View metadata, citation and similar papers at core.ac.uk brought to you by CORE REVIEW ARTICLE published: 17 Novemberprovided 2011 by Frontiers - Publisher Connector doi: 10.3389/fpls.2011.00080 Physiological limits to zinc biofortification of edible crops

Philip J. White 1* and Martin R. Broadley 2

1 The James Hutton Institute, Dundee, UK 2 and Crop Sciences Division, University of Nottingham, Loughborough, UK

Edited by: It has been estimated that one-third of the world’s population lack sufficient Zn for adequate Søren Husted, University of nutrition. This can be alleviated by increasing dietary Zn intakes through Zn biofortification Copenhagen, Denmark of edible crops. Biofortification strategies include the application of Zn-fertilizers and the Reviewed by: Javier Abadía, Consejo Superior de development of crop genotypes that acquire more Zn from the soil and accumulate it in Investigaciones Científicas, Spain edible portions. Zinc concentrations in roots, leaves, and stems can be increased through Ismail Cakmak, Sabanci University, the application of Zn-fertilizers. Root Zn concentrations of up to 500–5000 mg kg−1 dry Turkey matter (DM), and leaf Zn concentrations of up to 100–700 mg kg−1 DM, can be achieved *Correspondence: without loss of yield when Zn-fertilizers are applied to the soil. It is possible that greater Philip J. White, The James Hutton Institute, Invergowrie, Dundee DD2 Zn concentrations in non-woody shoot tissues can be achieved using foliar Zn-fertilizers. 5DA, UK. By contrast, Zn concentrations in fruits, seeds, and tubers are severely limited by low Zn e-mail: [email protected] mobility in the phloem and Zn concentrations higher than 30–100 mg kg−1 DM are rarely observed. However, genetically modified with improved abilities to translocate Zn in the phloem might be used to biofortify these phloem-fed tissues. In addition, genetically modified plants with increased tolerance to high tissue Zn concentrations could be used to increase Zn concentrations in all edible produce and, thereby, increase dietary Zn intakes.

Keywords: Arabidopsis, bean, cassava, maize, potato, rice, wheat, zinc

INTRODUCTION or distribution (Cakmak, 2004, 2009). Genetic biofortification is Zinc (Zn) is an essential element for human nutrition (White and predicated on increasing Zn acquisition from the soil and its accu- Broadley, 2005; Graham et al., 2007). Symptoms of Zn-deficiency mulation in edible portions. In most agricultural soils there is include stunting, diarrhea, and pneumonia in children, with the sufficient Zn to produce biofortified crops for many years, pro- latter two contributing significantly to infant mortality (Stein vided it becomes phytoavailable (Graham et al., 1999). Genetic et al., 2005). The US recommended daily allowance (RDA, or ade- biofortification strategies are, of course, ineffective if there is insuf- quate intake) of Zn is 8.0–13.0 mg and the UK guidance daily ficient Zn present in the soil. Most economic analyses suggest that reference nutrient intake (RNI) is 7.0–13.0 mg for adults (Depart- genetic strategies toward Zn biofortification are more practical, ment of Health (UK), 1991; Institute of Medicine (USA), 2001). enduring, and cost effective than dietary diversification, supple- Unfortunately, the diets of many people across the world lack suf- mentation, or food fortification programs for increasing dietary ficient Zn for their adequate nutrition (White and Broadley, 2009; Zn intakes of vulnerable populations (Horton,2006; Graham et al., Bouis and Welch, 2010; Stein, 2010; Sayre et al., 2011). This has 2007; Stein et al., 2007; Ma et al., 2008; Bouis and Welch, 2010; been attributed to sourcing produce from land with low Meenakshi et al., 2010; Stein, 2010). phytoavailability, eating crops with inherently low tissue mineral Several national and international projects are addressing Zn concentrations, or consuming processed foods. It has been esti- biofortification of edible crops (Graham et al., 2001, 2007; Cak- mated that almost one-third of the world’s population consumes mak, 2004; Pfeiffer and McClafferty, 2007; White and Broadley, less Zn than the US RDA and that Zn-deficiency contributes 1.9% 2009; Bouis and Welch, 2010; Stein, 2010; Sayre et al., 2011). of the overall burden of disease caused by major health risks world- The target Zn concentrations set by the HarvestPlus program wide (World Health Organization, 2002; Hotz and Brown, 2004). are 28 μgg−1 dry matter (DM) in polished rice, 38 mg kg−1 DM This has considerable socio-economic impacts (Stein, 2010). in wheat grain, 38 mg kg−1 DM in maize, 66 mg kg−1 DM in Dietary Zn intakes can be increased through a variety of inter- pearl millet, 56 mg kg−1 DM in beans, 34 mg kg−1 DM in cas- ventions (Stein, 2010). These include both agronomic and genetic sava roots, and 70 mg kg−1 DM in roots of sweet potatoes (Bouis biofortification of edible crops (Graham et al., 1999, 2001, 2007; and Welch, 2010). These target concentrations are considered White and Broadley, 2005, 2009; Cakmak, 2008; Khoshgoftar- to be conservative, and have been exceeded in breeding lines of manesh et al., 2009; Bouis and Welch, 2010; Martínez-Ballesta rice, wheat, and maize (Bouis and Welch, 2010). This article asks et al., 2010). Agronomic biofortification can be achieved by whether higher target Zn concentrations can be achieved and, increasing soil Zn phytoavailability or by applying Zn-fertilizers. more broadly, what the physiological limits to Zn biofortification This requires appropriate infrastructures, but can be very suc- of crops might be. It considers (a) the physiological requirements cessful in regions where mineral fertilizers are used to increase and tolerance of Zn in crop plants, (b) the uptake and distribution crop yields and Zn is added to these at the point of manufacture of Zn between and within plant organs, (c) agronomic strategies to

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increase Zn concentrations of edible tissues, (d) genetic variation in Zn concentrations of edible portions within plant species, and (e) transgenic strategies to increase Zn concentrations of edible produce.

