Review TRENDS in Plant Science Vol.12 No.12

Transgenic strategies for the nutritional enhancement of plants

Changfu Zhu, Shaista Naqvi, Sonia Gomez-Galera, Ana M. Pelacho, Teresa Capell and Paul Christou

Universitat de Lleida, Av. Alcalde Rovira Roure, 191, E-25198 Lleida, Spain

The nutrients in the human diet ultimately come from these are impractical in developing countries, where plants. However, all our major food crops lack certain poverty is widespread and over three billion people earn essential vitamins and minerals. Although a varied diet under US$2 per day. An alternative approach is to tackle provides adequate nutrition, much of the human popu- the problem of nutritionally poor staple crops at its source lation, particularly in developing countries, relies on by increasing their nutritional qualities through a strategy staple crops, such as or , which does not provide known as ‘biofortification’, which should translate into the full complement of essential nutrients. is improved diets [4]. a significant public health issue in most of the developing Several different tactics for biofortification have been world. One way to address this problem is through the adopted. Perhaps the simplest of these tactics relies on an enhancement of staple crops to increase their essential increase of the mineral content of plants through the nutrient content. Here, we review the current strategies addition of the appropriate mineral as an inorganic com- for the biofortification of crops, including mineral fertili- pound to the fertilizer. This method has been successful in zation and conventional breeding but focusing on trans- some instances [5] but depends on the crop species and genic approaches which offer the most rapid way to cultivar, the mineral itself and the quality and properties develop high-nutrient commercial cultivars. of the soil, making the strategy difficult to apply generally (Box 1). Another approach is to improve the nutrient content of plants by conventional breeding, sometimes in Nutrition and malnutrition in humans combination with mutagenesis. The identification of More than 50% of the human population worldwide has no mineral-dense cereal varieties and the use of marker- access to a healthy variety of fresh food [1]. In developing assisted selection to introgress (see Glossary) such traits countries, this reflects the reliance on a staple diet of into widely cultivated, adapted germplasm (see Glossary) cereals, such as rice or maize, and the lack of fresh fruit, have recently been reviewed [4] (Box 2). Mutagenesis has vegetables, meat and fish. Milled cereal grains are poor also been used to produce crops with higher nutrient levels, sources of lysine, vitamin A, folic acid, iron, zinc and including the lysine-enriched maize opaque-2 mutant [6]. selenium, which are essential for normal growth and One problem with these conventional breeding approaches metabolism; yet, about one-third of the world’s population is the time taken to identify useful traits and breed them (mostly in sub-Saharan Africa and South-east Asia) rely on into elite cultivars (see Glossary). cereals as their only source of nutrition [1]. Even in the Many transgenic strategies are also available to West, lifestyle choices and lack of education can lead to an enhance the nutritional value of crops; these strategies improper diet and, hence, deficiencies in some vitamins offer a rapid way to introduce desirable traits into and iron, although this can be addressed to a certain degree by dietary supplements and fortification programs. In developing countries, such interventions have not been Glossary successful because they rely on adequate and sustained Adapted germplasm: A collection of genetic resources for an organism, in the funding, efficient distribution to scattered populations and case of plants usually a seed collection, which has been adapted to a particular political stability [2]. Therefore, malnutrition and the environment. Bioavailable: The proportion of an ingested nutrient that can be absorbed and deficiency diseases resulting from this (e.g. beriberi, pella- used by the body. gra, , , anemia) remain a significant public- Constitutive: Expressed in all tissues all the time rather than in specific organs health challenge [3]. With a major focus of or developmental stages, or in response to a given stimulus. Elite cultivar: A variety of a crop or ornamental plant that has been selected for governments and non-governmental organizations alike, it its superior performance or characteristics. These characteristics are retained is timely to look at the strategies available to scientists to during propagation and set the cultivar aside from other members of the same improve the nutritional value of food. species. Expression stability: The tendency for gene expression levels in transgenic plants to remain stable over multiple generations (i.e. not affected by Strategies to address malnutrition epigenetic silencing phenomena). Introgress: The movement of a gene from one species into the gene pool of Dietary diversity and supplementation with vitamins and another by backcrossing an interspecific hybrid with one of its parents. minerals might be the best ways to tackle malnutrition but Permissive locus: A locus for transgene integration which is permissive for high transgene expression levels and transgene stability. Rhizosphere: The ecological zone that surrounds the roots of plants. Corresponding author: Christou, P. ([email protected]). www.sciencedirect.com 1360-1385/$ – see front matter ß 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.tplants.2007.09.007 Review TRENDS in Plant Science Vol.12 No.12 549

