Molecular Plant Advance Access published online on June 19, 2009
Molecular Plant, doi:10.1093/mp/ssp041
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Post-Translational Regulation of AtFER2 Ferritin in Response to Intracellular Iron Trafficking during Fruit Development in Arabidopsis
a Biochimie et Physiologie Moléculaire des Plantes (B&PMP), Unité Mixte de Recherche, CNRS, INRA, Université Montpellier 2, SupAgro, Place Viala, Bat. 7, F-34060 Montpellier, France
b Department of Biological Sciences, Dartmouth College, 6044 Gilman Hall, Hanover, NH 03755, USA
c Institut des Sciences du Végétal, CNRS, Bat 23, Avenue de la Terrasse, 91198 Gif-sur-Yvette, France
1 To whom correspondence should be addressed. E-mail gaymard{at}supagro.inra.fr.
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Abstract |
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 TOP Abstract INTRODUCTIONRESULTSDISCUSSIONMETHODSSUPPLEMENTARY DATAFUNDING |
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Ferritins are major players in plant iron homeostasis. Surprisingly, their overexpression in transgenic plants led only to a moderate increase in seed iron content, suggesting the existence of control checkpoints for iron loading and storage in seeds. This work reports the identification of two of these checkpoints. First, measurement of seed metal content during fruit development in Arabidopsis thaliana reveals a similar dynamic of loading for Fe, Mn, Cu, and Zn. The step controlling metal loading into the seed occurs by the regulation of transport from the hull to the seed. Second, metal loading and ferritin abundance were monitored in different genetic backgrounds affected in vacuolar iron transport (AtVIT1, AtNRAMP3, AtNRAMP4) or plastid iron storage (AtFER1 to 4). This approach revealed (1) a post-translational regulation of ferritin accumulation in seeds, and (2) that ferritin stability depends on the balance of iron allocation between vacuoles and plastids. Thus, the success of ferritin overexpression strategies for iron biofortification, a promising approach to reduce iron-deficiency anemia in developing countries, would strongly benefit from the identification and engineering of mechanisms enabling the translocation of high amounts of iron into seed plastids.
Key Words: Nutrient and metal transport • iron transport • ferritin • post-transcriptional control • biofortification • Arabidopsis
Received for publication March 20, 2009. Accepted for publication May 19, 2009.
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INTRODUCTION |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSSUPPLEMENTARY DATAFUNDING |
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Essential micronutrients for humans can all be supplied by an appropriate diet. However, micronutrient malnutrition affects more than half of the world population. In particular, populations with cereals-based diets often lack trace elements (Combs, 2001; Kennedy, 2003). Iron deficiency is considered as the most serious health constraint worldwide (WHO, www.who.int/en/). Recently, a new approach to cope with mineral malnutrition in the human population has emerged, and is termed bio-fortification (Graham et al., 2001; Bouis, 2003). It consists of increasing the bio-available content of nutrients in edible crops through genetic or transgenic strategies. The main target for such biofortification strategies is seeds, because they represent the basis of the diet of a majority of human beings around the world.
Although iron is very abundant in soil, its bio-availability for plants, and consequently to humans, is low. Thus, the biofortification strategies should coordinately enhance iron uptake from the roots, its transport and storage to edible parts, in a manner that would not alter the fertility and the yield production of the plants (Briat et al., 1999). Seed mineral content is generally very low; however, seed represents the basis of staple food. As metal content in seeds is a critical parameter of their nutritional quality, enhancing the micronutrient content of crop grains, such as rice, maize, or wheat, is one of the major objectives of this field of research.
Initial attempts to increase iron content in seeds have focused on overexpression of a single gene in order to increase iron uptake or storage (Goto et al., 1999; Vasconcelos, 2003; Ramesh et al., 2004; Vasconcelos et al., 2006). Increasing the ferritin content of seeds is currently considered as one of the most promising approaches to improve the iron content of staple foods (Theil et al., 1997). Iron contained in ferritins is highly bioavailable during digestion, which represents a basic requirement for the successful implementation of dietary improvement (Murray-Kolb et al., 2003; Davila-Hicks et al., 2004; Lonnerdal et al., 2006; Hoppler et al., 2008). Overexpressing ferritin under the control of the cauliflower 35S promoter led to the increase of iron content in leaves, but did not in seeds (Van Wuytswinkel et al., 1999; Drakakaki et al., 2000). An alternative strategy was to overexpress ferritin under the control of a strong, seed-specific chimeric promoter. This approach increased transgenic seed iron content by 1.5–3-fold (Goto et al., 1999). These relatively moderate gains in iron content reflect the complexity of iron translocation to the seeds. In other words, increasing the iron sink capacity of seeds is not sufficient to fill up this sink.