PLANT PHYSIOLOGY PHYSIOLOGICAL REQUIREMENTS AND TOLERANCE OF ZINC IN PLANTS Plants, like other living organisms, require Zn for the regulation of transcription and translation, the structural stability of pro- teins, the function of oxidoreductases and hydrolytic enzymes, and the control of enzyme activities (Broadley et al., 2007; Clemens, 2010; White, 2012b). However, excessive tissue Zn concentrations are toxic. Plant species differ in both their Zn requirements and their tolerance of high tissue Zn concentrations (Broadley et al., 2007; Fageria, 2009). Most crop plants require leaf Zn concen- trations greater than 15–30 mg kg−1 DM for maximal yield, and their growth is inhibited at leaf Zn concentrations greater than 100–700 mg kg−1 DM (Fageria, 2009; White and Brown, 2010). By contrast plant species that hyperaccumulate Zn, such as Noc- caea caerulescens (formerly Thlaspi caerulescens) and Arabidopsis halleri, not only tolerate more Zn in their tissues than congeneric FIGURE1|Frequency distribution of mean zinc (Zn) concentrations in species that do not hyperaccumulate Zn but also require greater leaf or non-woody shoot tissues of 365 species from 48 angiosperm families grown under controlled conditions at non-toxic substrate Zn leaf Zn concentrations for optimal growth (Hammond et al., concentrations (Broadley et al., 2007). Data from 1108 studies were 2006; Broadley et al., 2007; White, 2012b). The trait of Zn hyper- combined using residual maximum likelihood (REML) procedures. accumulation is defined as a leaf Zn concentration exceeding 10,000 mg kg−1 DM when plants are sampled from their natural habitat, although a figure of 3000 mg kg−1 DM might be a more observed in the Linaceae, Poaceae, and Solanaceae, and the highest realistic threshold (Reeves and Baker, 2000; Broadley et al., 2007). shoot Zn concentrations were observed in the , Ama- Only 15–20 species hyperaccumulating Zn have been reported, ranthaceae, and Salicaceae. These phylogenetic effects on shoot mostly in the Brassicaceae (Broadley et al., 2007; Verbruggen Zn concentration are also observed in surveys of plants grow- et al., 2009; Krämer, 2010; White, 2012b). Zinc uptake, deliv- ing in their natural habitats (Watanabe et al., 2007). Similarly, ery to the xylem and tolerance in shoot tissues is maximized in seeds of cereals generally have lower Zn concentrations than seeds plants that hyperaccumulate Zn (Broadley et al., 2007; White and of legumes (Figure 2). As a consequence, the occurrence of Zn- Broadley, 2009; Krämer, 2010; Hassan and Aarts, 2011; Rascio and deficiency disorders has increased in populations changing from Navari-Izzo, 2011; White, 2012b). To achieve this, genes encoding traditional diets dominated by pulses,,and fruits to diets enzymes synthesizing compounds enabling Zn uptake and xylem dominated by cereals (Graham et al., 2001). transport, and proteins catalyzing Zn uptake, vacuolar efflux and xylem loading are constitutively highly expressed in plants hyper- THE UPTAKE AND DISTRIBUTION OF ZINC BETWEEN AND WITHIN accumulating Zn (Hammond et al., 2006; Broadley et al., 2007; PLANT ORGANS Hanikenne et al., 2008; Roosens et al., 2008; Verbruggen et al., Zinc is unevenly distributed within the plant. When plants are 2009; Hassan and Aarts, 2011; Rascio and Navari-Izzo, 2011; Ó supplied Zn through the rhizosphere, tissue Zn concentrations Lochlainn et al., 2011). Shoot Zn concentrations are often an order generally decrease in the order root ≈ shoot > fruit, seed, tuber of magnitude greater than root Zn concentrations in plants that (Broadley et al., 2012). Consequently Zn concentrations are often hyperaccumulate Zn, although the exact ratio depends on soil greater in root crops and leafy vegetables than in grain, seed, fruit, Zn phytoavailability (Frey et al., 2000; Broadley et al., 2007). It is or tuber crops (Figure 2; White and Broadley, 2005, 2009; Pfeiffer believed that the formation of Zn-complexes and the transloca- and McClafferty, 2007). Within each organ, Zn is preferentially tion of Zn from the root to the shoot prevent the accumulation accumulated by specific cell types. For example Zn is often local- of toxic Zn concentrations in root tissues, thereby enabling plants ized in distinct regions within the root, such as the elongation that hyperaccumulate Zn to tolerate high Zn concentrations in the zone, and is concentrated in endodermal cells of dicotyledonous soil solution (Broadley et al., 2007; White, 2012b). species and in the pericycle of monocotyledonous species (Van Plant species can differ greatly in their tissue Zn concentrations Steveninck et al., 1994). The distribution of Zn within shoots and when grown under comparable conditions (Figure 1; Broadley leaves varies between plant species. For example, in N. caerulescens, et al., 2001, 2007). Shoot Zn concentrations are generally lower leaf epidermal cells, with the exception of guard cells, have greater in the Ericales and commelinoid monocotyledons, and higher Zn concentrations than leaf mesophyll cells and leaf Zn concen- in the , Caryophyllales, and non-commelinoid mono- trations are higher in older leaves (Vázquez et al., 1994; Küpper cotyledons. Amongst the well-replicated plant families studied by et al., 1999, 2004; Frey et al., 2000; Ma et al., 2005; Monsant et al., Broadley et al. (2007), the lowest shoot Zn concentrations were 2010), whereas in A. halleri, and in Arabidopsis murale,Znismore

