Zinc (Zn) is an essential element for human nutrition (White and Broadley, 2005; Graham et al., 2007). Symptoms of Zn-deficiency include stunting, diarrhea, and pneumonia in children, with the latter two contributing significantly to infant mortality (Stein et al., 2005). The US recommended daily allowance (RDA, or adequate intake) of Zn is 8.0–13.0 mg and the UK guidance daily reference nutrient intake (RNI) is 7.0–13.0 mg for adults (Department of Health (UK), 1991; Institute of Medicine (USA), 2001). Unfortunately, the diets of many people across the world lack sufficient Zn for their adequate nutrition (White and Broadley, 2009; Bouis and Welch, 2010; Stein, 2010; Sayre et al., 2011). This has been attributed to sourcing produce from land with low mineral phytoavailability, eating crops with inherently low tissue mineral concentrations, or consuming processed foods. It has been estimated that almost one-third of the world’s population consumes less Zn than the US RDA and that Zn-deficiency contributes 1.9% of the overall burden of disease caused by major health risks worldwide (World Health Organization, 2002; Hotz and Brown, 2004). This has considerable socio-economic impacts (Stein, 2010).

Dietary Zn intakes can be increased through a variety of interventions (Stein, 2010). These include both agronomic and genetic biofortification of edible crops (Graham et al., 1999, 2001, 2007; White and Broadley, 2005, 2009; Cakmak, 2008; Khoshgoftarmanesh et al., 2009; Bouis and Welch, 2010; Martínez-Ballesta et al., 2010). Agronomic biofortification can be achieved by increasing soil Zn phytoavailability or by applying Zn-fertilizers. This requires appropriate infrastructures, but can be very successful in regions where mineral fertilizers are used to increase crop yields and Zn is added to these at the point of manufacture or distribution (Cakmak, 2004, 2009). Genetic biofortification is predicated on increasing Zn acquisition from the soil and its accumulation in edible portions. In most agricultural soils there is sufficient Zn to produce biofortified crops for many years, provided it becomes phytoavailable (Graham et al., 1999). Genetic biofortification strategies are, of course, ineffective if there is insufficient Zn present in the soil. Most economic analyses suggest that genetic strategies toward Zn biofortification are more practical, enduring, and cost effective than dietary diversification, supplementation, or food fortification programs for increasing dietary Zn intakes of vulnerable populations (Horton, 2006; Graham et al., 2007; Stein et al., 2007; Ma et al., 2008; Bouis and Welch, 2010; Meenakshi et al., 2010; Stein, 2010).

Several national and international projects are addressing Zn biofortification of edible crops (Graham et al., 2001, 2007; Cakmak, 2004; Pfeiffer and McClafferty, 2007; White and Broadley, 2009; Bouis and Welch, 2010; Stein, 2010; Sayre et al., 2011). The target Zn concentrations set by the HarvestPlus program are 28 μg g⁻¹ dry matter (DM) in polished rice, 38 mg kg⁻¹ DM in wheat grain, 38 mg kg⁻¹ DM in maize, 66 mg kg⁻¹ DM in pearl millet, 56 mg kg⁻¹ DM in beans, 34 mg kg⁻¹ DM in cassava roots, and 70 mg kg⁻¹ DM in roots of sweet potatoes (Bouis and Welch, 2010). These target concentrations are considered to be conservative, and have been exceeded in breeding lines of rice, wheat, and maize (Bouis and Welch, 2010). This article asks whether higher target Zn concentrations can be achieved and, more broadly, what the physiological limits to Zn biofortification of crops might be. It considers (a) the physiological requirements and tolerance of Zn in crop plants, (b) the uptake and distribution of Zn between and within plant organs, (c) agronomic strategies to 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.

