivo. Intact chloroplasts (ICl) isolated from pfld5-8 specimens were resuspended in isotonic medium and incubated for 30 min on ice either in the absence (–) or presence (+) of 100 µg/mL thermolysin. The same treatment was applied to chloroplasts that had been osmotically shocked in 20 mM Tris-HCl and 1 mM EDTA (BCl). Samples corresponding to 10 µg of chlorophyll were analyzed by SDS-PAGE and immunoblotting with Fld antisera. The positions of the unprocessed (U) and mature (M) forms of the flavoprotein are indicated by arrowheads. (D) Fld is predominantly associated with the chloroplast stroma in pfld plants. Chloroplasts (Cl lanes) were isolated from pfld4-2, pfld5-8, and cfld1-4 plants and ruptured by osmotic shock. Stromal fractions (St lanes) were cleared by centrifugation, and thylakoids (Th lanes) were washed once (Wa lanes) with rupture buffer to remove bound soluble proteins. Samples corresponding to 5 µg of chlorophyll were loaded onto each lane. Extracts, chloroplasts, and samples for ultrastructural analysis were prepared from node-8 leaves of 8-week-old T3 plants cultured in soil under growth chamber conditions.

Plants Expressing a Cyanobacterial Fld in Chloroplasts Develop Increased Tolerance to Various Sources of Environmental Stress
Plastidic lines pfld5-8, pfld4-2, and pfld12-4 and cytosolic lines cfld1-4 and cfld2-1 were able to accumulate 70, 57, 7, 108, and 127 pmol of Fld per gram of leaf fresh weight, respectively, as estimated by integration of the immunoblot signals and comparison with pure standards (see Supplemental Figure 2D online). When cultured in a growth chamber (see Methods), no phenotypic differences were observed between wild-type and transgenic plants (see Supplemental Table 1 online). Photosynthetic activities also failed to reveal major changes in either maximal CO₂ assimilation rates (Figure 4C ) or the light intensity at which saturation was attained (see Supplemental Table 1 online). The only significant difference among the lines was in the fluorescence quantum yield of PSII photochemistry (_PSII), which was 2- to 2.5-fold higher in leaf discs from pfld5-8 and pfld4-2 plants, corresponding to a doubling in the electron transport rates through PSII (Figure 4C). These _PSII changes did not result from increases in the capture efficiency of excitation energy by PSII, nor did they lead to overreduction of the PETC. Indeed, all lines displayed similar maximum photochemical efficiency of PSII in the dark-adapted state (F_v/F_m), chlorophyll a/b ratios (Figure 4C), and relative reduction state of PSII reflected by (1 – qP) values (data not shown).

View larger version (83K): [in this window] [in a new window]   Figure 4. Fld Expression in Chloroplasts Increases Tolerance to MV and Environmental Stress Conditions. (A) Wild-type plants and T3 transformants expressing various levels of pfld and cfld Fld were grown for 6 weeks in hydroponia and exposed for 18 h to 30 µM MV at 25°C and 500 µmol·m–2·s–1. (B) Ultrastructural analysis of chloroplast damage in leaf cross sections of 6-week-old wild-type, pfld5-8, and cfld1-4 plants cultured in hydroponia and exposed to 30 µM MV. M, mitochondria; S, starch. Bars = 1 µm. (C) Effect of MV treatment on membrane integrity, pigment levels, and photosynthetic activities of wild-type and transformed plants (T3 generation) grown in soil for 8 weeks. Ion leakage, chlorophyll (Chl) degradation, Fv/Fm, and PSII were measured on leaf discs incubated for 18 h at 500 µmol·m–2·s–1 with the indicated concentrations of the herbicide. CO2 assimilation rates were determined on attached leaves (node 8) from plants sprayed with 30 µM MV or Silwet. Values shown are means ± SE of four to five experiments. The chlorophyll a and b contents of each line are given in Table 1. (D) Two-week-old seedlings from wild-type (left) and pfld5-8 (right) lines were illuminated for 18 h at 500 µmol·m–2·s–1 and 40°C in MS agar. (E) Leaves from 2-month-old wild-type (left) and pfld5-8 (right) plants grown in soil were exposed for 18 h to a focused light beam of 2000 µmol·m–2·s–1. (F) Seven-week-old wild-type (left) and pfld5-8 (right) plants were cultured for 20 d at 500 µmol·m–2·s–1 and 9°C in MS agar. (G) Seven-week-old wild-type (left) and pfld5-8 (right) plants cultured in hydroponia were exposed to UV-AB radiation for 24 h. For (D) to (G), homozygous transformants of the T3 generation and their wild-type siblings were exposed to the stress treatments as described in Methods. The panels illustrate characteristic phenotypes, with the arrows indicating sites of tissue damage ([E] and [G]).