Ferritin function in the physiology of plants has been recently documented (Ravet et al., 2009). It appears more related to the protection against free iron toxicity than to the establishment of a reserve pool through its storage capacity. Indeed, the vacuolar compartment emerged as the major iron source in seeds, and the metal transporters VIT1, NRAMP3, and NRAMP4 have been shown to play key roles in this function (Lanquar et al., 2005; Kim et al., 2006). A. thaliana VIT1 encodes a vacuolar transporter involved in iron influx. In the vit1 mutant seeds, iron is misallocated. Whereas iron is localized close to the vascular tissues in the wild-type, iron is detected in the peripheral tissues of the seed in vit1 mutant. Thus, an impairment in vacuolar iron loading alters the distribution of iron in the seed (Kim et al., 2006). AtNRAMP3 and AtNRAMP4 are also vacuolar iron transporters expressed in seed, which are responsible for iron remobilization from the vacuole during the germination (Lanquar et al., 2005). However, the use of ferritins as transgenic targets for biofortification remains pertinent because of its biochemical ability to sequester thousands of iron atoms in plastids in a safe and bioavailable form. It has become obvious that the success of iron biofortification relies on the knowledge of the mechanisms involved: (1) in the unloading of this metal into the seeds, and (2) in the cross-talks integrating these unloading transport mechanisms to the intracellular storage processes occurring downstream. In such a context, a recent study reports the whole plant mineral partitioning (Waters and Grusak, 2008). It highlighted the necessity to identify processes involved in seed filling, which undoubtedly represent the limitation step for seed iron content improvement.
The present work aims to document some of the molecular and physiological events involved in metals and, more specifically, iron loading into A. thaliana fruits and seeds, during the various stages of their development. This was achieved by measuring seed metals from wild-type Arabidopsis and ferritin mutant plants. The potential cross-talk between the vacuolar and plastidial seed compartments for iron store allocation has also been studied in various genetic backgrounds affecting iron homeostasis of both compartments (fer, nramp, and vit knockout mutants, and NRAMP and VIT overexpressors). This approach showed that ferritin stability depends on a proper allocation of iron from vacuole to plastid. Consequently, it can be concluded that the success of ferritin overexpression strategies for iron biofortification would be highly dependent on the control of the mechanisms enabling the translocation of high amounts of iron into seed plastids.
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RESULTS |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSSUPPLEMENTARY DATAFUNDING |
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Expression of Ferritin during Fruit Development in Various Genetic Backgrounds
In order to analyze ferritin expression and its potential involvement in iron loading during fruit and seed development in Arabidopsis thaliana, an experimental procedure was set up to sample fruit and seed from nine different development stages (Figure 1A and Methods section). Fruit growth was monitored by weighting the organs after desiccation (i.e. flower or silique). From flower formation to the initiation of silique elongation, only a slight increase in the fruit dry weight was observed and fruit dry weight increased rapidly and reached its maximum in mature siliques (Figure 1B). This step can be defined as the active growth phase of the fruit development (stages 3–6). Then, no change in dry weight was observed during the three last stages (Figure 1B). The formation of the fruit is also characterized by desiccation events leading to the proper maturation of dry seeds. In order to study the kinetics of water loss during this developmental process, the water content of the fruit during the whole period was considered (Figure 1C). At the beginning of the growth period (stages 3 and 4), fruit water content stayed above 80%. The desiccation of the fruit occurred gradually from the elongation of the fruit (stage 4 in Figure 1C). This result showed that fruit water loss started before completion of fruit growth and extended during all the growth period (compare Figure 1B and 1C). This result is consistent with the study describing seed development in A. thaliana Wassileskija ecotype (Baud et al., 2002).
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Ferritin abundance was determined for the various stages of the fruit development described above, by Western blot analysis using antibodies raised against recombinant AtFER1 protein (Dellagi et al., 2005). This antibody recognized the four different ferritin subunits (Ravet et al., 2009). In order to assign the various ferritin polypeptides detected during fruit development to each of the four Arabidopsis ferritin genes, ferritin abundance was determined both in wild-type plants and in various ferritin mutant plants. Samples were collected from Col, fer2, fer1-3-4, and fer1-2-3-4 mutant plants grown on soil without Fe supplementation.
Results of the immunoblots presented in Figure 2 indicated that two polypeptides of, respectively, 26.5 and 28 kDa were detected. The evidence for various ferritin subunits has been reported in the literature for different plant species including Arabidopsis (Laulhère et al., 1989; Lobréaux and Briat, 1991; Masuda et al., 2001; Ravet et al., 2009). In Arabidopsis flowers, the 28-kDa polypeptide can be mainly attributed to AtFER1 and the 26.5-kDa protein to AtFER3 and AtFER4 (Supplemental Figure 1). In dry seeds, only one 26.5-kDa isoform was detected that corresponded to AtFER2 (Ravet et al., 2009; Figure 2).
