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

Abstract

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.

Improving crop productivity is an important goal to meet the increasing demands for food that result from rapid population growth. Within this context, one of the most severe limitations for agricultural development is the availability of iron, required for the function of a plethora of metalloenzymes involved in fundamental processes such as respiration and photosynthesis. Iron is found in nature mostly as ferric oxides that are sparingly soluble at neutral pH, indicating that the main problem of Fe acquisition is not of abundance, but of the paucity of soluble amounts (1–4). Deficiency can be especially pronounced for plants grown in alkaline, calcareous soils that cover more than one-third of the planet's cultivable land and represent a major deterrent for agriculture (5). Iron availability is also a critical factor limiting the size of phytoplankton stocks and the rates of biological CO₂ uptake in the oceans, which accounts for approximately half of the photosynthetic fixation of atmospheric carbon on Earth (6). Buildup of Fe reserves within cells is further complicated because high Fe concentrations can wreak cellular havoc and lead to oxidative damage and growth inhibition (1, 2, 7). As a result of these confronting demands, organisms need to tightly regulate Fe uptake and storage within a limited range.

Iron-deprived plants usually develop interveinal chlorotic symptoms in young leaves and poor root formation. When severe, the deficiency leads to growth retardation, stasis, and death (1, 8). Chlorosis has been attributed to inhibition of chlorophyll synthesis, which requires the function of Fe-containing enzymes (9), but expression of chlorophyll-binding proteins and other photosynthetic components is down-regulated with relative independence of pigment levels (10). Chloroplasts are therefore primary targets of Fe deficiency, resulting in a decrease of photosynthetic activity and of plastidic pigments and proteins (11, 12). Both the utilization of ribulose-1,5-bisP by Rubisco and its regeneration by the Calvin cycle appear compromised in Fe-starved plants (12, 13).

Soil-based organisms have evolved various adaptive mechanisms to mitigate the consequences of Fe deficit. Most of them rely on optimization of metal uptake from scarcely available sources. Dicotyledoneous plants and nongraminaceous monocots display the so-called strategy I response, characterized by development of root hairs and transfer cells, increased proton extrusion to the rizosphere to improve Fe³⁺ solubility, and enhanced accumulation of ferric-chelate reductases and broad-range metal transporters in the root cell plasma membrane to favor Fe²⁺ intake (14–18). Grasses, on the other hand, have developed a different response, releasing phytosiderophores of the mugineic acid family into the surrounding soil (5).

Some photosynthetic microorganisms display still a different strategy to survive in Fe-restricted environments, based on the replacement of metalloproteins that become limiting by isofunctional counterparts. An example of such an adaptation is provided by some cyanobacteria and marine algae that, when exposed to Fe-deficient conditions, induce the expression of flavodoxins (Fld), small redox proteins harboring one molecule of flavin mononucleotide as prosthetic group (6, 19–21). These soluble electron carriers are able to replace Fe-containing ferredoxin (Fd) in many different processes, including photosynthesis (22). The Fe-sulfur cluster of Fd cannot be assembled in Fe-limited media, and genome-wide microarray analysis indicates that accumulation of Fd transcripts is repressed in both plants (10) and cyanobacteria (21). Fld expression relieves the burdens imposed by Fe deficit and restores normal rates of growth and reproduction. The importance of this flavoprotein in the dynamics of marine ecology is highlighted by its use as the most sensitive marker of Fe stress in the oceans (6, 19). Up-regulation of Fld levels is also considered as one of the factors determining colonization of Fe-poor waters by phytoplankton (23), and photosynthetic microorganisms lacking the Fld gene are usually restricted to Fe-rich coastal environments (24). The Fld gene is absent from the plant genome (25), indicating that this adaptive resource was lost somewhere in the transition between algae and plants, although at least some of the plant enzymes whose bacterial ancestors used Fld as a normal or occasional substrate have retained the ability to productively interact with this electron carrier (26).