Increased tolerance of Fld-expressing lines was not limited to MV toxicity. Two-week-old pfld5-8 and pfld4-2 plantlets tolerated exposure to 40°C, whereas wild-type seedlings were entirely bleached (Figure 4D; see Supplemental Figure 4A online). Similar results were obtained when the heat stress was applied on leaf discs of 2-month-old specimens grown in soil (Table 1 ). Transformants also exhibited increased tolerance to high light intensities, chilling, UV radiation, and water deficit (typical examples are shown in Figures 4E to 4G; see Supplemental Figures 4B to 4F online). Damage caused by all of these treatments in wild-type and cfld plants was reflected by extensive leaf bleaching and/or wilting (see Supplemental Figures 4A to 4F online), augmented electrolyte leakage, and declines in chlorophyll contents and photosynthetic capacities (Table 1). Tolerance of the pfld lines was accompanied by preservation of these functions, and in the case of plants subjected to water deprivation, by the maintenance of stomatal conductance (Table 1).

The tolerant phenotype of Fld-expressing plants was evident in assays conducted on specimens of different ages cultured on various supports (soil, agar, hydroponia), although the extent of damage and protection varied depending on the developmental stage of the plants and the growth conditions (see Supplemental Figure 4 online). In the case of MV, accessibility of the herbicide to the target tissues was also a significant factor. Visible tissue deterioration occurred faster and at lower MV concentrations when the reagent was applied directly to leaves (by spraying) or floating discs than when it was fed through the roots in hydroponia (cf. the different MV sensitivities between the stressed leaves in Figure 4A and the leaf discs in Supplemental Figure 3 online). In all cases, however, there was a good correlation between stress tolerance and the levels of expressed Fld. For instance, lines pfld5-8 and pfld4-2, which are the result of distinct insertional events but accumulate similar amounts of Fld in chloroplasts, displayed equivalent levels of tolerance to MV and other sources of stress, whereas the 10-fold lower level of Fld in pfld12-4 plants resulted in a susceptibility close to that of the wild type (Figure 4, Table 1; see Supplemental Figures 3 and 4 online). These results indicate that the tolerant phenotypes were attributable to expression of the transgene and not to the genome position where it was integrated after transformation.

The effects of MV on photosynthetic parameters (Figure 4C) indicate that components of the PETC were harmed or downregulated by the herbicide and that Fld was able to compensate for this loss, allowing substantial electron transport even under suboptimal photon capture conditions. The results shown in Figure 1A suggest that one of the missing components could be Fd. Indeed, analysis of Fd contents in transgenic lines confirmed its depletion under various stress conditions, with overall declines comparable to those of the wild-type plants (Figure 1A), whereas Fld levels were hardly affected by the treatments (Figure 1B). The collected results suggest that chloroplast Fd became limiting under stress conditions and that Fld expression was able to compensate for this loss.

Chloroplast-Targeted Fld Behaves as an Antioxidant in Stressed Transgenic Plants
The levels of H₂O₂ and superoxide were found to be equivalent in leaves of unstressed plants irrespective of Fld expression, but their buildup under adverse conditions was impaired in the lines accumulating Fld in chloroplasts, relative to wild-type and cfld plants (Figure 5 ). A similar trend was observed when measuring the formation of malondialdehyde (MDA), a proxy for membrane lipid peroxidation (Figure 5), indicating that the tolerance of Fld-expressing lines correlated with a decrease in ROS accumulation. Of the oxidants assayed, superoxide determination is less reliable because of the limited life span of this radical in living tissues. Even though its absolute contents should be considered with caution, differences between the various stressed lines were highly significant (P < 0.05).

View larger version (34K): [in this window] [in a new window]   Figure 5. Effect of Fld Expression on the Levels of Oxidants in Plants Exposed to Various Sources of Stress. Assays were conducted in leaf extracts from soil-cultured 2-month-old transgenic plants (T5 generation) and their wild-type siblings. Leaves from node 9 were incubated under growth chamber conditions (open bars) or exposed to 30 µM MV at 500 µmol·m–2·s–1 (closed bars), to light stress (LS) at 1200 µmol·m–2·s–1 (hatched bars), or to heat stress (HS) at 40°C (shaded bars). Exposure times were 18 h in all cases. Methods used to estimate ROS and MDA levels are detailed in Methods. Data shown are averages ± SE of five experiments. Values reported for pfld5-8 and pfld4-2 differed significantly from those of the other lines (P < 0.05). fw, fresh weight.

The total pool of ascorbate species, represented by the sum of ascorbic acid (ASC) and dehydroascorbic acid (DHA), displayed similar levels in leaves from unstressed plants of all lines assayed (see Supplemental Table 2 online). This pool experienced a mild decrease (20%) after treatment with MV, but ASC oxidation was partially prevented in the tolerant pfld specimens (Figure 6 ; see Supplemental Table 2 online for actual contents). In the absence of stress, the levels of GSSG were significantly lower in leaves of pfld5-8 and pfld4-2 plants, resulting in higher GSH:GSSG ratios, and this trend was maintained after MV application (Figure 6; see Supplemental Table 2 online for actual contents).