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carboxylic acids, such as citrate, malate, and oxalate, amino acids, such as histidine and asparagine,glutathione,phytochelatins,nico- tianamine (NA), and proteins. In addition, Zn is found as phos- phate salts, such as Zn3(PO4)2, and organic Zn-phytates. In the apoplast, Zn2+ binds to negatively charged cell-wall components, organic acids and phytosiderophores if these are present, and can be precipitated as phosphate or phytate salts when its concentra- tion in the apoplast becomes excessive (Van Steveninck et al., 1994; Broadley et al., 2007; Straczek et al., 2008; Terzano et al., 2008; Kopittke et al., 2011). In the cytosol, Zn can be complexed by proteins, glutathione, phytochelatins and NA, and Zn2+ concen- trations are vanishingly low (Broadley et al., 2007; Roosens et al., 2008; Clemens, 2010). The vacuoles of root and leaf cells contain Zn largely as Zn2+ and Zn-organic acid complexes (Broadley et al., 2007; Straczek et al., 2008; Sarret et al., 2009). Within the xylem, Zn is also present predominantly as Zn2+ and as complexes with carboxylic acids, such as citrate and malate (Welch, 1995; Broadley et al., 2007; Terzano et al., 2008). By contrast, phloem sap contains little Zn2+, and Zn is thought to be transported complexed with NA or small proteins (Welch, 1995; Curie et al., 2009; Waters and Sankaran, 2011). The forms of Zn present in tissues of plants that hyperaccu- FIGURE 2 | Variation in zinc (Zn) concentrations in roots, shoots, seeds mulate Zn depends on the plant species, the tissue studied, and and tubers of edible crops. Bars represent maximum and minimum the concentration of Zn in that tissue (Küpper et al., 2004; Sarret values obtained for large collections of cassava (Chávez et al., 2005), sweet et al., 2009; Monsant et al., 2011). More than 30% of the Zn in potato (Pfeiffer and McClafferty, 2007), carrot (Nicolle et al., 2004), chickpea (leaves; Ibrikci et al., 2003), (Broadley et al., 2010), roots of N. caerulescens is generally associated with cell walls, and Brassica rapa (Wu et al., 2007), spinach (Grusak and Cakmak, 2005), rice much of the remainder is complexed with histidine (Salt et al., (Yang et al., 1998), wheat (Graham et al., 1999), maize (Bänziger and Long, 1999; Monsant et al., 2010, 2011). The presence of Zn-phytate 2000), pearl millet (Velu et al., 2007), barley (P.J. White and I. J. Bingham is also observed occasionally (Monsant et al., 2011). About 80% cited in White and Broadley, 2009), sorghum (Reddy et al., 2005), bean of the Zn in the xylem sap of this plant species is Zn2+, with the (Islam et al., 2002), pea (Grusak and Cakmak, 2005), cowpea (Pfeiffer and McClafferty, 2007), chickpea (seed, M. A. Grusak cited in White and remainder complexed with carboxylic acids (Salt et al., 1999; Mon- Broadley, 2009), lentil (Pfeiffer and McClafferty, 2007), soybean (Raboy sant et al., 2011). In shoots of Zn-hyperaccumulator plants, such et al., 1984), peanut (Branch and Gaines, 1983), potatoes (White et al., as N. caerulescens and A. halleri, 20–50% of the Zn is present as 2009), and yam (Agbor-Egbe and Trèche, 1995) genotypes. Blue circles Zn2+, 40–99% is associated with vacuolar carboxylic acids, such as indicate Zn concentrations in the U.S. Department of Agriculture, citrate, malate, and oxalate, up to 45% can be associated with histi- Agricultural Research Service (2011). Red circles indicate target Zn concentrations proposed by the HarvestPlus program (Bouis and Welch, dine, and the remainder is largely bound to phosphate-groups and 2010). cell-wall components (Salt et al., 1999; Küpper et al., 2004; Sarret et al., 2009; Monsant et al., 2011). No major contributions of phy- tochelatins, metallothioneins, or other cysteine-rich peptides are uniformly distributed across the leaf, although mesophyll cells observed (Küpper et al., 2004; Sarret et al., 2009). have higher Zn concentrations than trichomes, which have greater In general, Zn enters plants from the soil solution and is trans- Zn concentrations than other epidermal cells (Küpper