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During fruit development, the pattern of ferritin protein accumulation appears very dynamic (Figure 2). Ferritins were abundant in both unfertilized and fertilized flowers, and during the first steps of the fruit elongation. During these steps, no ferritin subunits were detected in the fer1-3-4 mutant, indicating that the three isoforms AtFER1, AtFER3, and AtFER4 are highly expressed in flowers and young siliques. When the siliques initiated their growth phase (Figure 1B), ferritin protein abundance decreased. Among the three subunits expressed, AtFER1 (28-kDa isoform) exhibited the faster decrease (Figure 2A). During active growth phase, no ferritin was detected. During fruit desiccation, ferritin was detected when siliques had lost more than 50% of their water content. This step corresponds to the yellowing of the fruit. At this fruit developmental stage, ferritin was detected in the fer1-3-4 triple mutant but not in the fer2 mutant. Moreover, no ferritin was detected in the quadruple fer1-2-3-4 mutant plant. This result demonstrates that the ferritin that accumulates during silique desiccation corresponds to AtFER2.
Loading and unloading of iron into and from the vacuole have been reported as important determinants of the control of iron homeostasis in Arabidopsis thaliana seeds. AtVIT1 is a vacuolar iron influxer expressed in seed (Kim et al., 2006). The corresponding mutation leads to the mislocalization of Fe in this organ. AtNRAMP3 and AtNRAMP4 are vacuolar iron effluxers. They permit Fe remobilization from the vacuole during germination (Lanquar et al., 2005). These mutants are affected in Fe shuttling between the cytoplasm and the vacuole, potentially leading to major alterations in Fe localization and bioavailability. To investigate whether alterations in Fe transport across the vacuolar membrane influence ferritin synthesis during fruit development, we measured ferritin abundance at the various stages of fruit development in the vit1 and nramp3-4 mutants, comparatively to wild-type plants.
Col and WS fruits exhibited similar ferritin accumulation patterns (Figure 2). Ferritin accumulation in the vit1 fruit mutant was not altered compared to the control. In contrast, the nramp3-4 mutant accumulated less AtFER2 protein than the wild-type (Figure 2, lanes 7, 8, 9). However, AtFER1, 3 and 4 ferritin amounts were unchanged during the early fruit developmental stages, regardless of the genetic background. The decreased seed ferritin abundance observed in the nramp3-4 mutant is due neither to protein loading variations nor to a general impairment of seed protein pattern, as attested by a similar polypeptide profile of seed protein from Col or nramp3-4 stained with Coomassie blue (Supplemental Figure 2).
In summary, ferritin synthesis is controlled during the nine stages analyzed. Three distinct phases can be distinguished (Figure 2). In flowers and young siliques, AtFER1, AtFER3, and AtFER4 accumulated together (Supplemental Figure 1). Then, the abundance of these subunits decreased during fruit development, until no ferritin was detected during the fruit maturation phase. Finally, AtFER2 accumulated in the fruit when silique desiccation was initiated, and reached a maximum in dry seeds. AtFER2 accumulation at the late developmental stages of the fruit requires a functional vacuolar iron efflux transport system in the seed, since mutations in the AtNramp3 and AtNramp4 genes lead to a decrease in the accumulation of the seed-specific AtFER2 plastid ferritin isoform (Figure 2).
Metal Contents in Fruit during Its Development
Metal loading into fruits is a very dynamic process, which may result from both remobilization from older parts of the plant and direct uptake by the roots from the soil (Waters and Grusak, 2008). The alteration in AtFER2 accumulation in nramp3-4 (Figure 2) could be explained by the requirement for iron vacuolar sequestration during metal loading events. To address this point, we quantified the Fe content and concentration during the development of the fruit in various genetic backgrounds (Col and vacuolar iron transport mutants). Other metals (Mn, Cu, and Zn) were also quantified.
The changes in the content of various metals were very similar until the end of the growth phase (Figure 3). A slight increase in metal content was measured during the initiation steps of the fruit development. Then, a huge increase in all metals occurred during the growth phase of fruit development. During the maturation phase, the metal content remained the same. To determine the intensity of the metal fluxes during fruit development, we calculated the ratio between metal contents in the fruits between two successive stages (Figure 4). The influx into the organs was very similar for the different metals. This analysis indicated that a massive influx of metals into the flower occurs after fertilization. During initiation of silique elongation, no influx was detected. Then, coordinated with the fruit growth period, metal influxes increased to reach a maximum before complete silique elongation. During maturation of the fruit, metal influxes declined and finally stopped.