View larger version (19K): [in this window] [in a new window]   Figure 6. Effect of Fld Expression on the Levels of Antioxidant Metabolites in Plants Exposed to MV. Assays were conducted in leaf extracts from soil-cultured 2-month-old wild-type and transgenic plants (T5 generation) sprayed (closed bars) or not (gray bars) with 30 µM MV. Levels of ASC, DHA, GSH, and GSSG were estimated according to published procedures (Foyer et al., 1995). Means ± SE of three experiments are shown. Values reported for pfld5-8 and pfld4-2 differed significantly from those of the other lines (P < 0.05). Asterisks indicate values not determined.

View larger version (32K): [in this window] [in a new window]   Figure 7. Fld Expression Modulates the Redox State of Chloroplast 2-Cys Prx in Stressed Plants. (A) Fld accumulation in chloroplasts prevents the oxidation of 2-Cys Prx in plants subjected to environmental stress. Wild-type, pfld5-8, cfld1-4, and pfld4-2 specimens were grown in soil for 8 weeks and then exposed to 30 µM MV, low temperature (LT), or water deficit (WD) as described in Methods. Controls (Co) were incubated under growth chamber conditions for the same periods. Extracts were prepared from young leaves corresponding to node 8 by the freeze-clamp procedure, and samples corresponding to 8 µg of soluble protein were analyzed by nonreducing SDS-PAGE and immunoblotting. The positions of dimeric (Dim) and monomeric (Mo; overoxidized) Prx are shown by arrowheads. (B) Electrophoretic mobility of fully reduced and overoxidized Prx monomers. In vitro reduction of purified Prx was performed with the reconstituted system containing (+) or not (–) 10 µM Fld, essentially as described for Figure 2, except that the reaction was prolonged for 60 min. Stress treatments (MV, LT, WD) were applied as indicated for (A), and leaf extracts corresponding to 16 µg of total soluble protein were analyzed by SDS-PAGE and immunoblotting. N-ethylmaleimide derivatization was omitted, resulting in the accumulation of aggregates, as reported previously (König et al., 2002). Mr, fully reduced. (C) and (D) Discrimination between fully reduced and overoxidized Prx monomers. In vitro reduction of Prx (C) and stress treatments (D) were performed essentially as described for (A) and (B). After two-dimensional SDS-PAGE, Prx spots were visualized by immunoblotting. The arrowheads indicate the positions of overoxidized (Ox) and reduced (Rd; a mixture of disulfide and dithiol forms) Prx.

Prxs are not the only Fd/Trx targets in chloroplasts, raising the possibility that other subjects of reductive modulation could also be protected from oxidative inactivation in Fld-containing plants. Accordingly, we observed that the activation state of the chloroplast NADP(H)-dependent malate dehydrogenase (NADP-MDH) was largely preserved in pfld5-8 and pfld4-2 plants subjected to water deficit or MV toxicity, whereas it declined significantly in wild-type and cfld1-4 siblings (Figure 8 ; see Supplemental Table 3 online for specific activities). Target enzymes of central metabolic pathways, such as the Calvin cycle components fructose-1,6-bisphosphatase (FBPase) and phosphoribulokinase (PRK), were also protected from oxidative inactivation in stressed plants expressing Fld in plastids (Figure 8; see Supplemental Table 3 online for specific activities).

View larger version (23K): [in this window] [in a new window]   Figure 8. Activation States of Trx-Dependent Chloroplast Enzymes Are Preserved in Stressed Plants Expressing Plastid-Targeted Fld. Two-month-old wild-type plants and T5 transformants were sprayed with 30 µM MV (closed bars), exposed to water deficit (WD; shaded bars), or incubated under growth chamber conditions for the same periods (Control; open bars). Extracts were prepared from node-8 leaves as described in Methods. The activities of NADP-MDH, FBPase, and PRK were measured before (in vivo activity) and after reduction of the extracts with DTT (total activity). The activation states were calculated as the ratio between the in vivo and total activities for each condition. Means ± SE of three to five experiments are shown. Specific activities used for the calculations and statistical differences can be found in Supplemental Table 3 online.

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

The effects of Fd deprivation on plant physiology can be fully appreciated by analyzing the phenotypes of transgenic potato lines in which the levels of this iron–sulfur electron carrier were decreased by an antisense approach (Holtgrefe et al., 2003). When the Fd contents were between 50 and 80% of those found in wild-type plants, chlorophyll was progressively lost in a dynamic process and leaves turned yellowish with increasing age. In short-term experiments, the antisense lines experienced a significant decrease in CO₂ assimilation rates, the intersystem chain components were overreduced, and the distribution of electrons toward stromal acceptors was deeply perturbed, resulting in lower levels of Trx reduction (Holtgrefe et al., 2003). Depending on the amount of remaining Fd, severely depleted lines showed photoinhibition.

