Physiologia Plantarum 126: 407417.

2006

Copyright Physiologia Plantarum 2006, ISSN 0031-9317

R E VI EW

Progress in the genetic understanding of plant iron and zinc nutrition

Artak Ghandilyana, Dick Vreugdenhilb and Mark G. M. Aartsa,*
a b

Laboratory of Genetics, Wageningen University, Arboretumlaan 4, 6703 BD Wageningen, the Netherlands Laboratory of Plant Physiology, Wageningen University, Arboretumlaan 4, 6703 BD Wageningen, the Netherlands

Correspondence *Corresponding author, e-mail: mark.aarts@wur.nl Received 30 June 2005; revised 3 November 2005 doi: 10.1111/j.1399-3054.2006.00646.x

In this review, we describe the need and progress to improve the iron and zinc contents in crop plants by genetic means. To achieve this goal either by transgenic approaches or classical breeding, knowledge about the physiological and molecular mechanisms of mineral uptake and mineral homeostasis will be very helpful. The progress in our understanding of the molecular processes and genes is described, and the use of the identified genes by transgenic approaches is illustrated. Genetic mapping of the existing variation will allow marker-assisted breeding to exploit the available natural variation in crop plants. For this application, ultimately the knowledge of the genes underlying this quantitative variation, called quantitative trait loci (QTL), will be required. It is expected that research in this field in the model species Arabidopsis thaliana, where the molecular tools are available, might help in the identification of the allelic variation at QTL.

Introduction
Plant-derived foods contain a wide variety of vitamins, minerals and unique phytochemicals that may have beneficial or detrimental effects on human health and well-being. Besides a mixture of carbohydrates, lipids and proteins (amino acids), 17 mineral nutrients and 13 vitamins are essential components of the human diet (Grusak 1999). Deficiencies in bioavailable iron (Fe), zinc (Zn) and other essential cation minerals in human food, causing mineral malnutrition, affect a large proportion of the world population. Of all micronutrients which are essential for sustaining human health, Fe, Zn, iodine and vitamin A have been reported to be most at risk of malnutrition (Welch and Graham 2004). Key reasons for human mineral malnutrition

are the relatively low content of cation minerals in plantbased foods in combination with the abundance of antinutrient compounds that severelyreducetheir bioavailability. Even though essential minerals are generally sufficiently abundant in soils, most exist largely in forms that are not easily available to plants. To overcome this availability problem, plants have developed unique abilities to acquire minerals from various growth substrates, either or not in symbiosis with other rhizosphere organisms. To fulfil the mineral need and simultaneously avoid their possible toxic effects, plants have evolved a complex regulation network controlling mineral homeostasis, based on well-controlled uptake, translocation, redistribution and sequestration mechanisms. This network is

Abbreviations bHLH, basic helix-loop-helix; Ca, calcium; CaMV, cauliflower mosaic virus; Cd, cadmium; CDF, cation diffusion facilitator; EDX, energy dispersive X-ray; Fe, iron; G E, genotype by environment; HMA, heavy-metal ATPase; K, potassium; MAS, marker assisted selection; MG, magnesium; Mn, manganese; NA, nicotianamine; NAAT, nicotianamine aminotransferase; NAS, nicotianamine synthase; NRAMP, natural resistance associated macrophage protein; Ni, nickel; NIL, near isogenic line; PS, phytosiderophore; QTL, quantitative trait locus/loci; RIL, recombinant inbred line; WHO, World Health Organisation; YSL, yellow-stripe like; ZIP, ZRT/IRT-like protein; Zn, zinc.
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directed by many different genes. It includes various membrane transporters, reductive agents, specialized storage proteins, metal ligands with different substrate specificities and regulatory proteins such as transcription factors, protein kinases and receptors. At present, knowledge of the genes controlling specific steps of the cation mineral homeostasis network is still rudimentary, but increasing rapidly. Increased knowledge on metal homeostasis is an urgent step needed right now to be able to sufficiently understand plant mineral acquisition and storage. This knowledge is expected to efficiently improve crop yield, crop nutritional value and food safety, three items which are of major global concern. This review tries to address the current knowledge on metal homeostasis mainly related to the improvement of crop nutritional value by plant breeding and biotechnology.