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As shown in Figure 3 and Supplemental Figure 2, Mn, Fe, Zn content and concentrations (respectively 91, 50, 77 µg g–1 DW) were quite similar in dry seed, whereas Mn was very abundant compared to Fe and Zn in dry siliques (respectively, 325, 52, 88 µg g–1 DW). The efficiencies of metal loading into the seed were calculated as the percentage of metal translocated from entire desiccated siliques to the seeds (Figure 5). Whereas Fe, Zn, and Cu were efficiently allocated to the seed, Mn was not, with only 28% translocated to the seed. The same results were obtained by quantifying metal contents separately in hulls and seeds (not shown). Thus, whereas the loading of the different metals seems to be co-regulated in the fruit (Figure 4), it is not the case of the loading into the seed (Figure 5).
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To test whether alterations in intracellular metal distribution could result in an alteration of seed metal content, we measured the kinetics of metal accumulation in fruits from the vit1 and nramp3-4 mutants as presented in Figure 3 and Supplemental Figure 2 with Col. Under our conditions, no significant differences were observed in fruit and seed metal contents between the different genotypes analyzed, at any of the developmental stages considered (data not shown). This result is consistent with data showing that the metal content was unaffected in vit1 or in nramp3-4 dry seeds compared to wild-type seeds (Lanquar et al., 2005; Kim et al., 2006).
Iron regulates at the transcriptional level several genes including ferritins (Gaymard et al., 1996; Petit et al., 2001). It has been shown that the increase in iron accumulation in tissues is correlated with the increase in ferritin abundance (Ravet et al., 2009). In this study, during the stage of metal loading of the fruit (Figure 3), ferritins were not accumulated (Figure 2). Moreover, while the metal loading into the fruit and/or the seed of the nramp3-4 line is unchanged when compared to wild-type organs (data not shown), a decrease in AtFER2 ferritin abundance in seeds of this mutant was observed (Figure 2). These results indicate that there is no obvious correlation between the amount of iron and the amount of ferritins in fruits and seeds, raising the question of the level of regulation leading to this decrease in AtFER2 abundance in the nramp3-4 mutant.
AtFER2 Post-Transcriptional Regulation Revealed by Expression Analysis in the nramp3-4 Mutant Affected in Seed Iron Homeostasis
The pattern of ferritin accumulation during fruit growth (Figure 2) is not correlated with the pattern of metal loading (Figures 3 and Supplemental Figure 3). However, the developmental regulation of AtFER2 abundance during fruit development requires a functional vacuolar iron export system, as evidenced by a lower accumulation of AtFER2 protein in the nramp3-4 genetic background (Figure 2). Ferritin synthesis in Arabidopsis is mainly regulated at the transcriptional level. AtFer1, AtFer3, and AtFer4 mRNA abundance increase in response to iron treatment (Petit et al., 2001; Arnaud et al., 2006). To determine whether the decrease in AtFER2 protein amount was due to an altered transcript accumulation in nramp3-4 mutant, a time course for AtFer2 mRNA profiling was determined at the various fruit and seed developmental stages of this mutant, comparatively to wild-type Col plants (Figure 6A). AtFer2 mRNA abundance was not affected in nramp3-4 fruits and seeds when compared to wild-type, whatever the developmental stage considered. Furthermore, this result was confirmed by using a second Arabidopsis ecotype (WS), for which no difference in AtFer2 transcript abundance was observed between wild-type or nramp3-4 dry seeds (Figure 6B). The decrease in AtFER2 abundance in nramp3-4 mutant seeds originates therefore from a post-transcriptional regulation. Ferritin stability has been analyzed mainly in animals, using in vitro approaches, and it has been shown that the content of Fe stored in ferritin could modulate the protein stability (Theil and Hase, 1993). In plants, in vitro iron-exchange experiments revealed that a degradation of the protein occurs in response to iron release (Laulhere and Briat, 1993). Thus, it can be hypothesized that the lack of iron efflux from the vacuoles via the AtNRAMP3 and AtNRAMP4 transporters could lead to depletion of the plastid iron pool available for ferritin loading.
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In order to test this hypothesis, we attempted to alter Fe vacuolar transport, both for influx and efflux, by ectopically overexpressing the two vacuolar transporters, AtNRAMP4 and AtVIT1. Ferritin abundance was determined in dry seeds of Col, WS, 35S::Vit1#10 and #17, 35S::Nramp3#1 and #2 and 35S::NRamp4#1 and #2 (Figure 7). Whereas ferritin accumulation in seeds was similar in the various AtNRAMP3 and AtNRAMP4 overexpressor lines compared to Col, ferritin abundance was strongly decreased in the two 35S::VIT1 lines. These variations are not due to differences in protein loading of the various samples, as controlled by Coomassie blue staining (Figure 7). Together with the decreased ferritin level in the nramp3-4 mutant, this result supports the hypothesis that increased iron sequestration into the vacuole leads to a decrease in ferritin abundance in the plastids. Such a hypothesis is consistent with a tight regulation of the iron shuttling between the different sub-cellular compartments via a major vacuolar checkpoint. Perturbation of both influx and efflux from the vacuole is detrimental for the control of ferritin abundance, suggesting that vacuolar and plastidial iron homeostasis are interrelated.