Although the total Zn concentrations in many soils are sufficient to support mineral-dense crops (Graham et al., 1999) Zn uptake by plants is often limited by its phytoavailability and acquisition by roots (Broadley et al., 2007; White and Broadley, 2009). Agronomic strategies seek to improve Zn phytoavailability in the soil, for example by remedying soil alkalinity, adopting appropriate crop rotations or introducing beneficial soil microorganisms (Rengel et al., 1999; He and Nara, 2007; Fageria, 2009; White and Broadley, 2009), or to deliver phytoavailable Zn through the application of Zn-fertilizers to soil or foliage (Cakmak, 2004; Graham et al., 2007; Fageria, 2009; White and Broadley, 2009; Bouis and Welch, 2010). Common inorganic Zn-fertilizers include ZnSO₄, ZnO, and synthetic Zn-chelates (Fageria, 2009; White and Broadley, 2009). When Zn-fertilizers are applied to foliage, it is particularly important that the Zn compounds used are readily soluble, enter the leaf apoplast, and can be taken up by plant cells (Haslett et al., 2001; Cakmak, 2008; Brown, 2009). This avoids the accumulation of Zn salts on the surface of leaves or in the leaf apoplast, which can interfere with photosynthesis and cell function, and promotes the translocation of Zn from leaves to phloem-fed tissues.

Zinc concentrations in roots, leaves, and stems can be increased readily by applying Zn-fertilizers to the soil in plants growing on most, but not all, soils and by foliar application of Zn-fertilizers (Figure 3; Rengel et al., 1999; Cakmak, 2008; Fageria, 2009; Sagardoy et al., 2009; White and Broadley, 2009; Bouis and Welch, 2010). Thus, Zn concentrations in these tissues will be limited solely by Zn toxicity. When Zn-fertilizers are added to the soil, root tissues often exhibit higher Zn concentrations than shoot tissues, and it is likely that plant Zn accumulation and yield is limited by Zn toxicity to root cells under these conditions. Critical leaf Zn concentrations for most crop plants lie between 100 and 700 mg kg⁻¹ DM when Zn-fertilizers are applied to the soil (MacNicol and Beckett, 1985; Fageria, 2009). It is possible that, when Zn-fertilizers are applied foliarly, higher leaf Zn concentrations might be achieved without loss of yield (Cakmak et al., 1999; White et al., submitted).

Soil or foliar applications of Zn-fertilizers can also increase Zn concentrations in phloem-fed tissues, such as fruits, seeds, and tubers (Rengel et al., 1999; Cakmak, 2004, 2008; Fang et al., 2008; White et al., 2009; Cakmak et al., 2010a,b). However, increasing Zn concentrations in these tissues requires adequate Zn mobility in the phloem and, unless Zn-fertilizers are applied directly or they have functional xylem continuity, the mobility of Zn in the phloem will limit their Zn accumulation (Rengel et al., 1999; White and Broadley, 2005, 2009; Broadley et al., 2007; Stomph et al., 2009; Cakmak et al., 2010a,b; White et al., submitted). The relationship between seed Zn concentration and Zn phytoavailability often follows a saturation curve. For example, when rice is grown in quartz sand irrigated with a complete nutrient solution, Zn concentrations in brown rice reach a maximum of about 50–110 mg Zn kg⁻¹ DM depending upon variety (Figure 3; Jiang et al., 2008). Similarly, the relationship between tuber Zn concentration and foliar Zn application in “Maris Piper” potatoes grown in the field followed a saturation curve, reaching a maximum of about 30 mg Zn kg⁻¹ DM (Figure 3; White et al., submitted). In the phloem, Zn is thought to be transported either as Zn–NA or complexed with small proteins (White and Broadley, 2009). In cereals, grain Zn concentration is correlated with grain protein concentration (e.g., Zhao et al., 2009; Cakmak et al., 2010a,b; Gomez-Becerra et al., 2010), and the limit to grain Zn concentration can be increased by higher N-fertilizer applications (Hao et al., 2007; Kutman et al., 2010, 2011a,b; Shi et al., 2010). Similarly, there is a significant relationship between tuber Zn concentration and tuber N concentration among potato genotypes (White et al., submitted) and tuber Zn concentrations can be increased by N-fertilization (Hlusek et al., 1997). Nevertheless, Zn concentrations in phloem-fed tissues rarely exceed 30–100 mg kg⁻¹ DM.