Micronutrient malnutrition: Where are we and where should we go?

The Food and Agriculture Organization and the World Health Organization (WHO) have estimated the daily requirements of the various micronutrients in the human diet. Individuals between 25 and 50 years of age require 1015 mg Fe. For Zn, people require between 12 and 15 mg (Welch and Graham 2004). Although these do not seem to be such high amounts, micronutrient deficiencies severely affect one in every three humans worldwide, afflicting all age groups and populations, but especially the poor, and woman and children. Fe deficiency is the most common nutritional disorder in the world, affecting possibly between 4 and 5 billion people, with more than 2 billion people, mainly in developing countries, actually being anaemic (WHO; http://www.who.int/nut/ida.htm). Anaemia is used as an indirect indicator of Fe deficiency, as most anaemic disorders are due to lack of Fe. Zn deficiency may be equally prevalent in the human population. On the basis of intake figures, billions of people are at risk of Zn deficiency. However, Zn deficiency is not easily distinguished, and hence no accurate estimates are available for the number of people who are actually Zn deficient. Humans fulfil their dietary needs through food originating from diverse sources, the major part being derived from higher plants and animals. With a relatively low intake of meat and a high intake of legumes and whole grains, which contain high amounts of antinutritional compounds, the prevalent use of plant-based diets explains the reduced dietary Fe and Zn absorption (Hunt 2002). Practical methods to improve mineral absorption in humans include consumption of absorption enhancers and special food preparation techniques, e.g. germination, fermentation,
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soaking and other processing (Gibson 2004), but these methods are not always appreciated. Although the previous efforts to encourage more diverse diets and to supply extra minerals through supplementation or food fortification are still in practice and of great importance, recently research efforts have shifted towards biofortification. This generally means to increase the concentration of mainly Fe and Zn in agronomically important crops through plant breeding (Gregorio 2002). Varieties, landraces and wild species are being explored for their mineral levels, and this knowledge will be used to create new varieties with increased mineral content. As an example, the average Fe concentration in beans is currently about 55 mg kg1, but varieties exceeding 100 mg kg1 have been described (House et al. 2002). The Consultive Group on International Agricultural Research recently launched their Harvest Plus Challenge programme of which a major objective is to obtain new varieties of beans, rice, maize, wheat, cassava and sweet potato with a doubled Fe concentration and a 40% increase in Zn concentration (HarvestPlus; http://www.harvestplus.org/). An increase in mineral content is not always effective to increase the bioavailable content in food due to the presence of antinutritional compounds in plants. Therefore, in addition to focusing on increased mineral content, a decrease in the content of antinutrients should be achieved at the same time. Unfortunately, there are many plant substances that inhibit the bioavailability of Fe and Zn to humans when present in food. These include phytic acid (myo-inositol-1,2,3,4,5,6-hexakisphosphate), fibres (e.g. cellulose), polyphenols (e.g. tannins), haemagglutinins (e.g. lectins) and micronutrient-like competitive heavy metals (e.g. Cd) (Graham et al. 2001, Hurrell 2004, Welch and Graham 2004). Genetically reducing the concentration of polyphenols and other antinutrients in crops will additionally improve the Fe and Zn bioavailability. Most effort so far has been on reducing the phytic acid content in plants. Phytic acid or the salt thereof (phytate) constitutes the major storage form of phosphorous in the seed needed for proper germination. It can bind strongly to divalent cations, even while in the digestive tract, and thus limit the bioavailability of these cations. For cereals and legumes, low-phytic acid mutations have been isolated and used to breed first-generation low-phytate genotypes of maize (Zea mays), barley (Hordeum vulgare), rice (Oryza sativa) and soybean (Glycine max). Seed phytic acid could be reduced in these crops by 5095% (Raboy 2002). An evaluation of the efficacy of biofortified foods for improving human nutrition and health requires laboratory-based trials on experimental animals and is then followed by
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community-based trials (King 2002). Human nutrition studies of tortillas made from either low-phytate or highphytate corn showed that the fractional absorption of Zn is positively correlated with the reduction in phytic acid content (Hambidge et al. 2004).