Even though Fld-encoding genes were lost during the transition from algae to plants, we were able to demonstrate that incorporation of a bacterial Fld into tobacco chloroplasts could compensate for the decline in Fd levels, leading to increased tolerance to oxidants and to a wide range of adverse conditions (Figure 4, Table 1; see Supplemental Figures 3 and 4 online). The plastid-targeted flavoprotein exhibited antioxidant activity in stressed transformants, reflected by lower ROS accumulation (Figure 5) and reduced oxidative damage to sensitive enzymes (Figures 7 and 8), membranes, pigments, and photosynthesis (Figure 4, Table 1). These effects can be accounted for by Fld involvement in NADP⁺ photoreduction (see Supplemental Figure 1 online) and especially in Trx reduction as a substrate of FTR (Figures 2 and 7). Maintenance of high levels of reduced Trx could favor a number of dissipative and scavenging pathways, including (1) Prx regeneration to eliminate H₂O₂ and organic peroxides formed under stress (Figures 2 and 7), (2) export of the excess of reducing power via the malate valve (Figure 8), and (3) productive consumption of the NADPH surplus by the Calvin cycle (Figure 8). In addition, the ascorbate and glutathione pools were maintained at a more reduced state in pfld lines, in the latter case even in nonstressed plants (Figure 6). One of the chloroplast routes for ASC regeneration involves Fd, and it is possible that Fld can drive this reaction in vivo, although higher ASC reduction levels could also be a secondary consequence of Prx activity preservation in these lines. Antisense suppression of Prx is reported to cause extensive oxidation of the ascorbate pool in Arabidopsis (Baier et al., 2000). By contrast, the link between Fld expression in chloroplasts and glutathione reduction is unclear, because GSH formation does not require Fd. It is conceivable that this effect did not stem from an increase in glutathione reduction but from a decline in GSH oxidation attributable to lower ROS accumulation in Fld-expressing transformants (Figure 5).

Still other protective routes that require a steady provision of reduced Trx, such as the oxidative pentose phosphate pathway, ATP synthesis, and redox-modulated communication of the chloroplast with the nucleus and mitochondria (Balmer et al., 2003, 2004), could also be preserved in Fld-expressing plants, but support for this contention requires rigorous experimental proof. However, many features of the tolerance exhibited by the transformants suggest a prominent role for Fld in Trx metabolism and are consistent with the reported role of 2-Cys Prxs as floodgates that scavenge oxidants only above a certain threshold, preventing toxicity but allowing their involvement in signal transduction (Wood et al., 2003). The wild-type phenotypes (Table 1; see Supplemental Table 1 online) and ROS levels (Figure 5) displayed by unstressed transformants strongly suggest that the signaling functions played by stable oxidants were not affected by Fld accumulation. A comprehensive scheme of chloroplast Trx-dependent routes that might be subject to Fld stimulation is provided in Figure 9 .

View larger version (32K): [in this window] [in a new window]   Figure 9. Model for the Protective Mechanism of Fld in Chloroplasts. The electrons originating in the electron transport chain may be transferred from Fd or Fld to the Trx system via FTR. Trxs will then regenerate the active forms of key target enzymes (Prx, NADP-MDH, FBPase, PRK) through reduction of their critical Cys residues, resulting in the maintenance and/or stimulation of the Calvin cycle, the Prx cycle, the malate valve, and presumably other metabolic routes. Other Fd redox partners, such as FNR and the ascorbate regeneration cycle, are also shown. Fld expression is inconsequential in phenotypic terms for plants grown under normal conditions but becomes critical as Fd declines to limiting levels in the stressed organism. ASC, ascorbate; Cyt, cytochrome; DHA, dehydroascorbate; FBPase, fructose-1,6-bisphosphatase; F1,6BP, fructose-1,6-bisphosphate; F6P, fructose 6-phosphate; G3P, glyceraldehyde 3-phosphate; GSH and GSSG, reduced and oxidized glutathione; MDHA, monodehydroascorbate; NADP-MDH, NADP(H)-dependent malate dehydrogenase; OAA, oxalacetic acid; PC, plastocyanin; PRK, phosphoribulokinase; PSI and PSII, photosystems I and II; 4Q, plastoquinone; R5P, ribulose 5-phosphate; R1,5BP, ribulose-1,5-bisphosphate; SOD, superoxide dismutase.

Fld engagement in the Fd/Trx route, which is so prominent in chloroplasts, explains why plastid targeting was a mandatory feature for the manifestation of the protective effects observed. A similar role in the cytosol would be unlikely because the corresponding Trx reductases specifically use NADPH as the hydride donor to reduce h-type Trxs (Buchanan et al., 2002). The pleiotropic nature of the protection afforded concurs with the many different regulatory roles played by Trxs in the context of the chloroplast and the plant cell (Figure 9) and highlights the relevance of the Trx/FTR system for the development of the tolerant phenotypes.