We have introduced the Fld gene from the cyanobacterium Anabaena PCC7119 into the genome of tobacco and demonstrated that transgenic plants expressing this flavoprotein in chloroplasts developed generalized tolerance to various sources of oxidative and environmental stress (26). Here we show that these transformants were also able to grow on Fe-limited substrates. Increased tolerance to Fe starvation relied on productive interaction of Fld with Fd-dependent oxidoreductive pathways of the host, whereas other features of the plant response to this nutritional deficit were unaffected by Fld expression.

Results

Transgenic Plants Expressing a Plastid-Targeted Fld Display Increased Tolerance to Iron Deprivation.

In eukaryotic algae, Fld is nuclear-encoded and targeted to chloroplasts (6). We therefore analyzed the response to Fe limitation of transgenic tobacco plants in which Fld accumulated in plastids (pfld lines, for plastidic Fld) using WT siblings and transformants expressing the flavoprotein in the cytosol (cfld lines, for cytosolic Fld) as controls. Preparation of stable homozygous pfld and cfld tobacco lines expressing various levels of Fld has been described elsewhere (26). Most experiments were carried out with plants from lines pfld5-8, pfld4-2, and cfld1-4, containing Fld at 70, 57, and 108 pmol·g⁻¹ fresh weight, quantities comparable to 98 pmol·g⁻¹ fresh weight for endogenous Fd (26). When grown in Fe-replete media, transformed plants displayed WT phenotypes with respect to biomass accumulation (Table 1), flower development, and seed production (26). Seeds of WT and transformed plants germinated with similar efficiency in Murashige–Skoog (MS) agar medium lacking an Fe source, and supplemented with the Fe chelator ferrozine or with CaCO₃ (pH 8.0) [supporting information (SI) Fig. 6], suggesting that they contained enough reserves of the transition metal to sustain germination and early development.

Table 1.

Fld expression in chloroplasts partially prevents growth arrest and photosynthesis inhibition caused by iron deficit

Plants	Height, cm	No. of nodes	Internodal distance, cm	Fresh weight/plant, g	% H2O	Chl (a + b)/area, μg·cm−2	Carotenoids/area, μg·cm−2	CO2 maximum fixation rate, μmol of CO2 per m−2·s−1	Estimated PPFD at saturation, μmol·quanta·m−2·s−1	Fv /Fm	φPSII	NPQ
Control
WT	29.1 ± 1.2	14 ± 1	2.45 ± 0.10	46.7 ± 4.3	88.3 ± 1.1	36.28 ± 1.57	5.35 ± 0.14	8.45 ± 0.71	700	0.77 ± 0.03	0.34 ± 0.01	1.20 ± 0.03
pfld5-8	28.0 ± 1.5	14 ± 1	2.48 ± 0.15	44.1 ± 5.1	88.8 ± 0.7	40.58 ± 2.36	6.59 ± 0.22	9.12 ± 0.55	700	0.78 ± 0.02	0.31 ± 0.02	1.44 ± 0.02
cfld1-4	31.1 ± 1.7	14 ± 1	2.51 ± 0.13	48.5 ± 4.9	89.4 ± 1.3	31.61 ± 1.26	4.67 ± 0.09	9.80 ± 1.18	700	0.77 ± 0.04	0.37 ± 0.01	1.27 ± 0.03
Fe-deficient
WT	9.6 ± 0.9	8 ± 1	0.87 ± 0.06	16.3 ± 1.1	90.9 ± 1.3	15.01 ± 0.95	1.85 ± 0.09	0.86 ± 0.10 (10.2)	100	0.70 ± 0.04	0.29 ± 0.03	1.14 ± 0.04
pfld5-8	14.0 ± 0.7	12 ± 1	0.91 ± 0.05	20.6 ± 0.8	89.3 ± 1.1	20.02 ± 0.99	2.57 ± 0.05	3.65 ± 0.34 (40.0)	700	0.74 ± 0.04	0.32 ± 0.02	1.17 ± 0.04
cfld1-4	7.1 ± 0.9	6 ± 1	0.72 ± 0.09	11.4 ± 1.6	92.1 ± 1.4	5.83 ± 0.63	0.77 ± 0.04	1.59 ± 0.28 (16.2)	100	0.64 ± 0.05	0.19 ± 0.04	0.98 ± 0.08

WT and transformed plants were exposed to Fe deficit for 29 days in hydroponics as indicated in Materials and Methods. Analytical procedures and determination of photosynthetic parameters are also described there. Values presented are the means ± SE of 7–10 independent plants. PPFD, photosynthetic photon flux density; NPQ, nonphotochemical quenching.