be determined, we can already indicate the activity of a series of genes along the tract of Fe or Zn from soil to the edible organs. Mineral uptake Plants first need to mobilize Fe and Zn from soil particles so they become available for uptake. Two types of plant species are distinguished based on the way they acquire Fe. Strategy I plants, i.e. dicots and non-graminaceous monocots, will go through morphological modifications under Fe deficiency, including enhanced development of lateral roots and differentiation of specialized transfer cells, and induce physiological responses including increased acidification of the rhizosphere and secretion of phenolics and organic acids to chelate Fe. Reduction of ferric to ferrous Fe on the root surface is an obligatory process for Fe acquisition from soil in strategy I plants (Yi and Guerinot 1996). This reduction is performed by an Fe-deficiency inducible, plasma membrane-bound Fe (III) reductase. In Arabidopsis, this is encoded by the AtFRO2 gene (Guerinot and Yi 1994). Like AtFRO2, also the expression of AtFRO3 is strongly induced upon Fe deficiency, which suggests that the latter gene has a similar function (Wu et al. 2005). Strategy II plants, which are graminaceous monocots such as rice, corn and wheat, use a chelation strategy for primary acquisition of Fe from the soil. These plants secrete phytosiderophores (PSs), which are compounds of the mugineic acid family, that form stable Fe (III) chelates in soil (Roberts et al. 2004). Transporters

Target crop species for mineral improvement

On the basis of worldwide consumption levels, rice, wheat, maize, cassava and beans are the most important staple crops. Genetic variation for Fe and Zn concentrations in accessions of common beans, wheat, rice and maize has been reviewed by Welch and Graham (2004). To be able to perform breeding for mineral content, the availability of genetic variation is essential. However, for efficient breeding, also knowledge about the genetics of the observed variation and insight into the genotype by environment (G x E) interaction are relevant. Recent developments in quantitative genetics making use of molecular markers allow the determination of the map positions and the relative contribution of the different loci to the observed trait variation. An important application of this knowledge is via marker-assisted breeding (MAS), where molecular markers linked to the loci determining variation for the trait can be used to select the most favourable genotypes without a need of determining mineral levels by relatively complex and expensive assays in all breeding generations. For MAS, ultimately the genes underlying the variation of the trait are the best suited markers. These genes can be best identified using the knowledge on metal homeostasis that is rapidly accumulating for model species such as Arabidopsis thaliana.