Box 1. Biofortification through fertilizer application Box 2. Nutritional improvement through conventional breeding Although conceptually simple, the biofortification of crops through the application of fertilizers containing essential mineral micronu- All our staple crops show significant genetic variation in essential trients is complicated by several factors, such as the application nutrient content, which allows breeding programs to be used to method, soil composition, mineral mobility in the plant and its improve the levels of minerals and vitamins, and mutation accumulation site. Therefore, this strategy has been successful for strategies to be used for the introduction of new alleles. For some minerals but not for others, and the success rate varies example, different rice genotypes show a fourfold variation in iron according to the geographical location. and zinc levels [53] and up to 6.6-fold variation has been reported in For example, both iodine and selenium are mobile in soil and in beans and peas [54]. Several international organizations have plants, thus biofortification with iodine [50] and selenium [51] embarked on programs to study the genetic variation of mineral fertilizers has been used to increase mineral levels, with particularly content and the feasibility of breeding programs for nutritional encouraging results for selenium biofortification in Finland and enhancement. The Consultative Group on International Agricultural New Zealand. Because zinc (Zn) is also highly mobile, zinc fertilizers, Research (CGIAR) is foremost among these organizations having such as ZnSO4, can increase the yield of cereals and legumes in launched the HarvestPlus initiative in concert with the International Zn-deficient soils, and can also increase Zn concentration in the grain Center for Tropical Agriculture (CIAT) and the International Food (although some genotypes accumulate more zinc than others [4]). Policy Research Institute (IFPRI) to breed staple food crops high in By contrast, iron (Fe) has a low mobility in soil because Fe(II) (in mineral nutrients (http://www.harvestplus.org/index.html). the form of FeSO4) is rapidly bound by soil particles and converted Conventional breeding could also be useful for the enhancement into Fe(III); therefore, Fe fertilizers have little impact on Fe levels in of organic nutrients, although there has not been as much of a plants [52]. In addition, large quantities of metal applied to soils can coordinated international effort to achieve progress in this area. limit plant growth as well as affecting other soil organisms. In Even so, crosses between inbred lines have been used to identify nutrient-poor ecosystems, plant growth is limited by the lack of quantitative trait loci affecting carotene and tocopherol content in nitrogen, phosphorus and potassium, as a result, the application of maize [55], as well as carotene content in carrot and tomato [56], and NPK fertilizers that contain these three minerals together can these could be used in the future for nutritional improvement increase root growth and result in a higher transfer of micronu- programs. trients from the soil to the plant. However, this depends on the effect One of the significant disadvantages of conventional breeding on soil pH: NH4 causes acidification of the rhizosphere (see compared to transgenic strategies is its reliance on alleles already in Glossary), which enhances Fe and Zn transfer from the soil to the the species genepool. In some cases, this can be overcome by plant, whereas NO3 makes the soil more alkaline, reducing this crossing to distant relatives and thus introgressing traits into transfer rate. Foliar sprays of FeSO4 or iron chelates enable the commercial cultivars. Such a strategy could be used to increase direct uptake of Fe, which might be a suitable alternative method. selenium levels in , because modern bread show little However, even if the mineral is absorbed efficiently, it can increase variation in selenium content whereas wild wheats have much mineral levels in leaves but not necessarily in fruits or seeds, higher levels [57]. Alternatively, new traits can be introduced directly because the relative efficiency of mineral transport varies depending in the commercial cultivars by mutagenesis. This approach has been on the different plant organs [52]. used in several cereals and legumes to generate low-phytate One final point is that fertilizers must be applied varieties, which, therefore, have higher levels of bioavailable iron