To investigate the effects of Fe restriction on vegetative growth, plants were cultured for 4 weeks on MS agar containing 30 μM Fe⁺² (standard medium) and then subjected to acute Fe deficit by transfer to Fe-free MS agar supplemented with ferrozine or CaCO₃. After 15–20 days of treatment, specimens from all lines showed some degree of deterioration, which was severe in WT and cfld1-4 plants but mild in pfld5-8 siblings (Fig. 1A). Root development was impaired in the sensitive lines but proceeded normally in plants containing a plastid-targeted Fld (Fig. 1A). Similar results were obtained when 4-week-old specimens were Fe-starved for 29 days in hydroponic nutrient solution (Fig. 1B). Symptoms of Fe deprivation included growth arrest and leaf chlorosis (Fig. 1 A and B), as well as significant declines in chlorophyll and carotenoids (Table 1). The phenotype of pfld plants was confirmed by using various independent lines expressing different levels of Fld in chloroplasts or cytosol. SI Fig. 7 shows typical results obtained with plants from various lines exposed to CaCO₃ treatment in MS-agar. WT and cfld specimens displayed a significantly retarded growth when compared with pfld siblings, with the degree of tolerance correlating with plastidic Fld contents in chloroplasts (compare growth of pfld5-8 plants with that of pfld12-4 siblings, expressing 10 times less Fld). In general, cfld plants displayed lower growth rates and accumulated less biomass and leaf pigments than WT specimens under Fe deficit (Table 1). The reasons for this behavior are unclear. It is possible that the presence of Fld might negatively affect electron distribution in the cytosol.

Fig. 1.

Phenotypes of WT and Fld-expressing tobacco plants cultured under different conditions of iron availability. Plants were grown for 4 weeks in standard MS agar and then transferred to Fe-free MS agar supplemented with 10 mM CaCO₃ (pH 8.0) (A) or to hydroponic Hoagland solution containing either 30 μM FeSO₄-EDTA or 10 mM CaCO₃ (pH 8.0) (B). Plants were photographed 20 (A) or 29 (B) days after transfer. Other conditions are given in Materials and Methods. (C) Lots of 100 seeds were germinated and grown on soil watered daily with 10 mM CaCO₃ (pH 9.0). Pictures were taken at 5 (Top) and 7 (Middle) weeks of treatment. Graphs in Bottom show percentages of surviving plants after 7 weeks of Fe deficit (filled bars) relative to siblings grown under iron-sufficient conditions (open bars). Values presented are the means ± SE of three experiments.

To evaluate the behavior of Fld-expressing lines under a growth regime that more closely resembled field conditions, plants were grown autotrophically in alkaline soil (pH 9.0). Germination and early development proceeded normally in the basic matrix and after 5 weeks mortality was still marginal, although WT specimens and cfld1-4 transformants exhibited augmented chlorosis and growth retardation when compared with siblings of the pfld5-8 line (Fig. 1C). In the following days the pace of leaf bleaching increased dramatically in the sensitive lines, growth ceased, and most plants died after two further weeks (Fig. 1C). In contrast, pfld5-8 transformants survived the treatment and increased in weight and height, although they did exhibit stress symptoms, such as early flowering and slightly diminished (≈25%) seed production.

Plants Expressing Fld Exhibit a Normal Response to Iron Starvation.

Six-week-old plants grown in standard MS agar accumulated similar Fe levels irrespective of the presence of Fld in chloroplasts or cytosol (SI Table 2). On exposure to limited media, Fe contents declined to 25–50% of the control values in all lines, indicating that Fld expression did not contribute to Fe uptake, mobilization, or storage and that the tolerant pfld lines could still grow and reproduce with considerably lower Fe quotas.