It is conceivable that Fld could also interact with Fd-based pathways other than NADP⁺ or Trx reduction. Stimulation of amino acid and fatty acid synthesis, nitrogen and sulfur assimilation, and especially ascorbate regeneration (Figure 9) as well as the dissipative routes of cyclic electron flow and chlororespiration also might contribute to the stress tolerance observed in the transformants. Hagemann et al. (1999) have demonstrated that Fld accumulation critically contributes to enhanced cyclic electron transport around PSI in stressed cells of the cyanobacterium Synechocystis. The possibility that Fld could replace Fd in any of these chloroplast-based pathways has not been addressed experimentally and certainly deserves further research.

The approach explored in this work was to express Fld, the gene product from a photosynthetic prokaryote, in a higher plant, where it can engage in a multiplicity of reactions affecting general plant function. Within this context, Fld manipulation could provide fresh and powerful resources to investigate the requirements of photosynthesis in vivo, based on the use of a bacterial protein whose expression is free from the endogenous regulation and feedback loops that might complicate phenotype interpretation. The proposed strategy might still benefit from the functional complementation of Fd-deficient mutant or antisense plants. Plant life appeared to be irreversibly compromised when Fd protein contents were diminished below 50% of those present in nontransformed wild-type siblings (Holtgrefe et al., 2003). Fld expression could help to overcome this lower limit, potentially leading to the generation of transformed lines that could survive and develop with marginal levels of the iron–sulfur protein. Experiments aimed at this goal are currently under way. Finally, the broad-range stress tolerance displayed by Fld-expressing plants suggests that the use of this flavoprotein might have applications as a biotechnological tool to improve the field performance of crops grown in suboptimal environments.

	METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

 
Construction of Fld Expression Vectors and Tobacco Transformation
A DNA fragment encoding Anabaena PCC 7119 Fld was obtained by PCR amplification of a previously isolated gene (Fillat et al., 1991) with primers 5'-GACGAGCTCTCATAATGTCAAAG-3' and 5'-ACTGTCGACTTTTTACAAACCAAAT-3', containing SacI and SalI recognition sites, respectively. Underlined bases represent the initial and termination codons of the Fld gene. The amplified fragment was digested with the corresponding enzymes and ligated in-frame to the SacI end of a BamHI/SacI DNA, encoding the chloroplast-targeting transit peptide of pea (Pisum sativum) FNR (Serra et al., 1995). The fused gene was cloned between the cauliflower mosaic virus 35S promoter and polyadenylation regions of pDH51 (Pietrzak et al., 1986) to yield pfld. Fld DNA was also amplified using the same 3' end primer and the oligonucleotide 5'-AAGATCAGGATCCATAATGTCAAA-3', containing a BamHI site, as the 5' end primer. The PCR fragment was digested with BamHI/SalI and cloned in pDH51 to produce cfld. Both constructs (pfld and cfld, directing Fld expression to plastids and the cytosol, respectively) were excised from pDH51 plasmids with EcoRI and inserted into compatible sites of the binary vector pCAMBIA 2200 (Hajdukiewicz et al., 1994). Recombinant plasmids were introduced into the genome of tobacco (Nicotiana tabacum cv Petit Havana) through Agrobacterium tumefaciens–mediated leaf disc transformation (Gallois and Marinho, 1995).

Homozygous lines were selected by following kanamycin resistance and Fld levels in the progeny of self-pollinated primary transformants. The presence of the bacterial flavoprotein in cleared leaf extracts was analyzed by SDS-PAGE on 15% polyacrylamide gels, followed by immunoblot analysis with Fld antisera. Reactive bands were integrated using the Multi-Analyst Package 1.1 from Bio-Rad, and Fld contents were estimated by comparison with pure standards (see Supplemental Figure 2D online). The concentration of the purified protein was determined by the absorption of bound flavin mononucleotide, using ₄₅₄ = 8.8 mM^–1 cm^–1.

Plant Growth and Characterization
Seeds were germinated on Murashige and Skoog (MS) agar plates supplemented with 2% (w/v) sucrose and, in the case of transformants, 100 µg/mL kanamycin. After 4 weeks, seedlings were transferred to new MS agar or soil or grown hydroponically in nutrient medium (Geiger et al., 1999). Soil-cultured plants were watered daily with the same solution and illuminated in all cases at 200 µmol·m^–2·s^–1 and 25°C to provide a 14-h photoperiod (growth chamber conditions).

Stress treatments were performed with homozygous plants belonging to various generations (T3 to T6). Plants transferred to hydroponia were cultured for 2 to 4 weeks before exposure to adverse conditions. Most experiments were performed with specimens grown in soil for 8 to 9 weeks. These plants usually contained 10 to 11 nodes with internodal distances of 6 to 7 cm (Table 1). Treatments were performed on whole plants or on the youngest fully developed leaves, corresponding to nodes 8 or 9 (counting from the bottom) of wild-type and transformed specimens. To improve reproducibility and facilitate comparisons between lines, we always assayed, side-by-side, tissues of the same developmental stage (the same node) in wild-type and transformed plants that displayed similar height and number of nodes. Statistical analysis was conducted using a two-sided t test.