et al., 2000; ported either symplastically, following uptake by root cells, or Zhao et al., 2000; Tappero et al., 2007). In seeds of cereals, Zn is apoplastically, in regions of the root lacking a Casparian Band, preferentially accumulated in the husk, aleurone layers or embryo to the stele where it enters the xylem (White et al., 2002b; Broadley (Lin et al., 2005; Ozturk et al., 2006; Liu et al., 2007; Hansen et al., et al., 2007). Zinc is taken up by root cells as Zn2+ and, in some 2009; Persson et al., 2009; Cakmak et al., 2010a,b; Lombi et al., plant species, also as Zn-phytosiderophore complexes (Broadley 2011; Stomph et al., 2011). In potato tubers about 17% of total et al., 2007; Palmer and Guerinot, 2009; Puig and Peñarrubia, tuber Zn is present in the skin (Subramanian et al., 2011). These 2009; Verbruggen et al., 2009; White and Broadley, 2009; Clemens, distribution patterns reflect both local and long-distance transport 2010; Krämer, 2010; White, 2012b). Although some plasma mem- of Zn within the plant (Broadley et al., 2007; Stomph et al., 2011; brane Ca2+ channels are permeable to Zn2+ (Demidchik et al., Subramanian et al., 2011). The distribution of Zn within cereal 2002; White et al., 2002a), it is thought that most Zn2+ influx to seeds and tubers will reduce the potential dietary Zn intakes from the cytoplasm of root cells is mediated by ZRT-, IRT-like proteins these crops when, for example, polished grains or peeled tubers (ZIPs), in Arabidopsis thaliana principally AtZIP4 and AtIRT1, and are consumed. that yellow stripe like proteins (YSLs) catalyze the uptake of Zn- Plant tissues accumulate Zn in both soluble and insoluble forms phytosiderophore complexes in cereals and grasses (Broadley et al., (Broadley et al., 2007). In crop plants, much of the soluble Zn is 2007; Roosens et al., 2008; Curie et al., 2009; Palmer and Guerinot, complexed with organic compounds. These compounds include 2009; Verbruggen et al., 2009; White and Broadley, 2009; Waters

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and Sankaran, 2011; White, 2012b). In the cytosol of root cells, when plant tissues have sufficient Zn for their physiological Zn2+ is complexed by numerous proteins, including many that requirements (Broadley et al., 2007; White and Broadley, 2009; modulate enzymic activities or gene transcription, by glutathione, Waters and Sankaran, 2011). Recently, two members of the basic by phytochelatins, and by NA, and the cytosolic Zn2+ concentra- region/leucine zipper motif (bZIP) transcription factor gene fam- tion is likely to be extremely low (Broadley et al., 2007; Roosens ily, bZIP19 and bZIP23, were shown to coordinate the adaptation et al., 2008; Clemens, 2010). of A. thaliana to low Zn phytoavailability (Assunção et al., 2010). Members of the cation diffusion facilitator (CDF) family, such This general homeostatic regulation of tissue Zn concentrations as orthologs of the A. thaliana metal tolerance proteins AtMTP1 through Zn acquisition and distribution within the plant could and AtMTP3, and the Mg2+/H+ antiporter AtMHX, transport limit Zn accumulation by edible tissues. Zn2+ into the vacuole, whilst orthologs of the A. thaliana Zn- induced facilitator 1 (AtZIF1) protein transport Zn2+-complexes AGRONOMIC STRATEGIES TO INCREASE ZINC into the vacuole (Roosens et al., 2008; Palmer and Guerinot, 2009; CONCENTRATIONS OF EDIBLE CROPS Puig and Peñarrubia, 2009; White and Broadley, 2009; Clemens, Although the total Zn concentrations in many soils are sufficient 2010; Hassan and Aarts, 2011). It is thought that Zn is sequestered to support mineral-dense crops (Graham et al., 1999)Znuptake in the vacuole as an organic acid complex (Broadley et al., 2007). by plants is often limited by its phytoavailability and acquisi- Zinc is released from the root vacuole through NRAMPs, includ- tion by roots (Broadley et al., 2007; White and Broadley, 2009). ing orthologs of the AtNRAMP3 and AtNRAMP4 transporters of Agronomic strategies seek to improve Zn phytoavailability in the A. thaliana (Roosens et al., 2008; Verbruggen et al., 2009). Mem- soil, for example by remedying soil alkalinity, adopting appropri- bers of the heavy metal P1B-ATPase family, such as AtHMA2 and ate crop rotations or introducing beneficial soil microorganisms AtHMA4 in A. thaliana,loadZn2+ into the xylem (Roosens et al., (Rengel et al., 1999; He and Nara, 2007; Fageria, 2009; White 2008; Palmer and Guerinot, 2009; Puig and Peñarrubia, 2009; and Broadley, 2009), or to deliver phytoavailable Zn through Verbruggen et al., 2009; White and Broadley, 2009; Waters and the application of Zn-fertilizers to soil or foliage (Cakmak, 2004; Sankaran, 2011; White, 2012b). Within the xylem, Zn is trans- Graham et al., 2007; Fageria, 