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Iron Shuttling between Vacuole and Plastid Implies Ferritin-Dependent Protection against Oxidative Stress
Ferritins in seeds do not constitute the major iron source for the seedling development, since their iron content represents less than 5% of the total iron seed content. Nevertheless, they are involved in the protection against oxidative stress during germination, since fer2 seeds exhibit hypersensitivity to methylviologen (MV) (Ravet et al., 2009). In order to analyze the physiological impact of the iron shuttling between the vacuole and the chloroplast, we have determined oxidative stress sensitivity of seeds in various Arabidopsis lines altered in iron vacuolar transport and in ferritin expression. With the aim to increase iron efflux from the vacuole in the presence or in the absence of ferritins, we engineered transgenic plants overexpressing AtNRAMP4 in both Col and fer2 backgrounds. In addition to the two independent homozygous lines overexpressing AtNRAMP4 in Col, two independent homozygous lines overexpressing AtNRAMP4 in fer2, named 35S::NRamp4#1 fer2 and 35S::NRamp4#2 fer2, were selected. Germination of seeds from these various lines was assayed in the presence or absence of MV (Figure 8). MV acts as a pro-oxidant molecule, by reacting with free Fe to produce ROS. In the absence of MV, seed germination kinetics were similar, whatever the genotype considered (Figure 8A). In the presence of MV in the medium, fer2 seeds exhibited a delay of germination compared to Col seeds (Figure 8B), in agreement with our previous observations (Ravet et al., 2009). When germination rates of the two 35S::NRamp4 Col lines were compared to those of the 35S::NRamp4 fer2 lines, this delay was strongly increased (Figure 8C). This result showed that overexpression of AtNRAMP4 exacerbated the sensitivity to MV in ferritin-depleted seeds.
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These data suggest that increasing iron efflux from the vacuole in a genetic background lacking ferritin increases seed sensitivity to oxidative stress. Thus, ferritins play a role in the protection of seeds against oxidative stress, which can potentially occur during the dynamic iron shuttling between organelles during seed formation.
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DISCUSSION |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSSUPPLEMENTARY DATAFUNDING |
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One of the limitations for seed biofortification is the lack of knowledge of how minerals are loaded into seeds. A recent physiological study has described the partitioning of metals between vegetative parts and fruits in various Arabidopsis thaliana ecotypes (Waters and Grusak, 2008). Such basic physiological knowledge appears very informative in order to identify important developmental steps, which could be targeted in order to enrich the metal content of seeds. In this study, we have quantified ferritin composition and abundance, and metal contents and concentrations, in order to establish references of physiological parameters during fruit development, from unfertilized flowers to dry seeds in Arabidopsis thaliana. By manipulating the expression of the vacuolar iron transport systems AtVIT1, AtNRAMP3, and AtNRAMP4, we have shown that the control of iron fluxes between seed vacuoles and plastids is an important determinant of iron homeostasis in seeds. Disturbing these fluxes revealed a post-transcriptional control of the AtFer2 seed-specific ferritin gene and highlighted the important function of ferritins in protection against oxidative stress in seeds.
Metal Loading into the Fruit at Various Stages of Its Development
All the parameters analyzed exhibited highly dynamic variations during fruit development. Fruit development is characterized by gain of dry matter that corresponds to the fruit growth, and loss of water that corresponds to the desiccation events leading to dry seed maturation. We found that the kinetics of metal uptake were very similar, independently of the metal being considered (Figure 4). This suggests that (1) either the transporters involved in their allocation to the fruit are co-regulated, or (2) these metals are transported by the same mechanisms, independently of their nature. Metal uptake into the fruit occurred mainly during its active growth phase, from elongation of the fruit to its maturation—a period corresponding to the beginning of water loss (Figures 1, 3, and 4). Our work also indicates a control checkpoint of the specificity of metal transport from hulls into the seed. Whereas Fe, Zn, and Cu are efficiently loaded into the seeds, an important proportion of Mn remains in hull tissues (Figure 5). Mn is an essential metal, but can react with oxygen to produce ROS. Its content is high in fruit (Figure 3). The control of its accumulation in the seed is therefore likely a protection against potential toxicity of this metal. Taken together, these results indicate that there is no specificity for metal loading into the fruit, and that the step controlling metal loading into the seed occurs most likely by the regulation of hull to seed metal transport. The success of biofortification strategies is undoubtedly highly dependent on the identification of the transport systems involved in metal allocation into the seeds. Concerning iron, although it is very efficiently transported into the seeds under our conditions, Fe loading may constitute a limiting step, and would deserve to be improved through transgenic strategies (Waters and Grusak, 2008).