Plants were exposed to MV by spraying the aerial parts or through the nutrient solution, depending on whether they were cultured in soil or hydroponia, respectively. The spraying solution contained 0.005% (v/v) Silwet. Discs (12 mm diameter) were punched from expanded leaves (nodes 8 to 9) of 2-month-old plants grown in soil and floated topside up in 1 mL of water or MV. Plants and discs were illuminated at 500 µmol·m^–2·s^–1 for 18 h, unless stated otherwise, whereas controls were kept in the dark. Ion leakage was measured as the increase in conductance of the medium using a Horiba B-173 conductivity meter. At the end of the assay, samples were autoclaved to disrupt all cells, and total electrolyte contents were determined in the resulting solution.

To evaluate the tolerance to high temperatures, 2-week-old seedlings cultured in MS agar or leaf discs from 2-month-old plants grown in soil were exposed to 500 µmol·m^–2·s^–1 at 40°C for 12 h on MS agar plates. Chilling sensitivity was assayed in 7-week-old plants grown in MS agar that were cultured for 20 d at 9°C and 500 µmol·m^–2·s^–1 or in 2-month-old plants grown in soil and exposed to 4°C and 500 µmol·m^–2·s^–1 for 48 h. Treatments with intense illumination were performed in 2-month-old plants grown in soil by focusing a light beam of 2000 µmol·m^–2·s^–1 for 18 h at 25°C on the upper surface of a fully expanded leaf from node 8 with the aid of a light cannon. Plants of similar age were also subjected to a 3-d water deficit regime or to UV-C radiation. In the latter case, samples were exposed to a germicidal lamp (Phillips 36 W/636T8) placed at 60 cm in a laminar flow hood (CASIBA HI3) for 20 min and photographed after 3 d. Tolerance to UV-AB radiation was assayed by exposing 6-week-old plants cultured in hydroponia to a combination of UV-A (315 to 380 nm, 2.2 W/m²) and UV-B (280 to 315 nm, 1.0 W/m²) radiation for 24 h at 25°C. Leaf discs from 2-month-old plants grown in soil were also subjected to heating, high light intensity (1200 µmol·m^–2·s^–1), and UV-AB radiation essentially as described above, except that treatments were applied for 18 h in these cases.

Fixation, Substitution, and Embedding for Light and Transmission Electron Microscopy
For the primary fixation, 1-mm² sections of leaf tissue were incubated for 2.5 h at 25°C in 50 mM cacodylate buffer, pH 7.2, containing 0.5% (v/v) glutaraldehyde and 2.0% (v/v) formaldehyde, followed by one wash with buffer and two washes with distilled water. Samples for immunogold labeling studies were dehydrated stepwise by increasing the concentration of ethanol under progressive lowering of temperature in an automated freeze substitution unit (AFS; Leica). The steps used were as follows: 30% (v/v), 40% (v/v), and 50% (v/v) ethanol for 1 h each at 4°C; 60% (v/v) and 75% (v/v) ethanol for 1 h each at –15°C; 90% (v/v) ethanol and twice 100% (v/v) ethanol for 1 h each at –35°C. The samples were subsequently infiltrated with Lowycryl HM 20 resin (Plano) by incubating them in the following mixtures: 33% (v/v), 50% (v/v), and 66% (v/v) HM 20 resin in ethanol for 4 h each and then 100% (v/v) HM 20 resin overnight. Samples were transferred into gelatin capsules, incubated for 3 h in fresh resin, and polymerized at 35°C for 3 d under indirect UV light. Thin sections, 70 nm thick, were cut with a diamond knife and used for immunogold labeling with anti-Fld as the primary antibody and 10 nM protein A–gold as described by Teige et al. (1998).

Samples for ultrastructural analysis and light microscopy were transferred after primary fixation into a solution of 1% (w/v) OsO₄ for secondary fixation. After 1 h, samples were washed three times with distilled water. Dehydration at 25°C was performed stepwise by increasing the concentration of ethanol as follows: 30% (v/v), 50% (v/v), 60% (v/v), 75% (v/v), 90% (v/v), and twice 100% (v/v) ethanol for 1 h each. After additional dehydration with propylene oxide for 1 h, the samples were infiltrated with Spurr resin (Plano) as follows: 33% (v/v), 50% (v/v), and 66% (v/v) Spurr resin in propylene oxide for 4 h each and then 100% (v/v) Spurr resin overnight. Samples were transferred into embedding molds, incubated there for 3 h in fresh resin, and polymerized at 70°C for 24 h. Semithin sections with a thickness of 3 µm were mounted on slides and stained for 2 min with 1% (w/v) methylene blue and 1% (w/v) Azur II in 1% (w/v) aqueous borax at 60°C before light microscopic examination. For electron microscopic analysis with a Zeiss CEM 920A transmission electron microscope at 80 kV, ultrathin sections with a thickness of 70 nm were cut with a diamond knife and contrasted with a saturated methanolic solution of uranyl acetate and lead citrate before examination.