Key mineral homeostasis genes

Thus far, many mineral homeostasis genes were identified and further characterized by Arabidopsis-mutant screenings and yeast-mutant complementation studies. With the development of tools to analyse genome or transcriptome differences between plant species and between developmental or environmental states of plant organs or tissues, new molecular approaches are available to identify genes involved in mineral homeostasis. Currently, most of the transcriptome analyses have been performed for A. thaliana or closely related species, for which both custommade and commercial microarray platforms are available (Becher et al. 2004, Weber et al. 2004, Wintz et al. 2003, van de Mortel and Aarts, unpublished results). So far, genes involved in mineral uptake, cellular import and export and intracellular sequestration have been identified. Although the set of genes is far from complete and the functional relevance of many of the differentially expressed genes needs to
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Plants contain a wide range of cation transporters which have been extensively reviewed (Clemens et al. 2002, Hall and Williams 2003, Maser et al. 2001). For many of these transporters, their exact function is not known yet, although gradually more and more of their roles were revealed, especially for Arabidopsis. As far as Fe and Zn uptake and translocation are concerned, there are several identified transporters. ZRT/IRT-like protein (ZIP)like transporters (Guerinot 2000) are needed for uptake, such as AtIRT1 and AtIRT2 for Fe uptake into the roots (Connolly et al. 2002, Vert et al. 2001, Vert et al. 2002) and AtZIP1, AtZIP2, AtZIP3 and AtZIP4 for Zn uptake into the roots (Grotz et al. 1998). Strategy II plants take up Fe as a PS-metal chelate through Yellow Stripe-like (YSL) transporters (Roberts et al. 2004), but Zn appears to be taken up through ZIP-like transporters as in strategy I plants (Ramesh et al. 2003). Once in the roots, both minerals will be transferred to the xylem for further transport towards the above-ground organs. The heavy-metal
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ATPase (HMA) genes (AtHMA2, AtHMA3 and AtHMA4), which are members of the family of P1B ATPases (Axelsen and Palmgren 2001) and expressed in vascular tissue, appear to be the most likely candidates to perform this task at least for Zn (Eren and Arguello 2004, Hussain et al. 2004). For Fe, also the FRD3 gene plays a role (Green and Rogers 2004). Although a transport function has not been shown, the membranelocated FRD3 protein is expressed in root pericycle and vascular cylinder. In the absence of a functional FRD3 protein, the root to shoot Fe transport is severely inhibited. Fe and Zn are most likely chelated by nicotianamine (NA) during transport, not only in xylem, but especially in phloem transport (von Wiren et al. 1999). This is most evident in the NA synthase (NAS) mutant chloronerva of tomato, which shows an Fe-deficiency phenotype under normal growth conditions (Ling et al. 1999), and also in tobacco plants overexpressing the NA-converting enzyme NA aminotransferase (NAAT), which led to changes in distribution and content of metals including Fe and Zn. Surprisingly, overexpression of an NAS gene in tobacco, and thus raising the internal NA level, only caused marginal increase in Fe and Zn contents in tobacco leaves (Takahashi et al. 2003), clearly suggesting that NA is not the only factor limiting mineral translocation from the root. The NAmetal complexes are most probably transported across plasma membranes by YSL transporters. There are several YSL genes in Arabidopsis, but at least the Arabidopsis AtYSL2 gene (DiDonato et al. 2004) transports FeNA, most probably laterally through the plant veins. Not all the Fe and Zn taken up by roots are transported upwards. Some will remain in the roots to be used by root cells and probably also as storage in case demand from the above-ground parts increases. The minerals are then most probably stored in the vacuoles. The natural resistance associated macrophage protein (NRAMP) transporters, especially the orthologues of AtNRAMP3 (Thomine et al. 2003) and AtNRAMP4, are currently the most likely candidates to control the active (re)mobilization of metals to and from the vacuolar pool. In addition, there is a superfamily of cation diffusion facilitators (CDFs), of which AtMTP1 (also named ZAT) (van der Zaal et al. 1999) appears to be involved in storing Zn in the vacuole of predominantly leaf tissue (Desbrosses-Fonrouge et al. 2005, Kobae et al. 2004). Because CDFs mainly facilitate diffusion processes, also the function of AtMTP1 is most prominent under situations of high external and subsequently internal Zn levels. There are 12 CDFs in Arabidopsis, and for most of these their function is unresolved, but they are likely to differ in their metal specificity (e.g. Mn, Ni) and tissue-specific expression.
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Transcription factors Not much is known about the regulation of metal homeostasis by metal-responsive transcription factors. Although we know that many genes are differentially expressed in response to changes in Zn and Fe exposure (Becher et al. 2004, Colangelo and Guerinot 2004, Weber et al. 2004, Wintz et al. 2003, van de Mortel and Aarts, unpublished results), only one transcription factor controlling such response has been cloned. The FER gene of tomato encodes a basic helixloop-helix (bHLH) protein that is expressed at the root tip independently from Fe supply and is required for induction of Fe mobilization in tomato (Ling et al. 2002). The chlorotic fer mutant is not able to switch on strategy I responses upon Fe deficiency. FER acts upstream of the Fe uptake machinery encoded by LeIRT1 and LeFRO1, because these genes are also downregulated in the roots of the fer mutant (Li et al. 2004b, Ling et al. 2002). Recently, the FER-like regulator of Fe uptake gene in Arabidopsis was also cloned (respectively known as FIT1, FRU or AtbHLH29) (Colangelo and Guerinot 2004, Jakoby et al. 2004, Yuan et al. 2005). This gene can functionally complement the tomato fer mutant and appears to be the Arabidopsis orthologue of LeFER (Yuan et al. 2005). Overexpression of either LeFER or AtbHLH29 does not lead to a constitutive Fe uptake, indicating that additional regulatory mechanisms are active.