2009; White and Broadley, 2009; ported either as Zn2+ or as a complex with organic acids or NA, Bouis and Welch, 2010). Common inorganic Zn-fertilizers include and it is thought that YSL proteins load Zn-NA complexes into ZnSO4, ZnO, and synthetic Zn-chelates (Fageria, 2009; White and the xylem and orthologs of AtFRD3 load citrate into the xylem to Broadley, 2009). When Zn-fertilizers are applied to foliage, it is promote Zn transport (Broadley et al., 2007; Roosens et al., 2008; particularly important that the Zn compounds used are readily Waters and Grusak, 2008; Curie et al., 2009; Waters and Sankaran, soluble, enter the leaf apoplast, and can be taken up by plant cells 2011). (Haslett et al., 2001; Cakmak, 2008; Brown, 2009). This avoids Within the shoot,the uptake of Zn2+ and Zn-complexes by spe- the accumulation of Zn salts on the surface of leaves or in the cific cell types are facilitated by members of the ZIP and YSL fami- leaf apoplast, which can interfere with photosynthesis and cell lies, respectively (White and Broadley, 2009; Waters and Sankaran, function, and promotes the translocation of Zn from leaves to 2011). Members of these protein families are also thought to load phloem-fed tissues. Zn into the phloem (Curie et al., 2009; White and Broadley, 2009), Zinc concentrations in roots, leaves, and stems can be increased where it is transported as a complex with NA or small proteins readily by applying Zn-fertilizers to the soil in plants growing on (Curie et al., 2009; Puig and Peñarrubia, 2009; White and Broadley, most, but not all, soils and by foliar application of Zn-fertilizers 2009; Waters and Sankaran, 2011). In A. thaliana,AtYSL1,AtYSL3, (Figure 3; Rengel et al., 1999; Cakmak, 2008; Fageria, 2009; Sagar- and AtOPT3 have been implicated in delivering Zn from vascular doy et al., 2009; White and Broadley, 2009; Bouis and Welch, tissues to developing seeds (Waters and Grusak, 2008; Puig and 2010). Thus, Zn concentrations in these tissues will be limited Peñarrubia, 2009; Waters and Sankaran, 2011). The mobility of solely by Zn toxicity. When Zn-fertilizers are added to the soil, Zn in the phloem will determine Zn accumulation by phloem-fed root tissues often exhibit higher Zn concentrations than shoot tissues, such as fruits, seeds, and tubers. Although Zn is generally tissues, and it is likely that plant Zn accumulation and yield is considered to have a low mobility in the phloem (Fageria, 2009; limited by Zn toxicity to root cells under these conditions. Crit- White, 2012a), the translocation of Zn in the phloem of several ical leaf Zn concentrations for most crop plants lie between 100 plant species following the application of foliar Zn-fertilizers has and 700 mg kg−1 DM when Zn-fertilizers are applied to the soil been found to be nutritionally significant for their growth and (MacNicol and Beckett, 1985; Fageria, 2009). It is possible that, development especially when cultivated in substrates with low Zn when Zn-fertilizers are applied foliarly, higher leaf Zn concentra- phytoavailability (Haslett et al., 2001; Brown, 2009; Waters and tions might be achieved without loss of yield (Cakmak et al., 1999; Sankaran, 2011). White et al., submitted). The activity of transport proteins catalyzing Zn uptake, and Soil or foliar applications of Zn-fertilizers can also increase Zn the expression of the genes encoding proteins responsible for concentrations in phloem-fed tissues, such as fruits, seeds, and the mobilization of transition-metal elements from soil, their tubers (Rengel et al., 1999; Cakmak, 2004, 2008; Fang et al., 2008; uptake by plant roots, and their distribution within the plant are White et al., 2009; Cakmak et al., 2010a,b). However, increasing regulated in response to plant Zn status to ensure appropriate Zn concentrations in these tissues requires adequate Zn mobil- tissue Zn concentrations. Thus, ZIPs, YSLs, HMAs, MTPs, ZIF1, ity in the phloem and, unless Zn-fertilizers are applied directly FRD3,and enzymes involved in the synthesis of phytosiderophores or they have functional xylem continuity, the mobility of Zn and NA are upregulated during Zn-deficiency and downregulated in the phloem will limit their Zn accumulation (Rengel et al.,

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FIGURE3|(A–C)The effect of solution zinc (Zn) concentration on solution (Jiang et al., 2008). (D–F) The effect of foliar Zn fertilizer (A) grain yield and Zn concentrations in (B) shoots and (C) grain of applications on (D) tuber yield and (E) shoot and (F) tuber Zn two rice varieties, Handao297 (filled circles) and K150 (open concentrations in “Maris Piper” potatoes grown in the field (White circles), grown in quartz sand irrigated with a complete nutrient et al., submitted).