regularly and are, therefore, costly as well as potentially damaging and zinc [58]. to the environment. Overall, such strategies are applicable to specific crop and mineral scenarios but cannot be applied generally as a strategy to improve nutrition. deficient in lysine and threonine, whereas legumes such as peas and beans tend to lack methionine and cysteine. Because up to 50% of the world’s population relies on elite varieties. Transgenic strategies differ from other cereals or legumes as a staple diet, there has been great approaches in that novel genetic information is introduced interest in developing novel varieties with increased essen- directly into the plant’s genome. The chosen approach tial amino acid content to prevent deficiency diseases, such depends predominantly on whether the nutritional com- as . A simple approach is the expression of pound is synthesized de novo by the plant or obtained from recombinant storage proteins with desirable amino acid the environment. Organic molecules, such as amino acids, profiles. Early examples of this approach include the fatty acids and vitamins, are synthesized by the plant. expression of pea (Pisum sativum) legumin, which has a Increasing the nutritional value requires some form of high lysine content, in rice and wheat grains [8,9], and the metabolic engineering with the aim of increasing the amount of these desirable compound, decreasing the Table 1. Relative merits of biofortification strategies amount of a competitive compound or even extending an Mineral Conventional Transgenic fertilization breeding/mutagenesis approaches existing metabolic pathway to generate a novel [7]. Advantages: Advantages: uses Advantages: rapid; By contrast, mineral nutrients are obtained by the plant simple; intrinsic properties of unconstrained by from the environment; therefore, mineral enhancement inexpensive; rapid crop; few regulatory genepool; targeted involves strategies to increase uptake, transport and/or enhancement. constraints. expression in edible accumulation in harvestable tissues. Different biofortifica- organs; applicable directly to elite tion strategies are compared in Table 1. The following cultivars. sections discuss case studies that show how different Disadvantages: Disadvantages: Disadvantages: transgenic approaches have been used to enhance the only works with depends on existing regulatory landscape; nutrient content of crops. minerals; not gene pool; takes a long political and socio- suitable for iron; time; traits might need economic issues very dependent on to be introgressed from relevant to transgenic Essential amino acids crop and cultivar; wild relatives; possible plants; possible Most crops are deficient in certain essential amino acids not possible to intellectual property intellectual property that cannot be synthesized de novo by humans; for target edible constraints. constraints. example, cereal grains such as rice and wheat tend to be organs. www.sciencedirect.com 550 Review TRENDS in Plant Science Vol.12 No.12 expression of sunflower seed albumin, which is rich in in metabolism, cardiovascular health, inflammatory methionine, in the laboratory model lupin [10]. Similarly, responses, blood pressure regulation, etc. Many of the AmA1 from the Prince’s feather (Amaranthus hypochon- involved in fatty acid biosynthesis and degra- driacus), which encodes seed albumin, was expressed in dation have been characterized, and much research has potato and was shown to double the protein content and been carried out on transgenic approaches for the modifi- increase the levels of several essential amino acids [11]. cation of oil and fat content in plants. This can be applied to This approach does not always work well, as shown by the enhance levels of the essential fatty acids linoleic acid and expression of sunflower-seed albumin in rice [12] and a-linolenic acid, and to synthesize the very-long-chain chickpea [13]. There was little impact on the content of polyunsaturated fatty acids (VLC-PUFAs) arachidonic sulfur-containing amino acids, which was the goal of the acid (ARA), eicosapentenoic acid (EPA) and docosahexe- studies, perhaps because of the regulatory mechanism that noic acid (DHA), which are usually sourced from fish oils enables seed storage-protein composition to be adjusted in [21]. response to sulfur and nitrogen levels [14]. Synthetic A breakthrough study was published in 2004 [22] in proteins (i.e. proteins designed from first principles) can which three transgenes were expressed constitutively also be expressed to boost the levels of particular amino in Arabidopsis, leading to the production of EPA and acids. For example, a synthetic protein matched to human ARA in vegetative tissues at levels of 3.0% and 6.6% of amino acid requirements was expressed in cassava [15]. total fatty acids. The final transgenic lines were generated The inability of heterologous proteins to change by three rounds of transformation with the genes encoding abruptly and predictably the essential amino acid content D9-elongase, D8-desaturase and D5-desaturase, thereby of target crops