Mineral distribution
Although minerals are distributed over all tissues of the plants, there are locations that accumulate more than others. Zn, Fe and Mn are not equally partitioned among plant organs. We find that Zn and Fe contents are higher in roots compared to rosette leaves of 3-week-old Arabidopsis thaliana plants, whereas Mn contents are higher in leaves (A. Ghandilyan and M. Aarts, unpublished data). Little information is available on the percentage of the total amount of micronutrients in the plant that is ultimately stored in the seeds. For pea, 75% of total Fe is present in seed, whereas for rice it might be as low as 4% (Grusak 2000). Partitioning within the plant also depends on the growth stage of the crop (Akman and Kara 2003) and the position of the grain within the plant (Calderini and Ortiz-Monasterio 2003). When considering the seed, which after all is the predominant source of human dietary Fe and Zn from plants, one of the major differences between monocots and dicots is the presence of an aleurone layer covering the endosperm in the monocot seeds. The aleurone layer is generally relatively rich in micronutrients compared with the rest of the grain. In rice, these layers are
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largely polished off during processing, further reducing the already low overall content of micronutrients. However, there are differences between rice cultivars for loss of minerals during polishing (Gregorio et al. 2000); hence, selection on high mineral retaining genotypes should be possible. In order to further understand the spatial distribution of minerals in plant tissues, including grains, of different species, various combinations of X-ray analysis and electron microscopy techniques can be employed, such as energy dispersive X-ray (EDX) analysis (Lott and West 2001, Otegui et al. 2002) or extended X-ray absorption fine structure (Kupper et al. 2004). Using EDX, we determined preferential accumulation of various minerals in the outer layers of rice grains (A. van Aelst and D. Vreugdenhil, unpublished results). It will be very interesting to find out whether this is in any way correlated with the expression of any of the previously indicated metal homeostasis genes.

The use of natural variation to discover new genes affecting Fe and Zn contents
As became evident from mutant studies, a plethora of genes affect the accumulation of micronutrients and many may still be undetected. For plant breeding, knowledge of the genes underlying the natural variation for Fe and Zn contents will be essential for the application of molecular markers. A powerful forward genetic approach to unravel such polygenic phenomena is quantitative trait locus/loci (QTL) analysis. This analysis is based on the genetic variation which exists between accessions of one species and is generally performed on genetic segregating populations like F2s or recombinant inbred line (RIL) populations (Koornneef et al. 2004). QTL analysis provides knowledge on the chromosomal locations of the important loci without any prior knowledge on the genes involved and reveals their possible genetic effects leading to phenotypes of interest. It is an unbiased investigation of the genes affecting a certain trait, meaning that genes corresponding to structural as well as regulatory aspects of the process under investigation can be identified. The ability to identify, manipulate and potentially clone individual genes involved in quantitatively inherited characters, combined with the demonstrated conservation of numerous linkage blocks among members of plant families, e.g. in Graminae, emphasizes the contribution of map-based genetic analyses both to applied and to basic crop research (McCouch and Doerge 1995). After fine-mapping of the identified QTL intervals using near isogenic lines (NILs) or other introgression lines, which differ only by a small introgression of one genotype in the background of another, candidate genes can be assigned to the QTL once the genomic sequence of the fine-mapped locus has been identified. Knowledge of the
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complete genomic sequence of the target species is very useful at this stage, as is the availability of knock-out mutants to confirm candidate genes, but this is not essential. If the complete genomic sequence is not known, positional cloning is a subsequent strategy to obtain genomic DNA covering the QTL region. This requires the availability of a genomic library. Obviously, the QTL region should be fairly small (generally below 200 kb, but depending on the gene density) to limit the number of candidate genes. Final proof of cloning the gene underlying the QTL is generally provided by complementing the recessive genotype after transformation of the allele of the other genotype. Alternatively, obtaining a loss-of-function allele for a candidate gene with the same phenotype as one of the two parental genotypes, which also turns out to have a loss-offunction allele for the same gene, is equally sufficient. The feasibility of this strategy was first proven with the positional cloning of the EDI locus of A. thaliana, representing a novel allele of the CRY2 gene encoding the blue-light photoreceptor (El-Assal et al. 2001). QTL affecting the same or similar traits in different species may have a common origin. Using a comparative genomic approach, we can reveal conserved relationships of QTL affecting important agronomic traits in a plant species leading to possible syntenic relationships of QTL. This strategy can be a useful and feasible approach to map and clone QTL in crops with a large genome. For instance, because colinearity is strong between oilseed rape and Arabidopsis, rape genes may be cloned using Arabidopsis genome information (Gale and Devos 1998b). Evidence of large intergenomic colinearity among cereals stresses the strength of comparative gene cloning (Devos and Gale 1997, Gale and Devos 1998a); thus for maize, genes may be cloned using rice genome information. In several studies, QTL were already identified for accumulation of various cation minerals (Zn, Fe, Mn, K, Ca and Mg) and the antinutrient phytate in different plant tissues. QTL involved in seed cationic mineral content in Arabidopsis were identified using immortal segregating RIL populations. Some of the QTL appeared to be involved in controlling accumulation of more than one mineral (Vreugdenhil et al. 2004). In addition, five genomic regions affecting the quantity of phytate and inorganic phosphor in seeds and leaves of Arabidopsis were found (Bentsink et al. 2003). Because the same segregating population was used for QTL analysis of phytate content as for mineral accumulation, it showed that the loci affecting Fe and Zn accumulation were largely different from those controlling the phytate content of seeds. It is conceivable that this is also true for crops, suggesting it should be possible to breed for lowphytate genotypes, without decreasing the levels of Fe
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and Zn. In a recent review, we listed QTL for mineralrelated traits in Arabidopsis (Vreugdenhil et al. 2005).