In dicots such as tobacco, Fe deficit causes induction of many genes, including those encoding ferric-chelate reductases (FRO) and broad-range metal transporters belonging to the IRT and NRAMP families (14–18). Semiquantitative RT-PCR revealed that IRT1, FRO1, and NRAMP1 transcripts accumulated in WT tobacco roots in response to Fe deficiency (Fig. 2, lanes 1 and 2). Induction was not influenced by the presence of Fld in plastids or cytosol (Fig. 2, lanes 3–6). Expression of FRO1 in leaves of Fe-starved pfld5-8 and cfld1-4 lines also followed WT response patterns (Fig. 2).

Fig. 2.

Expression analysis of iron-responsive genes in plants exposed to iron limitation. Four-week-old plants were cultured for an additional 4 weeks in standard MS medium (+ Fe) or in MS agar containing 10 mM CaCO₃ (− Fe). RNA was isolated from roots and leaves of WT (lanes 1 and 2), pfld5-8 (lanes 3 and 4), or cfld1-4 (lanes 5 and 6) plants and subjected to RT-PCR as indicated in Materials and Methods using gene-specific primers for FdI, IRT1, FRO1, NRAMP3, and actin.

Fld Accumulation Prevents the Decline of Photosynthetic Components in Iron-Limited Media.

Evaluation of Fd (Fig. 3) and Fd-encoding RNA contents (Fig. 2) indicates that expression of this electron carrier was repressed in Fe-deficient plants of all lines, whereas Fld levels were not affected (Fig. 3). Other photosynthetic components, such as the light-harvesting chlorophyll-binding protein and Rubisco, were also significantly reduced in the absence of Fe (Fig. 3), in agreement with previous observations (10–13). The presence of Fld in chloroplasts, but not in the cytosol, almost entirely prevented this decline (Fig. 3).

Fig. 3.

Fld accumulation in chloroplasts prevents decrease of photosynthetic components in leaves of iron-starved plants. Plants were grown and exposed to Fe deficit as indicated in the legend to Fig. 2. Extracts were prepared from young leaves of plants grown in standard medium (lanes 1–3) or exposed to Fe-free MS containing CaCO₃ (lanes 4–6) or ferrozine (lanes 7–9). Cleared lysates corresponding to 8 mg of fresh weight were subjected to SDS/PAGE and blotted onto nylon membranes for immunodetection of Fd, Fld, light-harvesting chlorophyll-binding protein (LHCP), and Rubisco large (LSU) and small (SSU) subunits.

Fld Expression Protects Photosynthesis from Inactivation Caused by Iron Deficiency.

Light-saturated photosynthesis was inhibited by 85–90% during acute Fe deprivation in WT plants together with a steep decrease in the photosynthetic photon flux density at which saturation was attained (Table 1). Similar results were obtained with cfld plants, whereas expression of plastid-targeted Fld restored the saturation photosynthetic photon flux density value and partially relieved inactivation of photosynthesis to ≈40% of the activities found under Fe-sufficient conditions (Table 1). Inhibition of photosynthesis was not accompanied by alterations of F_v/F_m or nonphotochemical quenching, indicating little damage from photoinhibition (Table 1). The quantum efficiency of photosystem II (φ_PSII) failed to exhibit significant changes upon metal deficiency (Table 1). The results concur with previous studies indicating that neither light absorption nor the structure of plant photosystems is seriously affected by the lack of Fe (11) and suggest that inhibition of photosynthesis occurred at a different stage, most likely during CO₂ fixation.