probably reflects the limited free amino acid reconstructing a bacterial pathway that is not present in pool, which provides the substrates for protein synthesis plants. The elevated accumulation of VLC-PUFAs seemed [12,13,15]. In all higher plants, lysine, threonine and to have no deleterious effect on plant growth. A similar methionine are synthesized from aspartic acid via a path- study achieved somewhat lower enhancement by introdu- way that is highly branched and under complex feedback cing genes from the conventional biosynthesis pathway control. Two key enzymes are aspartate kinase (AK), (D6-desaturase, D6-elongase, D5-desaturase) and expres- which functions early in the pathway and is inhibited sing them under a seed-specific promoter [23]. This boosted by both lysine and threonine, and dihydrodipicolinate the total level of D6-desaturated fatty acids to >25%, but synthase (DHPS), which functions in the lysine-specific EPA and ARA each accounted for <1% of this fatty acid branch and is inhibited by lysine alone (Figure 1a). Feed- pool, which suggests a bottleneck during elongation; this back-insensitive versions of the bacterial enzymes have bottleneck will be the target for future experiments. been expressed in plants with promising results: the free Recent progress has also been made in the reconstitu- lysine content of Arabidopsis seeds was increased either tion of the DHA biosynthetic pathway in transgenic plants. by expressing a bacterial, feedback-insensitive DHPS For example, transgenic Arabidopsis seeds with total fatty transgene or by knocking out the lysine catabolism path- acids containing up to 0.5% DHA were produced by expres- way, resulting in 12-fold or fivefold gains in lysine, respect- sing a bifunctional zebrafish D6/D5-desaturase and a dip- ively. However, where both the transgene and knockout teran D6-elongase in the seed, producing EPA for were combined in the same Arabidopsis line, increases of subsequent conversion into DHA by enzymes from the alga 80-fold over wild-type levels were achieved [16]. Similarly, Pavlova salina [24]. Another significant achievement was the expression of DHPS in maize increased levels of free the production of DHA in soybean seeds at levels of up to lysine from <2% to almost 30% of the free amino acid pool, 3% of total fatty acids using a similar strategy to that with concomitant increases in threonine. Analogous outlined above, but using a D6-elongase from the fungus approaches have increased the lysine levels in canola Mortierella alpina and adding an v-3 microsomal desatur- and soybean [17]. Expression of a feedback-insensitive ase from the fungus Saprolegnia diclina to maximize the subunit of rice anthranilate synthase in rice resulted in accumulation of n-3 VLC-PUFAs [25]. To date, the most the accumulation of tryptophan to twice the wild-type successful results have come from a study in which the level in the grain [18]. VLC-PUFA biosynthetic pathway was reconstituted in It might be possible to combine the heterologous protein Brassica junacea and was supplemented with a v-3 desa- and amino acid pool approaches in a single plant line to turase from Phytophtora infestans,aD12-desaturase from boost the levels of essential amino acids and to include Calendula officinalis and an acyltransferase from Thraus- them simultaneously in heterologous storage proteins. tochytrium aureum [26]. This could also be combined with strategies to regulate In each of these studies, there has been a striking the storage-protein profile in cereals to favor more nutri- increase in flux through the VLC-PUFA biosynthetic path- tious proteins, such as the creation of a new version of way, leading to the accumulation of ARA and EPA. How- maize opaque-2 by RNA interference using a construct ever, the levels of DHA are still low, and this might reflect targeting the genes encoding the 22-kDa zein storage the poor efficiency with which EPA is elongated and/or protein [19] and the elevation of methionine levels in maize desaturated, which, perhaps, indicates a competition be- kernels by increasing the stability of Dzs10 mRNA [20]. tween alternative pathways within the plant. Further investigation of pathway competition, feedback and Essential and very-long-chain fatty acids branching will allow the development of plants with Fatty acids are another target for biofortification because higher levels of DHA and other essential fatty acids in some of them are essential nutrients with diverse roles the future. www.sciencedirect.com Review TRENDS in Plant Science Vol.12 No.12 551