Approaches to improve plant mineral content

Genetic modification With the identification and the functional analysis of an increasing number of metal homeostasis-related genes and the concurrent elucidation of metal homeostasis, the time has come to use a more systematic genetic modification approach to improve the mineral content of plants in contrast to the empirical approach which has been used up until now and which was much more direct towards functional analysis of the identified genes. In general, we discern two major targets for biotechnological improvement of mineral content: leaves and seeds. The first mainly for vegetable crops, such as lettuce, cabbage or spinach, the second mainly for grain crops such as cereals and legumes. For the transport of Fe and Zn to the leaves, serious limitations are the mineral uptake into the roots and the loading into the xylem for transport to the above-ground organs. For transport to the seeds, which is mainly through phloem (Guerinot 2001), additional limitations are the loading into phloem, chelation in the phloem, unloading from the phloem and symplastic import into the grain. For Fe accumulation, we have to distinguish strategy I and strategy II plants. Most of the efforts to increase the Fe uptake in roots by genetic modification have focused on strategy I plants. Especially, the overexpression of the high-affinity Fe-transporting IRT1 protein or any of its orthologues has been tried (Connolly et al. 2002). Major limitation for this approach is that the IRT1 protein is controlled by the Fe status of the plant. The protein can only be found in the roots under Fe-deficient conditions (Connolly et al. 2002). At the moment, it looks like this post-translational control mechanism acts on more ZIP family members (Ramesh et al. 2004). If so, it would impose a severe limitation to improve Fe (II) or Zn uptake by plants, because this seems to be almost exclusively controlled by ZIP proteins. Even if transcription factors directly controlling expression of ZIP transporters will be found, it would not be useful to enhance their expression, because the limitation is not transcriptional but post-translational. For improving Fe uptake in strategy II plants, there is still the possibility to improve the production of PSs and the expression of the PSFe (III)transporting YSL proteins. Transgenic rice expressing the barley naat-A and naat-B genes, encoding enzymes involved in the biosynthesis of PSs, showed tolerance to low Fe availability when grown in a calcareous soil
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(Takahashi 2003). The transformants also secreted more PSs than non-transformants under Fe-deficient conditions (Takahashi et al. 2001). Because it is unclear whether a similar post-translational control acts on YSL transporters as it does on ZIP transporters, it is unclear whether an increased release of chelators to the rhizosphere might help to increase mineral uptake rates. For loading Fe and Zn into the xylem, the most likely transporters appear to be AtHMA4 orthologues (Papoyan and Kochian 2004, Verret et al. 2004) and YSL transporter orthologues (Schaaf et al. 2005). Overexpression of AtHMA4, a Zn efflux transporter, in Arabidopsis increased the Zn content in the shoot by two-fold (Verret et al. 2004). As AtHMA4 can transport Cd as well (Mills et al. 2005), also the Cd content is increased, but only when available in substantial amounts. Upon overexpression of AtHMA4 in the shoot, Zn may end up in the shoot apoplast rather than inside the cells. Although this may be beneficial to the plant to avoid high intracellular Zn concentrations, it may be disadvantageous when trying to load the shoots with Zn for further loading into the grain by phloem transport. Additional phloem companion-cell-specific overexpression of HMA4, or HMA2 which appears to perform a very similar function (Hussain et al. 2004), may be more successful. Because HMA genes have not been shown to transport Fe, overexpression of HMA genes is unlikely to have a positive effect on Fe loading into the xylem. Instead, overexpression of YSL transporters, of which some family members transport FeNA chelates (Schaaf et al. 2004), seems to be more appropriate. Which transporter should be chosen for this approach remains to be discovered. In Arabidopsis, there are already eight genes putatively encoding YSL transporters (Schaaf et al. 2005). Overexpression of NAS also has a positive effect on root to shoot transport, especially for Fe and Zn, as was found by overexpression of the HvNAS1 gene from barley that approximately doubled the Fe and Zn concentrations in young leaves of tobacco (Takahashi et al. 2003). Remarkably, overexpression of the AtNAS1 gene in tobacco only marginally increased the young leaf Fe (1.5-fold) and especially Zn content (approximately 1.2 fold), although in both cases, NA was overproduced (Douchkov et al. 2005). Because Fe and Zn are largely transported through phloem as NA-chelates, NA overproduction could also have an effect on increasing the seed Fe and Zn contents. This indeed is the case. Overexpression of the HvNAS1 gene in tobacco approximately doubled the seed Fe and Zn concentrations (Takahashi et al. 2003). Unfortunately, the exact location of Fe and Zn in the seed was not examined. Other attempts to increase grain Fe content included the overexpression of ferritin. When ferritin from bean or soybean is overexpressed in the rice