To further investigate the effects of Fe deficit on photosynthetic metabolism we measured the levels of central metabolites in leaves from iron-deprived and control plants expressing Fld or not. A summary of the most relevant results obtained with WT and two independent pfld lines is schematically shown in Fig. 4 as ratios of metabolite contents between Fe-starved and Fe-replete plants. Actual amounts for these plants and a cfld line used as a control are given in SI Tables 3 and 4. A ratio lower than unity indicates that Fe deficit causes a decline of the particular metabolite in the plants. In WT specimens Fe limitation caused a decrease in the levels of sucrose, total monosaccharides (mostly fructose and glucose), UDP-Glc, and adenine nucleotides (50%, 20%, 55%, and 45%, respectively) (Fig. 4). Contents of these metabolites, in contrast, were maintained (sucrose and monosaccharides) or even increased up to 2-fold (UDP-Glc) for the highly expressing pfld5-8 line, whereas pfld4-2 plants, which accumulate lower Fld levels, showed intermediate amounts (Fig. 4). A decrease in the contents of ribulose 5-P, 3-P-glyceric acid, hexoses-P, and P-enol pyruvate of 60%, 85%, 75%, and 40%, respectively, was observed for the WT leaves, whereas the levels of these intermediates were either maintained (3-P-glyceric acid and P-enol pyruvate), or increased up to 1.6-fold in pfld5-8 plants. The contents for pfld4-2 specimens were decreased but remained still above the values recorded for the WT (Fig. 4).

Fig. 4.

Relative metabolite changes in iron-starved and control plants. Four-week-old plants were transferred to hydroponic Hoagland solution supplemented with either FeSO₄-EDTA or CaCO₃ (pH 8.0). Leaf material was harvested after 29 days, and the corresponding metabolites were measured as described in Materials and Methods. Depicted are the ratios ± SE of metabolite contents between Fe-starved and -replete plants of WT (green bars), pfld5-8 (blue bars), and pfld4-2 (light blue bars) lines (n = 8–10 independent plants). The graph was created by using the visualization system Vanted (38).

Chloroplast-Targeted Fld Supports Amino Acid Metabolism in Iron-Starved Transgenic Plants.

Amino acid metabolism requires Fd at two critical steps: nitrite reduction by nitrite reductase and ammonium incorporation into Glu by Glu-oxoglutarate aminotransferase. It is conceivable that Fld could replace Fd as electron donor for these two reactions, as it occurs with other Fd partners (26), therefore compensating Fd loss under Fe limitation.

Fig. 4 shows the relative changes in the levels of leaf amino acids as a network based on the carbon source. When Fe was provided ad libitum total amino acids accumulated to similar levels in all lines (data not shown). Nitrogen assimilation into Glu declined in Fe-starved WT specimens to ≈65% of the control values, but this decrease was prevented in pfld siblings (Fig. 4). Gln contents did not exhibit major differences in all lines and conditions assayed, probably reflecting the fact that Fd not only acts as electron donor in the provision of ammonium for Gln synthesis but also for the Glu-oxoglutarate aminotransferase-mediated conversion of Gln into Glu. Therefore, rates of both Gln generation and Gln consumption should be affected by Fe deficit and Fld expression, apparently to similar extents. The other synthetic route coming from the Krebs cycle produces Asp, a crucial amino acid involved in recycling and maintenance of ammonium assimilation. Asp contents decreased by 50% in Fe-deficient WT plants while remaining almost unaltered in the transgenics. The levels of Asn, Lys, Thr, and Ile, all derived from the Asp pathway, increased up to 1.6-fold in the tolerant pfld lines, whereas they were maintained or slightly decreased in the WT. Ser contents did not change in the WT and increased slightly (1.3-fold) in transformants whereas Gly, which utilizes 3-P-glyceric acid as carbon backbone, augmented its level by ≈1.6-fold in the three lines assayed (Fig. 4). Cys slightly decreased in all lines, although to a lesser extent in the transformants. Increased Glu and Gly contents led to significantly higher glutathione amounts in the pfld lines. Finally, the levels of Ala, which is synthesized from pyruvate, did not change in the WT and increased up to 1.6-fold in transgenic lines (Fig. 4). Amino acid levels determined in Fe-replete and Fe-starved cfld1-4 plants were similar to those obtained with WT siblings (SI Table 4).