Ferritin Expression Pattern during Fruit Development
The pattern of ferritin abundance fluctuated during fruit development. Ferritins were present at high levels in flowers and in seeds, whereas they were undetectable during fruit growth (Figure 2). Ferritins in flowers have been shown to protect them against potential iron overload (Ravet et al., 2009). Surprisingly, the absence of ferritin correlated with the phase of maximum Fe loading into the fruit. The physiological significance of such an absence of ferritins during fruit active growth (Figures 1 and 2) will certainly need to be considered in future biofortification approaches, because overexpressing ferritins during this period may be detrimental.
Seed Intracellular Iron Trafficking and Ferritin Post-Transcriptional Regulation
Our work clearly shows that alteration of iron transport into the seed vacuolar compartment leads to a decrease in AtFER2 ferritin abundance in seeds (Figure 2). This observation is restricted to the nramp3-4 mutant and to the AtVit1 overexpressor genetic backgrounds. A decrease in ferritin abundance does not occur in the vit1 mutant (Figures 2 and 7), nor in the AtNRAMP3 or AtNRAMP4 overexpressors (Figure 7). This decrease in AtFER2 protein abundance is not correlated with any changes in AtFer2 mRNA abundance (Figure 6). This result reveals a translational or post-translational regulation of the AtFER2 ferritin, related to the control of seed vacuole iron homeostasis. The most direct explanation for these observations is that iron accumulation in the seed plastids controls ferritin protein stability. Such ferritin stabilization by iron has been reported previously, in mammals and in plants, by in vitro approaches. Iron exchange experiments have shown that iron release from ferritins mediated by reducing agents leads to its rapid degradation, suggesting that iron stabilizes the protein (Laulhere and Briat, 1993; Theil and Hase, 1993). Moreover, this observation has also been seen in vivo in the maize ys1 mutant, which is deficient in root iron uptake (Fobis-Loisy et al., 1996). Although the ferritin mRNA content in leaves of ys1 is similar to that of the wild-type plants, ferritin protein abundance is strongly decreased in the mutant. It has also been reported that ferritin mRNA abundance is almost the same in plants treated with an excess of iron or with exogenous ABA; however, ferritin protein abundance only increased in response to the iron treatment, and not to the ABA treatment (Lobréaux et al., 1993). These data and the present report suggest that ferritin is stabilized by the formation of the iron core into the protein shell. Thus, alteration of iron efflux from the vacuole probably leads to a depletion of iron into the plastid and consequently leads to protein destabilization.
Ferritin accumulation in seeds is likely dependent on cross-talk between vacuoles and plastids, requiring a preliminary vacuolar efflux prior to safe iron transport into plastids. Such a model fits well with the function of the AtNRAMP3 and AtNRAMP4 transporters evidenced in this work, establishing that they are not only required for iron mobilization during germination (Lanquar et al., 2005), but that this function is also important in the control of iron homeostasis in developing seeds.
In conclusion, this study lends further support for the necessity of obtaining an in-depth knowledge of the various parameters controlling iron loading and storage in fruits and seeds, in order to use it for improving biofortification strategies. For example, the use of ferritin as a biotechnology target to enrich seeds with iron has so far met with limited success, since the observed increase in seed iron was never higher than 1.5–3-fold (Goto et al., 1999; Qu et al., 2005). Indeed, despite the ability to produce high steady-state levels of ferritin mRNA in transgenic seeds, very little ferritin protein was produced in these plants (Van Wuytswinkel et al., 1999). This observation can be explained by our result showing a post-transcriptional regulation of the seed AtFER2 protein during fruit maturation. This regulation is likely due to the requirement of sufficient plastid iron to stabilize the ferritin protein. Such a requirement is itself dependent on the iron fluxes in and out of seed vacuoles, an important sub-cellular compartment, essential to controlling seed iron trafficking during fruit development, and impacting on iron homeostasis in seed plastids. Success of seed iron biofortification through biotechnology approaches using ferritin will clearly require also managing the improvement of iron loading in seed plastids, where this protein is localized.