Genetic strategies to increase Zn concentrations in edible portions seek to exploit genetic variation in the acquisition of Zn from the soil, Zn accumulation in edible portions and tolerance to high tissue Zn concentrations (Pfeiffer and McClafferty, 2007; White and Broadley, 2009). There is considerable genetic variation in Zn concentration in most edible crops (Figure 2). Edible roots often have low Zn concentrations, but researchers have reported >14-fold variation in root Zn concentrations among 600 cassava genotypes (Chávez et al., 2005) and 2.2-fold variation among 20 carrot genotypes (Nicolle et al., 2004). Zinc concentrations in edible greens are often relatively high and trials have indicated 2.5-fold variation in leaf Zn-concentration among 19 chickpea genotypes (Ibrikci et al., 2003), 6.7-fold variation in leaf Zn-concentration among 111 Brassica rapa genotypes (Wu et al., 2007), 12.3-fold variation in leaf Zn-concentration among 327 spinach genotypes (Grusak and Cakmak, 2005), and over 26-fold variation among 339 Brassica oleracea genotypes (Broadley et al., 2010). Absolute Zn concentrations and genetic variation in Zn concentrations are often lower in seeds than in leaves. Nevertheless, between 2.2- and 11.6-fold variation in seed Zn concentrations have been observed in large core collections of cereal germplasm (e.g., Yang et al., 1998; Graham et al., 1999; Bänziger and Long, 2000; Gregorio et al., 2000; Reddy et al., 2005) and between 1.8- and 6.6-fold variation in seed Zn concentrations in large core collections of legume germplasm (Raboy et al., 1984; Islam et al., 2002; Grusak and Cakmak, 2005; Pfeiffer and McClafferty, 2007; White and Broadley, 2009). Fruits generally have low Zn concentrations, and studies report about twofold variation among three to six cultivars in strawberries (Hakala et al., 2003), apples (Iwane, 1991), and plantains (Davey et al., 2007). Similarly, although tuber Zn concentrations are relatively low, 2.4-fold and 3.1-fold variation has been reported among 26 potato and 23 yam genotypes, respectively (Agbor-Egbe and Trèche, 1995; White et al., 2009). These data indicate that breeding for increased Zn concentrations is, in principle, feasible for most edible crops. Indeed, genetic loci (QTL) affecting Zn concentrations in cereal grain (Distelfeld et al., 2007; Stangoulis et al., 2007; Genc et al., 2009; Lonergan et al., 2009; Peleg et al., 2009), bean seeds (Guzmán-Maldonado et al., 2003; Cichy et al., 2005, 2009; Gelin et al., 2007; Blair et al., 2009, 2010, 2011), brassica leaves (Wu et al., 2008; Broadley et al., 2010), and potato tubers (N. K. Subramanian et al., unpublished data) have been identified.

It has been speculated that the constitutive expression of a suite of Zn-deficiency inducible responses through the overexpression of bZIP19 and bZIP23 transcription factors could be used to increase Zn accumulation in edible portions of crop plants (Assunção et al., 2010). Specific targets for the manipulation of root Zn concentrations include transport proteins in the plasma membrane and tonoplast of root cells that facilitate the uptake and sequestration of Zn in the vacuole, together with enzymes involved in the synthesis of compounds that bind Zn²⁺ in the rhizosphere, cytoplasm, and vacuole. Targets for the manipulation of shoot Zn concentrations include (a) transport proteins in the plasma membrane of root cells that facilitate Zn uptake and delivery to the xylem, or root structural modifications that facilitate apoplastic movement to the xylem, (b) enzymes involved in the synthesis of compounds that facilitate Zn movement through the root symplast or apoplast and in the xylem, (c) transport proteins in the plasma membrane and tonoplast of shoot cells that facilitate Zn uptake and vacuolar Zn sequestration, and (d) enzymes involved in the synthesis of compounds that detoxify Zn²⁺ within and outside shoot cells. Targets for the manipulation of Zn concentrations in phloem-fed tissues will additionally include transport proteins in the plasma membrane of shoot cells that facilitate the loading of Zn into the phloem and compounds that facilitate Zn movement in the phloem.

Most published studies describing GM strategies that increase Zn concentrations in plant tissues have been performed on “model” plants such as A. thaliana and, even when studies have been performed on crop species, data on commercial yields are rarely presented. Yield per plant can have a substantial effect on the tissue concentrations of mineral elements through dilution effects caused by plant growth (Jarrell and Beverly, 1981; Davis, 2011). Higher-yielding genotypes often have lower Zn concentrations in their edible tissues than lower-yielding genotypes (Monasterio and Graham, 2000; Garvin et al., 2006; Murphy et al., 2008; White et al., 2009; Zhao et al., 2009). Thus, it is important to consider whether any increase in tissue Zn concentration is simply a consequence of slower growth or reduced yields.