Fld Sustains Thioredoxin (Trx)-Dependent Activation Pathways in Iron-Deprived Transgenic Plants.

Several enzymes of the Calvin cycle require reduced thiols for full manifestation of activity, a task performed by various Trx isoforms present in chloroplasts. Trx reduction is in turn linked to Fd via the Fd-Trx reductase FTR (27). Not unexpectedly, therefore, some key components of the cycle are reported to be especially vulnerable to Fe limitation (12, 13). The ability of Fld to sustain Trx reduction in vitro and in planta has been documented (26). Fig. 5 shows that Fru-1,6-bisphosphatase and especially phosphoribulokinase (PRK) became extensively inactivated in Fe-starved WT plants. Under similar restrictions the activation states of the two enzymes were preserved in pfld5-8 and pfld4-2 lines (Fig. 5).

Fig. 5.

The activation state of Trx-dependent chloroplast enzymes is preserved in iron-restricted plants expressing plastid-targeted Fld. Plants were exposed (filled bars) or not (open bars) to Fe deprivation as indicated in the legend to Fig. 2. Extracts were prepared from young leaves, and the activities of Fru-1,6-bisphosphatase and PRK were measured before (in vivo activity) and after full reduction of the extracts with DTT (total activity) using published procedures (26). The activation state is calculated as the ratio between the in vivo and total activity for each condition. Means ± SE of three to five independent plants are provided.

Discussion

Manipulation of Fe use and acquisition represents a particularly demanding challenge for plant breeders and geneticists because of the complex interactions of this metal with living organisms: “nutritious, noxious and not readily available” (1). So far, strategies pursued to increase tolerance to Fe deficit have focused on engineering traits that improve plant access to elusive metal pools in the surrounding soil (28). We show here that expression of a bacterial Fld in chloroplasts could partially relieve some of the symptoms of Fe starvation while remaining unnoticed, in phenotypic terms, during normal growth. Iron-deprived plants expressing a plastid-targeted Fld retained higher amounts of photosynthetic pigments and proteins (Table 1 and Fig. 3) and carried out photosynthesis at rates that permitted growth and reproduction (Fig. 1 and Table 1). The tolerant lines accumulated less Fe in metal-limited media (SI Table 2), and yet they were able to complete their life cycles, whereas WT and cfld plants exhibited severe growth penalties and eventually died (Fig. 1C). Development of the tolerant phenotype relied on substitution of the activities of indigenous Fd counterparts as they declined for lack of Fe (Figs. 3–5) without interfering with physiological responses related to metal uptake and accumulation (Fig. 2).

It is noteworthy that all lines accumulated similar amounts of Fe irrespective of Fld contents or location (SI Table 2). This behavior contrasts with those of algae and cyanobacteria, in which expression of Fld and other replacement proteins lacking Fe (i.e., Cu-containing plastocyanin, Ni-containing superoxide dismutase, and modified photosystems and reaction centers) results in organisms that take up and grow on lower amounts of Fe, indicating that adaptation to in situ metal levels is a significant factor in phytoplankton speciation (29). Our results suggest that plants have lost these substitutive adaptations during their evolution, although the tolerance displayed by pfld transformants indicates that the compensatory functions of Fld are still operative in plant chloroplasts despite the evolutionary divergence between the donor and host organisms.

Chloroplast Fd plays a pivotal role in the physiology of the plant cell by acting as a mobile electron shuttle for the distribution of reducing equivalents generated during photosynthesis to various essential metabolic, regulatory, and dissipative pathways. Part of the Fd reduced by photosystem I delivers electrons to Fd-NADP⁺ reductase for NADP⁺ photoreduction, but a substantial fraction is used as electron donor to other plastidic enzymes such as nitrite reductase, sulfite reductase, Glu-oxoglutarate aminotransferase, fatty acid desaturase, and the Fd-Trx reductase FTR (30). Then, the function and regulation of metabolisms so essential to plant life as carbon fixation and allocation, nitrogen and sulfur assimilation, amino acid synthesis, and fatty acid desaturation depend on steady provision of reduced Fd (31). As could be anticipated by this multiplicity of functions, transgenic plants in which Fd contents were diminished by expression of an antisense RNA exhibited a pleiotropic phenotype including lower CO₂ assimilation rates, stunted growth, and a combination of other handicaps (31).