1999; White and Broadley, 2005, 2009; Broadley et al., 2007; either as Zn–NA or complexed with small proteins (White and Stomph et al., 2009; Cakmak et al., 2010a,b; White et al., sub- Broadley, 2009). In cereals, grain Zn concentration is correlated mitted). The relationship between seed Zn concentration and with grain protein concentration (e.g., Zhao et al., 2009; Cak- Zn phytoavailability often follows a saturation curve. For exam- mak et al., 2010a,b; Gomez-Becerra et al., 2010), and the limit ple, when rice is grown in quartz sand irrigated with a complete to grain Zn concentration can be increased by higher N-fertilizer nutrient solution, Zn concentrations in brown rice reach a maxi- applications (Hao et al., 2007; Kutman et al., 2010, 2011a,b; Shi mum of about 50–110 mg Zn kg−1 DM depending upon variety et al., 2010). Similarly, there is a significant relationship between (Figure 3; Jiang et al., 2008). Similarly, the relationship between tuber Zn concentration and tuber N concentration among potato tuber Zn concentration and foliar Zn application in “Maris Piper” genotypes (White et al., submitted) and tuber Zn concentrations potatoes grown in the field followed a saturation curve, reach- can be increased by N-fertilization (Hlusek et al., 1997). Nev- ing a maximum of about 30 mg Zn kg−1 DM (Figure 3; White ertheless, Zn concentrations in phloem-fed tissues rarely exceed et al., submitted). In the phloem, Zn is thought to be transported 30–100 mg kg−1 DM.

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GENETIC VARIATION IN ZINC CONCENTRATIONS OF EDIBLE and vacuole. Targets for the manipulation of shoot Zn concentra- CROPS tions include (a) transport proteins in the plasma membrane of Genetic strategies to increase Zn concentrations in edible por- root cells that facilitate Zn uptake and delivery to the xylem, or tions seek to exploit genetic variation in the acquisition of Zn root structural modifications that facilitate apoplastic movement from the soil, Zn accumulation in edible portions and tolerance to the xylem, (b) enzymes involved in the synthesis of compounds to high tissue Zn concentrations (Pfeiffer and McClafferty, 2007; that facilitate Zn movement through the root symplast or apoplast White and Broadley, 2009). There is considerable genetic varia- and in the xylem, (c) transport proteins in the plasma membrane tion in Zn concentration in most edible crops (Figure 2). Edible and tonoplast of shoot cells that facilitate Zn uptake and vacuo- roots often have low Zn concentrations, but researchers have lar Zn sequestration, and (d) enzymes involved in the synthesis reported >14-fold variation in root Zn concentrations among of compounds that detoxify Zn2+ within and outside shoot cells. 600 cassava genotypes (Chávez et al., 2005) and 2.2-fold variation Targets for the manipulation of Zn concentrations in phloem-fed among 20 carrot genotypes (Nicolle et al., 2004). Zinc concen- tissues will additionally include transport proteins in the plasma trations in edible greens are often relatively high and trials have membrane of shoot cells that facilitate the loading of Zn into indicated 2.5-fold variation in leaf Zn-concentration among 19 the phloem and compounds that facilitate Zn movement in the chickpea genotypes (Ibrikci et al., 2003), 6.7-fold variation in phloem. leaf Zn-concentration among 111 Brassica rapa genotypes (Wu Most published studies describing GM strategies that increase et al., 2007), 12.3-fold variation in leaf Zn-concentration among Zn concentrations in plant tissues have been performed on 327 spinach genotypes (Grusak and Cakmak, 2005), and over 26- “model” plants such as A. thaliana and, even when studies have fold variation among 339 Brassica oleracea genotypes (Broadley been performed on crop species, data on commercial yields are et al., 2010). Absolute Zn concentrations and genetic variation rarely presented. Yield per plant can have a substantial effect on the in Zn concentrations are often lower in seeds than in leaves. tissue concentrations of mineral elements through dilution effects Nevertheless, between 2.2- and 11.6-fold variation in seed Zn caused by plant growth (Jarrell and Beverly, 1981; Davis, 2011). concentrations have been observed in large core collections of Higher-yielding genotypes often have lower Zn concentrations in cereal germplasm (e.g., Yang et al., 1998; Graham et al., 1999; their edible tissues than lower-yielding genotypes (Monasterio and Bänziger and Long, 2000; Gregorio et al., 2000; Reddy et al., 2005) Graham, 2000; Garvin et al., 2006; Murphy et al., 2008; White et al., and between 1.8- and 6.6-fold variation in seed Zn concentra- 2009; Zhao et al., 2009). Thus, it is important to consider whether tions in large core collections of legume germplasm (Raboy et al., any increase in tissue Zn concentration is simply a consequence of 1984; Islam et al., 2002; Grusak and Cakmak, 2005; Pfeiffer and slower growth or reduced yields. McClafferty, 2007; White and Broadley, 2009). Fruits generally Overexpressing genes encoding Zn-transporters catalyzing have