Overexpressing genes encoding Zn-transporters catalyzing Zn²⁺ influx to root cells often increases root Zn concentrations but reduces leaf Zn concentrations. This has been observed when overexpressing OsZIP4 or OsZIP5 in rice (Ishimaru et al., 2007; Lee et al., 2010) and when overexpressing AtZIP1 in cassava (Sayre et al., 2011). Overexpressing a root plasma membrane Zn-transporter, AtZIP1, in barley had no effect on leaf Zn concentration, but reduced seed weight and increased seed Zn concentrations from 31 to 61–85 mg kg⁻¹ DM (Ramesh et al., 2004). Overexpression of the ZIPs TcZNT5 and TcZNT6 in A. thaliana had no consistent effects on root or shoot Zn concentrations (Wu et al., 2009). Overexpressing genes encoding proteins that transport Zn²⁺ into the vacuole, such as AtMTP1 (AtZAT1), AtMTP3, or TgMTP1, increases Zn uptake and root Zn concentrations, but rarely shoot Zn concentrations, when expressed in roots, and increases Zn uptake and leaf Zn concentrations, but rarely seed Zn concentrations, through systemic induction of Zn-deficiency responses when expressed in the shoot (van der Zaal et al., 1999; Arrivault et al., 2006; Gustin et al., 2009). The expression of AtMTP1 (AtZAT1) increased Zn concentrations in roots of cassava about fourfold to 40 mg kg⁻¹ DM (Sayre et al., 2011). The ferric reductase defective 3/manganese accumulator 1 (frd3 = man1) null mutants of A. thaliana, which constitutively express genes allowing increased rhizosphere Fe(III) reductase activity (AtFRO2), Zn uptake (AtIRT1), and tissue NA concentrations, have greater shoot Zn concentrations, but similar seed Zn concentrations, to wild-type plants (Rogers and Guerinot, 2002). Similarly, a mutant of A. thaliana (opt3.2) with reduced expression of AtOPT3 that shows constitutive expression of AtFRO2 and AtIRT1 has higher Zn concentrations in leaves, stems, and seed than wild-type plants (Stacey et al., 2008). Overexpressing enzymes involved in the synthesis of phytosiderophores, either HvIDS3 (encoding a dioxygenase, referred to as Iron-Deficiency Specific clone 3) alone or both HvNAS1 (encoding nicotianamine synthase) and HvNAAT (encoding nicotianamine aminotransferase) increased concentrations of Zn in seeds of paddy-grown rice from 11.2 mg kg⁻¹ DM to 13.4 and 15.3 mg kg⁻¹ DM, respectively (Suzuki et al., 2008). Leaf Zn concentrations can be increased in A. thaliana by reducing the expression of AtHMA2, which is thought to catalyze Zn²⁺ efflux across the membranes of root cells (Eren and Argüello, 2004) or by overexpressing the gene encoding AtHMA4, which is thought to load Zn into the xylem (Verret et al., 2004).

The overexpression of genes encoding nicotianamine synthase (NAS) often leads to increased Zn concentrations in leaves and grain. The overexpression of HvNAS1 in tobacco increased leaf Zn concentrations from 16 to 39 mg kg⁻¹ DM and seed Zn concentrations from 20 to 35 mg kg⁻¹ DM (Takahashi et al., 2003). The overexpression of OsNAS3 in rice also increased leaf and seed Zn concentrations (Lee et al., 2009; Johnson et al., 2011). Seed Zn concentrations of glasshouse-grown plants were increased from ∼40 to 79 mg kg⁻¹ DM (Johnson et al., 2011) and those of paddy-grown plants were increased from 16 to 35 mg kg⁻¹ DM (Lee et al., 2009). Constitutive overexpression of OsNAS2 in rice resulted in an increase in seed Zn concentrations from ∼23 to ∼60 mg kg⁻¹ DM (Lee et al., 2011) and from ∼40 to 95 mg kg⁻¹ DM (Johnson et al., 2011) in two independent studies. Constitutive overexpression of OsNAS1 in rice resulted in an increase in seed Zn concentrations from ∼40 to 59 mg kg⁻¹ DM (Johnson et al., 2011). The maximum Zn concentrations in polished grain of transgenic rice expressing Pvferritin in the endosperm and AtNAS1 throughout the plant was 34 mg kg⁻¹ DM, about 50% greater than in wild-type plants (Wirth et al., 2009). Earlier studies of plants expressing only Gmferritin found ∼56 mg Zn kg⁻¹ DM in brown rice (Vasconcelos et al., 2003). The overexpression of genes increasing the production of glutathione, phytochelatins, and total thiols resulted in increased leaf Zn concentrations in Indian mustard (Brassica juncea) grown on soils with high Zn phytoavailability (Bennett et al., 2003). Seed Zn concentrations can be increased in wheat by increasing the expression of a NAC transcription factor (NAM-B1) that accelerate senescence and increase remobilization of mineral elements from leaves to developing grain (Uauy et al., 2006).