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endosperm, it increases the maximum Fe concentration about 23-fold compared with non-transformed rice (Qu le et al. 2005). Interestingly, constitutive overexpression of the soybean ferritin gene, driven by the corn ubiquitin promoter, only marginally increases the Fe concentration in leaves of rice and wheat and has no effect on Fe in seed (Drakakaki et al. 2000). Until now, genetic modification has not tremendously improved the Fe or Zn content in vegetative tissue or in seeds. The main reason is that despite the increase in molecular genetic research on metal homeostasis in the last years, still not enough is known about which genes should be targeted. Another reason is that in most cases the strong constitutive CaMV 35S promoter has been used to express target genes. This promoter does not allow for tissue-specific, temporal or mineral content dependent finetuning of especially transporter gene expression. This could lead to conflicting transport situations where enhanced constitutive expression of a transporter can lead to enhanced mineral uptake in root epidermal or endodermal cells but at the same time prevents the loading of xylem due to retention in, for example, pericycle cells. In the future, more emphasis should be put on specifically targeting the (ectopic) expression of metal homeostasis genes, as was already done for the expression of ferritin in cereal endosperm. QTL analysis and marker-assisted breeding To breed for increased Fe and Zn contents, we need the presence of genetic variation for this trait. A wide range of rice, wheat, bean, maize and cassava germplasm has been studied indicating there is enough variation to allow breeding for nutritional improvement (Banziger 2000, Beebe et al. 2000, Chavez et al. 2000, Gregorio et al. 2000, Ortiz-Monasterio and Graham 2000). After evaluating genetic variation for mineral content in rice grains, aromatic grains were usually found to be higher in Fe concentration and often also higher in Zn, compared with nonaromatic varieties. The linkage between aromaticity and high Fe density was rarely lost, which is an encouraging prospect for the improvement of nutritional value as aromaticity is an easy selection marker (Graham et al. 1999). It is also encouraging that the high Fe and Zn traits in rice are expressed in different environments, although some evidence of G x E interactions were observed (Graham et al. 1999). For wheat, approximately 1500 accessions from germplasm resources have been screened (1% of the total available number). The best lines have 1.5 to two times more Fe and Zn contents in the grain than average. Crosses between high yielding, disease-resistant varieties and lines with high Fe and Zn contents are at an advanced stage of development. There is also a highly significant positive correlation between the concentrations of Fe and Zn
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across different genotypes of wheat lines, indicating the possibility to improve the concentration of both elements simultaneously. This co-segregation of genetic factors for increased Fe and Zn contents, suggesting that common mechanisms regulate Fe and Zn accumulation, is very useful for breeding purposes. Additional research has also shown that there is no negative correlation between grain yield and Fe and Zn concentrations in wheat grain (Graham et al. 1999). In general, there is abundant genetic variation for the concentrations of Fe, Zn and other minerals in the germplasm of major crops to justify selection. Despite some reported G x E interactions, mineral traits are generally stable across environments in the abovementioned crops, and it is feasible to combine highmicronutrient traits with high yield (Gregorio 2002). To facilitate breeding by MAS, knowledge on the genetic basis of Zn and Fe content is important. In rice, most genetic studies dealing with minerals related to the response of the plant to nutrient deficiencies or toxicities in soil. A range of QTL has been found, and in one case, i.e. tolerance of rice to P-deficiency, a clear candidate gene has been identified (Wissuwa 2005). Because a wide range of variation in micronutrient content is present in wheat, maize, rice or other crops, and because mapping populations are available or will soon be ready, a comprehensive QTL analysis is only a matter of time. Only for beans, a few QTL have been described for Fe and Zn contents, as well as loci affecting antinutrients (Guzman-Maldonado et al. 2003). QTL map ping of grain quality traits from the interspecific crosses of cereals is an important approach being pursued by several groups (Aluko et al. 2004, Li et al. 2004a, Quarrie et al. 2005, Septiningsih et al. 2003, Xiao et al. 1998, Yu et al. 1997). These same populations can be used for QTL analysis of seeds mineral content. Although to our knowledge no cloning of a mineral homeostasis QTL has been described so far, the limitations to clone QTL in crop plants are gradually being overcome, and the approach is becoming feasible. Not only genetic but also the genomic physical maps of the crop plants and mapping populations including NILs are becoming available. In tomato, a QTL for fruit size has already been cloned and the underlying gene was identified (Frary et al. 2000). In addition, a gene underlying a QTL controlling plant height in rice was identified and the candidate gene was selected and cloned (Ishimaru et al. 2004). Also in rice, a QTL controlling regeneration ability was recently identified and isolated using a mapbased cloning strategy (Nishimura et al. 2005), as well as the gene controlling an important QTL for grain yield in rice (Ashikari et al. 2005). These examples all show that cloning and identification of genes based on QTL
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are no longer limited to model species like Arabidopsis, but also a reality for crops.