low Zn concentrations, and studies report about twofold Zn2+ influx to root cells often increases root Zn concentra- variation among three to six cultivars in strawberries (Hakala tions but reduces leaf Zn concentrations. This has been observed et al., 2003), apples (Iwane, 1991), and plantains (Davey et al., when overexpressing OsZIP4 or OsZIP5 in rice (Ishimaru et al., 2007). Similarly, although tuber Zn concentrations are relatively 2007; Lee et al., 2010) and when overexpressing AtZIP1 in cas- low, 2.4-fold and 3.1-fold variation has been reported among sava (Sayre et al., 2011). Overexpressing a root plasma mem- 26 potato and 23 yam genotypes, respectively (Agbor-Egbe and brane Zn-transporter, AtZIP1, in barley had no effect on leaf Zn Trèche, 1995; White et al., 2009). These data indicate that breed- concentration, but reduced seed weight and increased seed Zn ing for increased Zn concentrations is, in principle, feasible for concentrations from 31 to 61–85 mg kg−1 DM (Ramesh et al., most edible crops. Indeed, genetic loci (QTL) affecting Zn con- 2004). Overexpression of the ZIPs TcZNT5 and TcZNT6 in A. centrations in cereal grain (Distelfeld et al., 2007; Stangoulis et al., thaliana had no consistent effects on root or shoot Zn concentra- 2007; Genc et al., 2009; Lonergan et al., 2009; Peleg et al., 2009), tions (Wu et al., 2009). Overexpressing genes encoding proteins bean seeds (Guzmán-Maldonado et al., 2003; Cichy et al., 2005, that transport Zn2+ into the vacuole, such as AtMTP1 (AtZAT1), 2009; Gelin et al., 2007; Blair et al., 2009, 2010, 2011), bras- AtMTP3, or TgMTP1, increases Zn uptake and root Zn concen- sica leaves (Wu et al., 2008; Broadley et al., 2010), and potato trations, but rarely shoot Zn concentrations, when expressed in tubers (N. K. Subramanian et al., unpublished data) have been roots, and increases Zn uptake and leaf Zn concentrations, but identified. rarely seed Zn concentrations, through systemic induction of Zn-deficiency responses when expressed in the shoot (van der GENETIC MODIFICATION STRATEGIES FOR ZINC Zaal et al., 1999; Arrivault et al., 2006; Gustin et al., 2009). The BIOFORTIFICATION OF EDIBLE CROPS expression of AtMTP1 (AtZAT1) increased Zn concentrations in It has been speculated that the constitutive expression of a suite of roots of cassava about fourfold to 40 mg kg−1 DM (Sayre et al., Zn-deficiency inducible responses through the overexpression of 2011). The ferric reductase defective 3/manganese accumulator 1 bZIP19 and bZIP23 transcription factors could be used to increase (frd3 = man1) null mutants of A. thaliana, which constitutively Zn accumulation in edible portions of crop plants (Assunção et al., express genes allowing increased rhizosphere Fe(III) reductase 2010). Specific targets for the manipulation of root Zn concen- activity (AtFRO2), Zn uptake (AtIRT1), and tissue NA concentra- trations include transport proteins in the plasma membrane and tions, have greater shoot Zn concentrations, but similar seed Zn tonoplast of root cells that facilitate the uptake and sequestration concentrations, to wild-type plants (Rogers and Guerinot, 2002). of Zn in the vacuole,together with enzymes involved in the synthe- Similarly, a mutant of A. thaliana (opt3.2) with reduced expres- sis of compounds that bind Zn2+ in the rhizosphere, cytoplasm, sion of AtOPT3 that shows constitutive expression of AtFRO2

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and AtIRT1 has higher Zn concentrations in leaves, stems, and Zn-fertilizer applications. These Zn concentrations approach that seed than wild-type plants (Stacey et al., 2008). Overexpressing found in raw beefsteak, which approximates 200–250 mg Zn kg−1 enzymes involved in the synthesis of phytosiderophores, either DM (U.S. Department of Agriculture, Agricultural Research Ser- HvIDS3 (encoding a dioxygenase, referred to as Iron-Deficiency vice, 2011). By contrast, Zn concentrations in fruits, seeds, and Specific clone 3) alone or both HvNAS1 (encoding nicotianamine tubers are severely limited by Zn transport in the phloem. This synthase) and HvNAAT (encoding nicotianamine aminotrans- might be a consequence of the need to maintain low Zn2+ con- ferase) increased concentrations of Zn in seeds of paddy-grown centrations in phloem sap to avoid cellular toxicity. Increased rice from 11.2 mg kg−1 DM to 13.4 and 15.3 mg kg−1 DM, respec- production of compounds that chelate Zn2+, such as NA, can tively (Suzuki et al.,2008). Leaf Zn concentrations can be increased increase Zn concentrations in the phloem and its delivery to in A. thaliana by reducing the expression of AtHMA2, which is phloem-fed tissues. Agrochemicals that increase the concentra- thought to catalyze Zn2+ efflux across the membranes of root cells tions of Zn-complexes in the phloem might be used to increase (Eren and Argüello, 2004) or by overexpressing the gene encoding Zn concentrations in fruits, seeds, and tubers. Thus, application AtHMA4, which is thought to load Zn into the xylem (Verret et al., of Zn-fertilizers, especially in combination with nitrogen fertiliz- 