Zinc concentrations in roots, leaves, and stems can be increased greatly by applying Zn-fertilizers (Figure 3). The accumulation of Zn in these tissues appears to be limited by Zn toxicity. In some crops, such as cereals and beans, root Zn concentrations of between 500 and 5000 mg kg⁻¹ DM have been reported without loss of yield (Reichman, 2002). Leaf Zn concentrations ≥100 mg kg⁻¹ DM, and perhaps up to 700 mg kg⁻¹ DM, can be achieved without loss of yield in Zn-tolerant crops when Zn-fertilizers are applied to the soil (Fageria, 2009). It is possible that greater Zn concentrations in non-woody shoot tissues might be achieved using foliar Zn-fertilizer applications. These Zn concentrations approach that found in raw beefsteak, which approximates 200–250 mg Zn kg⁻¹ DM (U.S. Department of Agriculture, Agricultural Research Service,2011). By contrast, Zn concentrations in fruits, seeds, and tubers are severely limited by Zn transport in the phloem. This might be a consequence of the need to maintain low Zn²⁺ concentrations in phloem sap to avoid cellular toxicity. Increased production of compounds that chelate Zn²⁺, such as NA, can increase Zn concentrations in the phloem and its delivery to phloem-fed tissues. Agrochemicals that increase the concentrations of Zn-complexes in the phloem might be used to increase Zn concentrations in fruits, seeds, and tubers. Thus, application of Zn-fertilizers, especially in combination with nitrogen fertilizers, can increase Zn concentrations in seeds of cereals to about 100 mg kg⁻¹ DM and Zn concentrations in seeds of legumes to about 120 mg kg⁻¹ DM (see Agronomic Strategies to Increase Zinc Concentrations of Edible Crops).

Since there is appreciable variation in Zn concentrations of edible tissues of food crops, conventional breeding for increased Zn concentrations appears feasible (see Genetic Variation in Zinc Concentrations of Edible Crops). It is possible that breeding can increase Zn-tolerance in root and leaf crops and increase Zn mobility in the phloem in fruit, seed, and tuber crops. Improving Zn-tolerance in root and leaf crops and increasing phloem Zn mobility in fruit, seed, and tuber crops might also be addressed through GM technologies (see Genetic Modification Strategies for Zinc Biofortification of Edible Crops). Several transgenic crop plants have been created that have greater Zn concentrations in their edible tissues than conventional varieties. These include cassava roots with about 40 mg Zn kg⁻¹ DM (Sayre et al., 2011), brown rice with 56–95 mg Zn kg⁻¹ DM (Vasconcelos et al., 2003; Johnson et al., 2011), and barley grain with 85 mg Zn kg⁻¹ DM (Ramesh et al., 2004). Nevertheless, it is possible that the general homeostatic regulation of tissue Zn concentrations through Zn acquisition and distribution within the plant could be a major constraint to increasing Zn concentrations in edible portions (see Plant Physiology). A critical role of Zn in plant cells is the control of transcription, translation, and protein activity through cytoplasmic Zn²⁺ concentration (Broadley et al. 2007). The key to Zn-homeostasis in plant cells is likely, therefore, to pivot on cytoplasmic Zn²⁺ concentration. Increasing tissue Zn concentrations will require increased biosynthesis of Zn-complexes and effective sequestration of Zn in non-vital compartments, such as the vacuole, to avoid unwanted perturbations in cytoplasmic Zn²⁺ concentration. For this, lessons might be learnt from Zn-hyperaccumulator plants.

To increase Zn concentrations in edible crops, future research should focus on (i) integrating agronomic and genetic strategies to increase Zn transport to phloem-fed tissues and (ii) identifying the mechanisms effecting Zn-homeostasis in plant cells and strategies to manage subcellular Zn compartmentalization.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.