Activity Determinations
Light-dependent CO₂ assimilation and stomatal conductance were determined on attached leaves from nodes 8 or 9 of 2-month-old plants grown in soil using an LI-6200 portable photosynthesis system from Li-Cor. The CO₂ concentration of the air entering the leaf chamber and the temperature were adjusted to 360 ppm and 20°C, respectively. PPFD ranging from 50 to 1000 µmol·m^–2·s^–1 was supplied by a controlled halogen light source. Chlorophyll a fluorescence measurements were performed at 25°C on dark-adapted leaves of the same plants using a Qubit Systems pulse-modulated fluorometer. The F_v and F_m parameters were determined after 30 min in the dark, and the light-adapted values (F_v' and F_m') were measured after 30 min of illumination with 500 µmol·m^–2·s^–1. Photosynthetic parameters (F_v/F_m, _PSII, and nonphotochemical and photochemical quenching) were calculated as described (Baker and Rosenqvist, 2004).

NADP⁺ photoreduction, using water as the electron donor, was measured at 25°C in 3 mL of 50 mM HEPES-KOH, pH 8.0, 5 mM MgCl₂, 330 mM sorbitol, 0.5 mM NADP⁺, thylakoids corresponding to 20 µg of chlorophyll, and 20 µM Fld. Illumination (2400 µmol·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 (₃₄₀ = 6.2 mM^–1 cm^–1).

SOD, APX, and GR were extracted from leaf tissue using the conditions and homogenization solutions reported by Gupta et al. (1993). Procedures used to assay their activities are also described there. The patterns of SOD and APX isoenzymes were analyzed by resolving cleared extracts corresponding to 45 and 170 mm² of leaf tissue, respectively, by nondenaturing PAGE and activity staining (Beauchamp and Fridovich, 1971; Mittler and Zilinskas, 1993).

Samples used for the estimation of the in vivo enzymatic activities of chloroplast NADP-MDH, FBPase, and PRK were prepared by the freeze-clamp method. At the end of the treatment or illumination periods, young leaves from node 8 of both control and transgenic plants grown in soil were harvested under light, ground to a fine powder at the temperature of liquid nitrogen, and extracted in the medium of Scheibe and Stitt (1988) supplemented with 5 mM DTT. Activities were assayed immediately after extraction using the conditions of Kossmann et al. (1994) for FBPase and PRK and the conditions of Scheibe and Stitt (1988) for NADP-MDH. Total activities of the three enzymes were determined after reduction with 50 mM DTT for 1 h at 25°C.

In vitro Prx reduction was performed in a reconstituted system consisting of 0.5 mM NADPH, 0.5 µM pea FNR, 1 µM spinach (Spinacia oleracea) FTR, 5 to 10 µM Trx f, m, or x from spinach, 1 µM 2-Cys Prx B from Arabidopsis thaliana, and the indicated concentrations of either pea Fd or Anabaena Fld in 30 mM Tris-HCl, pH 8.0. After 15 min at 30°C, the reaction was quenched with 10 mM N-ethylmaleimide and the redox state of Prx was evaluated by nonreducing SDS-PAGE and immunoblotting (König et al., 2002). Rates of H₂O₂ reduction were measured in the same reconstituted medium described above, except that Prx and Fld were 4 and 10 µM, respectively. The assay was initiated by the addition of H₂O₂ to 50 µM, and the rates of H₂O₂ consumption were determined by reaction with xylenol orange as recommended by the manufacturer (Bioxytech H2O2-560; Bio-Stat).

Two-Dimensional Gel Electrophoresis
To detect the levels of inactivated Prx in vivo, leaf tissue was collected at the end of the treatment and/or illumination periods, freeze-clamped, and extracted essentially as described above for the chloroplast enzymes. Aliquots corresponding to 8 to 16 µg of total soluble protein, derivatized or not with 10 mM N-ethylmaleimide, were resolved by nonreducing SDS-PAGE on 15% polyacrylamide gels followed by immunoblot detection (König et al., 2002).

For two-dimensional gel electrophoresis, lysates corresponding to 15 µg of protein were applied to precast vertical isoelectric focusing gels (pH 3 to 7; Novex, Invitrogen). Focusing, processing, and reduction of the isoelectric focusing strips and second-dimension SDS-PAGE on 15% polyacrylamide gels were performed as recommended by the manufacturer. Prx spots were visualized after transfer to nitrocellulose membranes and reaction with specific antisera.