2004). ers, can increase Zn concentrations in seeds of cereals to about The overexpression of genes encoding nicotianamine synthase 100 mg kg−1 DM and Zn concentrations in seeds of legumes to (NAS) often leads to increased Zn concentrations in leaves and about 120 mg kg−1 DM (see Agronomic Strategies to Increase Zinc grain. The overexpression of HvNAS1 in tobacco increased leaf Concentrations of Edible Crops). Zn concentrations from 16 to 39 mg kg−1 DM and seed Zn con- Since there is appreciable variation in Zn concentrations of centrationsfrom20to35mgkg−1 DM (Takahashi et al., 2003). edible tissues of food crops, conventional breeding for increased The overexpression of OsNAS3 in rice also increased leaf and Zn concentrations appears feasible (see Genetic Variation in Zinc seed Zn concentrations (Lee et al., 2009; Johnson et al., 2011). Concentrations of Edible Crops). It is possible that breeding can Seed Zn concentrations of glasshouse-grown plants were increased increase Zn-tolerance in root and leaf crops and increase Zn mobil- from ∼40 to 79 mg kg−1 DM (Johnson et al., 2011) and those of ity in the phloem in fruit, seed, and tuber crops. Improving Zn- paddy-grown plants were increased from 16 to 35 mg kg−1 DM tolerance in root and leaf crops and increasing phloem Zn mobility (Lee et al., 2009). Constitutive overexpression of OsNAS2 in rice in fruit, seed, and tuber crops might also be addressed through GM resulted in an increase in seed Zn concentrations from ∼23 to technologies (see Genetic Modification Strategies for Zinc Bio- ∼60 mg kg−1 DM (Lee et al., 2011) and from ∼40 to 95 mg kg−1 fortification of Edible Crops). Several transgenic crop plants have DM (Johnson et al., 2011) in two independent studies. Consti- been created that have greater Zn concentrations in their edible tis- tutive overexpression of OsNAS1 in rice resulted in an increase sues than conventional varieties. These include cassava roots with in seed Zn concentrations from ∼40 to 59 mg kg−1 DM (Johnson about40mgZnkg−1 DM (Sayre et al., 2011), brown rice with 56– et al., 2011). The maximum Zn concentrations in polished grain of 95 mg Zn kg−1 DM (Vasconcelos et al., 2003; Johnson et al., 2011), transgenic rice expressing Pvferritin in the endosperm and AtNAS1 and barley grain with 85 mg Zn kg−1 DM (Ramesh et al., 2004). throughout the plant was 34 mg kg−1 DM, about 50% greater than Nevertheless, it is possible that the general homeostatic regulation in wild-type plants (Wirth et al., 2009). Earlier studies of plants of tissue Zn concentrations through Zn acquisition and distrib- expressing only Gmferritin found ∼56 mg Zn kg−1 DM in brown ution within the plant could be a major constraint to increasing rice (Vasconcelos et al.,2003). The overexpression of genes increas- Zn concentrations in edible portions (see Plant Physiology). A ing the production of glutathione, phytochelatins, and total thiols critical role of Zn in plant cells is the control of transcription, resulted in increased leaf Zn concentrations in Indian mustard translation, and protein activity through cytoplasmic Zn2+ con- (Brassica juncea) grown on soils with high Zn phytoavailability centration (Broadley et al., 2007). The key to Zn-homeostasis (Bennett et al., 2003). Seed Zn concentrations can be increased in in plant cells is likely, therefore, to pivot on cytoplasmic Zn2+ wheat by increasing the expression of a NAC transcription factor concentration. Increasing tissue Zn concentrations will require (NAM-B1) that accelerate senescence and increase remobilization increased biosynthesis of Zn-complexes and effective sequestra- of mineral elements from leaves to developing grain (Uauy et al., tion of Zn in non-vital compartments, such as the vacuole, to 2006). avoid unwanted perturbations in cytoplasmic Zn2+ concentra- tion. For this, lessons might be learnt from Zn-hyperaccumulator CONCLUSION plants. Zinc concentrations in roots, leaves, and stems can be increased To increase Zn concentrations in edible crops, future research greatly by applying Zn-fertilizers (Figure 3). The accumulation of should focus on (i) integrating agronomic and genetic strategies Zn in these tissues appears to be limited by Zn toxicity. In some to increase Zn transport to phloem-fed tissues and (ii) identify- crops,such as cereals and beans,root Zn concentrations of between ing the mechanisms effecting Zn-homeostasis in plant cells and 500 and 5000 mg kg−1 DM have been reported without loss of strategies to manage subcellular Zn compartmentalization. yield (Reichman, 2002). Leaf Zn concentrations ≥100 mg kg−1 DM, and perhaps up to 700 mg kg−1 DM, can be achieved without ACKNOWLEDGMENTS loss of yield in Zn-tolerant crops when Zn-fertilizers are applied This work was supported by the Rural and Environment Sci- to the soil (Fageria, 2009). It is possible that greater Zn concentra- ence and Analytical Services Division (RESAS) of the Scottish tions in non-woody shoot tissues might be achieved using foliar Government through Workpackage 7.2 (2011–2016).

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