Conclusions
In the end, it is difficult to choose between the two options we presented for plant mineral improvement. Both plant breeding and genetic modification offer good opportunities to increase the Fe and Zn contents of edible parts of the major crop species. For a classical breeding approach, the search for suitable starting material is most important. Initially, limited knowledge on which genes play a role in the desired traits is not a problem, although certainly the availability of molecular selection markers can tremendously speed up the breeding process. New varieties will anyhow be generated after several rounds of selection and testing at various sites over different years. This will take its time, but there is no doubt that steady progress will be made. With the current increase in knowledge on the physiological and molecular processes underlying plant mineral accumulation, interesting alleles of relevant genes will be found by QTL analysis and the DNA information of identified genes will be converted into molecular markers. With the increasing availability of more accurate genetic markers, advanced breeding tools can be used, selecting for the presence or absence of specific alleles of genes known to play a role in, for example, mineral uptake or translocation to the grain. In order for genetic modification to be efficient and effective, it is clear that a much better understanding of Zn and Fe homeostasis is essential. This should not only focus on identifying more, or rather all, genes involved in the process but also on understanding the cellular and tissue localization of the proteins they encode and their interactions. Eventually, such information will justify a well-designed transgenic approach in which the expression of several genes will be affected, and in the long run, the introduction of superior heterologous alleles from other species can lead to varieties which will perform even better than what ever can be obtained by classical breeding. This, however, is unlikely to happen in the next 20 years. Acknowledgements We thank Professor Maarten Koornneef (Wageningen University, Wageningen, the Netherlands and the Max Planck Institute for Plant Breeding, Cologne, Germany) for critically reading the manuscript and for his valuable suggestions.

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