The decline of photosynthetic proteins in Fe-deprived plants has been extensively documented and appears to be a typical symptom of this nutritional deficit (10–13), although the mechanistic bases for this repression have not been studied in detail. Repression occurs at the level of transcript accumulation (10, 12), indicating that Fe starvation alters the expression patterns of these genes. Although most photosynthetic components are down-regulated under Fe deprivation (10), our results suggest that different mechanisms are implied. Fd expression responds directly to the Fe status, and therefore its repression cannot be relieved by the presence of Fld (Fig. 2 and 3). Expression of other chloroplast proteins, in contrast, apparently responds to secondary (probably redox-based) signals triggered by alterations in chloroplast electron distribution due to Fd decline. This repression might be overcome as Fld takes over Fd functions in the transgenic plants and restores adequate rates of electron flux within and from the photosynthetic electron transport chain.

Replacement of declining Fd by Fld could facilitate NADP⁺ photoreduction and Trx-dependent activation of redox-sensitive enzymes of the Calvin cycle (Fig. 5). Arulanantham et al. (13) have reported that ribulose-1,5-bisP regeneration catalyzed by PRK, rather than photon capture, NADP⁺ reduction, or ribulose-1,5-bisP carboxylation, was the key step affected by Fe availability. These results were later challenged by Winder and Nishio (12), who reported that photosynthesis inhibition correlated with Rubisco decline in Fe-restricted Beta plants, and by Paul et al. (32), who observed that CO₂ fixation rates were not significantly affected until PRK activity declined below 10% of the WT levels in antisense tobacco plants. However, more detailed investigations on the same lines showed that PRK did exert significant control on photosynthesis when the transformants were grown under a low-irradiation regime similar to that used in our experiments (33). It is thus likely that both the decrease in Rubisco levels (Fig. 3) and PRK inactivation (Fig. 5) could contribute to the phenotypes exhibited by Fe-stressed WT and cfld plants. Maintenance of Rubisco and PRK activity by chloroplast-targeted Fld might explain the higher photosynthetic rates exhibited by pfld lines (Table 1). On the other hand, the effects on amino acid contents suggest that Fld was able to engage in Fd-dependent reactions of this pathway.

Fd is an early and critical target of Fe deprivation in algae and photosynthetic prokaryotes, but other ferroproteins with higher sensitivity to Fe deficit could have been recruited during the evolution of terrestrial plants. Indeed, Fld might have become dispensable precisely because Fd replacement ceased to be of selective advantage. Observations reported here indicate otherwise: Fd decline still compromised plant survival under Fe deficit, and Fld expression allowed plants to grow on low Fe and Fd levels. In addition to the benefits derived from Fld engagement in Fd-dependent routes, the presence of this flavoprotein probably permits redistribution of the limited amount of available Fe to other demanding metabolic pathways, improving the general welfare of the host organism. It is not clear, then, why a genetic trait that confers such obvious advantages was not selected during the evolution of terrestrial plants. Fld has been found in the plastids of eukaryotic algae belonging to all major families (19), suggesting that the gene was lost in the green algal precursor or in the primitive plants. Fld loss might be related to ecological adaptations, but a systematic appraisal of this possibility will require an extensive survey of Fld-proficient and -deficient clones between algal groups and habitats.

Whatever the reasons for Fld loss in higher plants, its reintroduction into chloroplasts restored some of the selective advantages that allowed photosynthetic microorganisms to thrive in Fe-limited environments. Engineering this gene, either alone or in combination with other traits optimizing Fe intake from limited sources, could open fresh possibilities to increase the ability of agronomically important crops to grow in otherwise noncultivable lands.

Materials and Methods

Plant Materials and Iron Deprivation Treatment.