Analytical Procedures
Intact chloroplasts were isolated from wild-type and transgenic plants using Percoll gradient centrifugation (Wu et al., 1991). Chlorophyll in leaves and chloroplasts was determined spectrophotometrically in acetone extracts (Lichtenthaler, 1987). Limited proteolysis of isolated chloroplasts (corresponding to 2 mg chlorophyll/mL) was performed by incubation for 30 min with 100 µg/mL thermolysin in ice-cold 50 mM HEPES-KOH and 330 mM sorbitol. The reaction was stopped by the addition of EDTA to 10 mM, and the plastids were reisolated by centrifugation through silicone oil layers (Serra et al., 1995). Digestion of broken chloroplasts was performed under the same conditions, but the plastids were previously shocked by incubation for 10 min at 20°C in 20 mM Tris-HCl, pH 8.0, 1 mM EDTA, and the reisolation step was omitted. Detection of Fld, Fd, Rubisco LSU, PFP, and the cytosolic isoform of NAD-MDH in whole leaf and chloroplast extracts was performed by SDS-PAGE and immunoblotting using 15% polyacrylamide gels and polyclonal antisera.

For the determination of ROS and antioxidant metabolites, attached leaves from node 9 of soil-cultured 2-month-old plants were sprayed with 30 µM MV and illuminated at 500 µmol·m^–2·s^–1. Leaves were incubated at 40°C under the same light intensity or irradiated with 1200 µmol·m^–2·s^–1. Incubations were prolonged for 18 h in all cases, and controls were kept in the growth chamber for the same period. At the end of the treatments, leaves from the susceptible lines displayed mild symptoms of wilting, whereas chlorosis was only marginal. The tissue was ground in liquid nitrogen, and the contents of superoxide and H₂O₂ were estimated in cleared lysates by measuring lucigenin- and luminol-dependent chemiluminiscence, respectively (Bolwell et al., 1998; Dat et al., 1998). MDA levels were determined by HPLC after reaction with thiobarbituric acid (Templar et al., 1999). Ascorbate and thiols were assayed in leaf extracts by colorimetric methods using the extraction procedures and conditions reported by Foyer et al. (1995).

Accession Number
Sequence data from this article can be found in the GenBank/EMBL data libraries under accession number S68006.

Supplemental Data
The following materials are available in the online version of this article.

Supplemental Figure 1. Purified Anabaena Fld Mediates NADP⁺ Photoreduction by Tobacco Thylakoids.

Supplemental Figure 2. Accumulation of Fld in Transgenic Tobacco Plants.

Supplemental Figure 3. MV Toxicity in Leaf Discs from Wild-Type and Fld-Expressing Plants.

Supplemental Figure 4. Increased Tolerance of Transgenic Tobacco Plants Expressing Bacterial Fld in Chloroplasts to Different Sources of Environmental Stress.

Supplemental Figure 5. Effect of Fld Accumulation on the Patterns of Leaf Antioxidant Enzyme Isoforms.

Supplemental Figure 6. The Levels of Components of the Trx/FTR Pathway Are Not Affected by Fld Expression or Stress.

Supplemental Table 1. Phenotypic Analysis of Wild-Type and Transgenic Tobacco Plants Expressing Fld in Chloroplasts.

Supplemental Table 2. Effect of Fld Expression on the Levels of Antioxidant Enzymes and Metabolites in Plants Exposed to MV.

Supplemental Table 3. Fld Expression Prevents Overoxidation of 2-Cys Prx in Stressed Tobacco Plants.

Supplemental Table 4. Effect of Fld Expression on the Activation State of Trx-Dependent Chloroplast Enzymes in Plants Subjected to Stress Conditions.

	Acknowledgments

 
We are indebted to our colleagues from the National University of Rosario, Argentina: José Pellegrino (Instituto de Fisiología Experimental), María Mamprin (Pharmacology Unit), and Ramiro Rodriguez (Instituto de Biología Molecular y Celular de Rosario) for their assistance in ROS, thiol, and MDA determinations, respectively; Eligio Morandi (Agronomy School) for his invaluable help with the water-deficit and UV-C experiments; and Mercedes Leiva for statistical analyses. We also thank Karl-Josef Dietz (Bielefeld University, Germany) for the generous gift of 2-Cys Prx B from Arabidopsis and its specific polyclonal antiserum; Peter Schürmann (Neuchâtel University, Switzerland) for spinach Trx f, Trx m, and FTR; Myroslava Miginiac-Maslow (Université de Paris-Sud, France) for Trx x; and Florencio Podestá (Centro de Estudios Fotosintéticos y Bioquímicos, National University of Rosario, Argentina) for the antibodies against pineapple MDH and potato PFP. This research was supported by grants from Agencia Nacional de Promoción Científica y Tecnológica, the Consejo Nacional de Investigaciones Científicas y Técnicas, and the Fundación Antorchas, Argentina.

	Footnotes

 
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.plantcell.org) is: Néstor Carrillo (carrillo{at}ibr.gov.ar).

^[W] Online version contains Web-only data.

Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.106.042424.

Received March 10, 2006; Revision received May 15, 2006. accepted June 8, 2006.

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