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METHODS |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSSUPPLEMENTARY DATAFUNDING |
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Plant Material, Growth Conditions, and Tissue Sampling
Plants were sown on soil (Humin substrate N2) in pots as single plants. Arabidopsis accessions used were Columbia (Col) and Wassileskija (WS) and different mutant lines— fer2, fer1-3-4, fer1-2-3-4, vit1, and nramp3-4#2 in the Col background, and nramp3-4#1 in the WS background.
fer2 and fer1-3-4 mutants have been previously described in Ravet et al. (2009). The quadruple mutant was obtained by crossing the triple mutant fer1-3-4 with the fer1-2-4 mutant. The homozygous fer1-2-3-4 mutant was selected by PCR using the specific primers described in Ravet et al. (2009).
vit1 and nramp3-4#1 mutants have been previously described (Kim et al., 2006; Lanquar et al., 2005). The mutants nramp3#2 (N_523049) and nramp4#2 (N_503737) in Col background were provided by the Salk Institute (www.salk.edu/). Homozygous mutants were selected by PCR screening using the following primers: NR3-F 5'-CTA CAT GTT GAT GAA CGC TCG-3', NR3-R 5'-GAG CAG TAA TAA CAA CAC CAG-3’; NR4-F 5'-GAC GAA GAA GAA GAC GCT GAT TAC-3’; NR4-R 5'-CCA GCA TTT GCA AGA CCG ATG GTG-3'. Homozygous single mutants were crossed to obtain the double mutant nramp3#2–nramp4#2. The homozygous double mutant was selected by PCR screening using the same primers as described above, and was named nramp3-4#2.
For AtNRAMP4 overexpression in Col and fer2 mutant, AtNRAMP4 cDNA was amplified using Advantage cDNA Taq polymerase (Clontech) with NR4-BamH1S 5'-CGG GAT CCA TGT CGG AGA CTG ATA G-3’ and NR4-SalR 5'-GTG TGC ACT CAC TCA TCA TCC CTC-3'. The products were cloned between the BamHI and SalI restriction sites in pFP101 (Bensmihen et al., 2004). The resulting plasmid pFP101–AtNRAMP4 was transferred into the GV3101 Agrobacterium strain (Koncz and Shell, 1986). The transformation of Col and fer2 was performed using the floral dip procedure (Clough and Bent, 1998). Primary transformants and homozygous seeds were selected on the basis of GFP fluorescence under an epifluorescence binocular microscope (Leica), with excitation between 450 and 490 nm and a long pass emission filter at 515 nm. Two overexpressing lines in each background were selected for further analysis.
For AtVIT1 overexpression, the coding region of AtVit1 was amplified from genomic DNA with primers VIT1forward 5'-AGA TCT ATG TCG TCG GAG GAA GAT AAG ATT AC-3’ and VIT1reverse 5'-CTG CTA ATG TTG CAC AAC TTT AGC CAA AC-3’ using ExTaq polymerase (Takara) and subcloned in Teasy vector (Promega). The plasmid was digested with EcoRI and treated with Klenow fragment (Promega). The linearized plasmid was digested with BglII and the AtVit1 insert was isolated. The AtVit1 fragment was cloned in pCAMBIA1302 plant expression vector using BglII and PmlI restriction sites. The resulting construct pCAMBIA1302–AtVit1 was introduced into the AGL1 Agrobacterium strain. The transformation of Col was performed using the floral dip procedure (Clough and Bent, 1998). T1 transformants were selected on B5 media (Sigma) containing 50 µg ml–1 hygromycin and transferred to soil. Segregation analysis of the hygromycin resistance was used to screen T2 plants with a single T-DNA insertion and T3 homozygous lines. Two overexpressing lines were selected for further study.
Plants were grown in an air-conditioned glasshouse under shadecloth, with supplemental lighting of a long-day condition (16 h light/8 h dark photoperiod). These conditions provided light at approximately 100 µmol m–2 s–1, with brief periods of up to 250 µmol m–2 s–1. Plants were irrigated as needed (usually once a week) with water.
Flowers, fruits, and seeds were collected as described in Figure 1A. Flowers were harvested both before (stage 1) and after fertilization (stage 2). Silique development was analyzed as six successive stages: fruit initiation (stage 3), fruit elongation (stage 4), fruit maturation (stage 5), mature fruit (stage 6), fruit desiccation (stage 7), and desiccated fruit (stage 8). Dry seeds (stage 9) were harvested after total desiccation of the siliques. The developmental stages described above were defined on the basis of morphological characters. Silique elongation, silique maturation, and the silique mature stages corresponded to the elongation steps of the fruit. At stage 3, fruits measured less than 1/5 of their final length, at stage 4 their size was 
of their final length, and at stage 5 they reached their final length. Stage 6 corresponded to completely developed fruits. The yellowing of the fruit defined in stage 7 and stage 8 was represented by the fully browned silique. The organs were collected at the same time for the different genotypes analyzed.