Design and preparation of homozygous pfld and cfld tobacco lines (Nicotiana tabacum cv Petit Havana) are described elsewhere (26). Seeds were usually germinated and grown for 4 weeks in MS agar containing 2% (wt/vol) sucrose and 30 μM FeSO₄-EDTA (standard medium) and then transferred to Fe-free MS agar containing 100 μM ferrozine or 10 mM CaCO₃ (pH 8.0). Plants were illuminated at 200 μmol·quanta·m⁻²·s⁻¹ and 25°C to provide a 14-h photoperiod. For the hydroponic assays 4-week-old seedlings were grown in Hoagland broth supplemented with either 30 μM FeSO₄-EDTA or 10 mM CaCO₃ (pH 8.0) and grown at 25°C and 400 μmol·quanta·m⁻²·s⁻¹ with a 14-h photoperiod. The nutrient solution was changed every day to prevent evaporation and alterations in pH. For the soil-based experiments, lots of 100 seeds were germinated and grown on soil, watered daily with 10 mM CaCO₃ (pH 9.0). Pigments, proteins, metabolites, and photosynthetic parameters were determined in the two youngest fully expanded leaves from plants at the same developmental stage. Transcript levels and Fe contents were determined in whole roots or aerial parts.

RT-PCR Analysis.

Total RNA was extracted from 1 g of plant material with the TRIzol reagent (Invitrogen, Paisley, U.K.). Synthesis of cDNA was carried out on 1 μg of RNA by using the SuperScript III RT kit and oligo(dT)_12–18 primers of Invitrogen, as indicated in the instruction manual. Semiquantitative RT-PCR analysis was performed as described (18), with the optimal number of cycles determined for each gene to analyze expression in the exponential phase. Primers were designed to encode highly conserved regions of the IRT1, FRO1, and NRAMP1 genes (15–18). Oligonucleotides and cycle numbers used are given in SI Table 5. Amplification products were analyzed by electrophoresis on 1% agarose gels and stained with ethidium bromide.

Analytical Procedures.

Chlorophyll was determined spectrophotometrically (34). For estimation of Fe contents, 4-week-old plants grown in MS agar were exposed or not to Fe deficit (10 mM CaCO₃, pH 8.0) for an additional 4 weeks. Iron was determined by atomic absorption spectrometry after mineralization of the samples (7). To measure the contents of specific proteins, extracts were prepared from young leaves of each plant, resolved by SDS/PAGE on 15% polyacrylamide gels, blotted onto nylon membranes, and decorated with the corresponding polyclonal antisera raised in rabbits.

Metabolite Measurements.

Soluble sugars were determined as described by Chen et al. (35). To measure the levels of phosphorylated metabolites, two frozen leaf discs were homogenized in liquid nitrogen followed by addition of 1 ml of a 1:1 methanol:chloroform solution. Samples were mixed thoroughly and centrifuged for 5 min at 18,000 × g. Supernatants were transferred to new tubes, dried, resuspended in 0.2 ml of water, and filtered through microtiter plate filter (microcon 10 kDa pore size; Millipore, Billerica, MA). The collected filtrate was used for ion chromatography coupled to mass spectrometry analysis according to published procedures (35). Soluble amino acids were determined as described (36). Numerical analysis was carried out by using Empower Pro software (Waters, Milford, MA).

Activity Determinations.

Light-dependent CO₂ assimilation and other photosynthetic parameters were measured according to Chen et al. (35). Chlorophyll a fluorescence measurements were carried out at 25°C on dark-adapted leaves of the same plants by employing a pulse-modulated fluorometer (Qubit Systems, Kingston, Canada). 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·quanta·m⁻²·s⁻¹. Photosynthetic parameters (F_v/F_m, φ_PSII, and nonphotochemical quenching) were determined and calculated according to Baker and Rosenqvist (37). Samples used for the estimation of the in vivo activities of Fru-1,6-bisphosphatase and PRK were prepared and assayed as described before (26).

Abbreviations

Fd

ferredoxin

Fld

flavodoxin

MS

Murashige–Skoog

Trx

thioredoxin

PRK

phosphoribulokinase.