Plant tissues were dried in an 80°C oven during 48 h prior to determining dry weight. Plant tissues corresponding to the same developmental stage were either pooled and grounded in liquid nitrogen in order to estimate the metal or metabolite concentrations measured or studied as an entire organ in order to obtain the organ contents in these compounds.
Metal Content Measurements
Samples ground in liquid nitrogen were digested in 1 ml nitric acid (Merck) and diluted in 10 ml of de-ionized water. One milliliter of this sample was then diluted to 9.65 ml in de-ionized water. 300 µl of pure nitric acid and 50 ml of indium solution were added in the mineralization solution. Indium was used as an internal standard. Metal contents were determined by ICP–MS.
Measurement of total iron content was monitored as previously described (Lobréaux and Briat, 1991). Samples (10–20 mg DW) were digested according to Beinert's procedure (Beinert, 1978). Iron concentration was measured by absorbance of Fe2+–BPDS (1%) at 535 nm, using thioglycolic acid as a reducing agent. Determination of iron content was carried out using a range of standard Fe solutions (Carlo Erba).
The relative influx of metals between two successive stages was calculated as the ratio between the metal content in stage (n + 1) and that in stage (n).
The efficiency of metal translocation into the seed was calculated as the percentage of the metal content measured in the whole desiccated fruit that is then detected in dry seeds.
Quantitative RT–PCR
Total RNA from dry seeds was extracted using the Spectrum Total Plant RNA kit, according to the manufacturer's recommendations. cDNA synthesis, quality control, and quantitative RT–PCR were performed in a Light-Cycler (Roche Diagnostics) according to Girin et al. (2007). PCR amplification of AtFer2 was performed with the 3’ UTR gene-specific primers, F2-F 5'-AGA ACA ATG ATG TTC AGC TGG-3’ and F2-R 5'-AGA AGC TGA ACT CTC CTT CC-3'. Quantitative RT–PCR was analyzed using Light-Cycler 3 data-analysis software (Roche). Relative transcript levels (RTL) were calculated using the difference between the crossing time of the target gene and the threshold crossing time of the control gene for the respective templates, thus normalizing the target gene expression to the control gene expression (Arrivault et al., 2006). Gene expression was monitored in three independent biological experiments, and results were standardized using SAND gene (At2g28390) (Czechowski et al., 2005; Remans et al., 2008).
Immunodetection of Ferritins
Total protein extracts were prepared from samples as previously described (Lobréaux et al., 1992). Immunodetection of ferritin was performed using a rabbit polyclonal antiserum raised against purified AtFER1 protein (Dellagi et al., 2005). To monitor ferritin content by Western blot in the different mutants used in this study, the specificity of the serum directed against the AtFER1 subunit towards the four Arabidopsis ferritin subunits was determined. The four recombinant ferritin subunits were produced in E. coli, and the serum was used in Western blots to detect serial dilutions of the four purified recombinant proteins. Qualitatively, it reacted with all four subunits. Quantitatively, the detection was equivalent for AtFER1, AtFER2, and AtFER3, but was about 10 times lower for AtFER4 (data not shown).
Germination Assays
All the seed pools used for germination analysis were obtained from 10 plants grown under the same conditions and co-harvested. Seeds were first stratified for 1 week in a controlled storage closet at 4°C with a relative humidity of 30% under darkness. Then, seeds were sown on de-ionized water agar medium (6 g L–1) with or without 6 µM methylviologen (MV, Sigma Aldrich). Plates were placed at 4°C in the dark for 2 d, then transferred to a growth chamber at 23°C under long-day conditions (16 h light/8 h dark photoperiod) at 120 µmol m–2 s–1. Efficiency of germination was evaluated as radicle protrusion.
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSSUPPLEMENTARY DATAFUNDING |
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Supplementary Data are available at Molecular Plant Online.
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSSUPPLEMENTARY DATAFUNDING |
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This work was funded by the Institut National de la Recherche Agronomique and Centre National de la Recherche Scientifique, by the ANR-Blanche DISTRIMET No. 25383 from the Agence Nationale de la Recherche. The work of K.R. was supported by a thesis fellowship from the Ministère de l'Education Nationale, de l'Enseignement Supérieur et de la Recherche (France).
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Acknowledgements |
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We thank the Salk Institute Genomic Analysis Laboratory (SIGNAL) for providing the sequence-indexed Arabidopsis T-DNA insertion mutants and the Nottingham Arabidopsis Stock Centre (NASC) for providing seeds. We acknowledge Dr Jean-Luc Seidel (Laboratoire de Chimie des Eaux, CNRS Montpellier) for his help in ICP–MS-based metal measurements. No conflict of interest declared.
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