Figure 1. Manipulation of amino acid and vitamin synthesis pathways in plants. (a) Synthesis of aspartate-family amino acids in plants. Aspartate is converted to 3-aspartylphosphate by AK, which is then converted to 3-aspartic semialdehyde by ASAD. This can be converted into homoserine by HSD; HSD is then converted to phosphohomoserine by HSK and threonine by TS. Alternatively, phosphohomoserine can be converted through several enzymatic steps into methionine. 3-Aspartic semialdehyde can be converted into 2,3-dihydrodipicolinate by DHDPS and then through multiple steps into lysine. In the unmodified pathway, lysine inhibits DHDPS and AK, whereas threonine inhibits HSD and AK. Block arrows show enzymatic steps (broken blocks indicate multiple steps). Thin broken lines indicate feedback inhibition. Abbreviations: AK, aspartate kinase; ASAD, aspartic semialdehyde dehydrogenase; DHDPS, dihydrodipicolinate synthetase; HSD, homoserine dehydrogenase; HSK, homoserine kinase; TS, threonine synthetase; (b) Enzymatic steps and metabolic products in the b-carotene biosynthesis pathway that are absent in cereal grains. In plants, the first committed step in the synthesis of carotenes is the joining of two GGDP molecules to form the precursor phytoene. The conversion of phytoene into b-carotene requires three additional activities: phytoene desaturase, b-carotene desaturase and lycopene b-cyclase. Cereal grains, such as rice, accumulate GGDP but lack the subsequent enzymes in the pathway, so the genes for all three enzymes are required to form b-carotene. Abbreviation: GGDP, geranylgeranyl diphosphate. (c) In plants, tocopherol synthesis requires input from two metabolic pathways. The shikimate pathway (left) generates homogentisic acid, which forms the aromatic ring of tocopherol, whereas the side chain is derived from phytyldiphosphate, a product of the MEP pathway. These precursors are joined together by HPT to form the intermediate MPBQ. MPBQ is the for two enzymes, tocopherol cyclase and MPBQ methyltransferase. Tocopherol cyclase produces d-tocopherol, whereas MPBQ methyltransferase introduces a second methyl group to form another intermediate, 2,3-dimethyl-5-phytylbenzoquinol. The action of tocopherol cyclase on 2,3-dimethyl-5-phytylbenzoquinol produces g-tocopherol. Both g-tocopherol and d-tocopherol are substrates for g-TMT, producing a- and b-tocopherol, respectively. Abbreviations: HPT, homogentisic acid prenyltransferase; MEP, methylerythritol phosphate; MPBQ, 2-methyl-6-phytylbenzoquinol; g-TMT, g-tocopherol methyltransferase.

Vitamins A and E An initial breakthrough was the development of a rice Vitamin A deficiency is prevalent in the developing world line expressing a daffodil (Narcissus pseudonarcissus) phy- and is probably responsible for the deaths of two million toene synthase, enabling the accumulation of the vitamin A children annually. In surviving children, vitamin A precursor phytoene in the endosperm [27],followedshortly deficiency is a leading, but avoidable, cause of blindness thereafter by the original ‘Golden Rice’ variety, expressing [see WHO report (1995) Global prevalence of Vitamin A two daffodil enzymes and one from Erwinia uredovora, deficiency; http://www.who.int/nutrition/publications/vad_ which reconstituted the entire pathway and enabled the global_prevalence/en/index.html]. Humans can synthesize rice endosperm to accumulate b-carotene, resulting in its vitamin A if provided with the precursor molecule b-caro- eponymous golden color [28]. In the best lines, the grain tene (also known as provitamin A), a pigment found in many contained >1.5 mgofb-carotene per gram of dry weight. plants but not in cereal grains. Therefore, a strategy was Additional Golden Rice varieties have been generated that devised to introduce the correct metabolic steps into rice contain only two recombinant enzymes (daffodil phytoene endosperm to facilitate b-carotene synthesis (Figure 1b). synthase and Erwinia phytoene desaturase [29])and,most www.sciencedirect.com 552 Review TRENDS in Plant Science Vol.12 No.12 recently, the ‘Golden Rice II’ variety in which the daffodil approach is required for minerals because they are not phytoene synthase gene is replaced with its more efficient synthesized in the plant but obtained from the immediate maize homolog, resulting in a 23-fold improvement in environment. Transgenic strategies to increase the mineral b-carotene content (up to 37 mggÀ1) [30]. This has led to content of crop plants have concentrated mainly on iron and similar progress in other crops, including, most recently, zinc (which are most frequently deficient in human diets) ‘yellow potato’ [31], ‘orange cauliflower’ [32,33], carrots with and have used two distinct approaches: (i) increasing the enhanced b-carotene in the taproot [34] and tomatoes with efficiency of uptake and transport to harvestable tissues, the b-carotene metabolic pathway transferred to the plas- and (ii) increasing the amount of bioavailable (see Glossary) tids (which enable higher expression levels) [35]. A recently mineral accumulating in the plant, i.e. how much is acces- developed potato variety containing the phytoene synthase, sible after digestion [4]. phytoene desaturase and lycopene b-cyclase from Erwinia One consideration particular to iron is that, although herbicola contained 114 mg carotenoids per gram of dry Fe(III) is the most abundant form of iron in the soil, plants weight and 47 mg b-carotene per gram of dry weight [36]. cannot absorb iron in this state. Two different pathways These studies show that investigations into alternative are used to convert Fe(III) into Fe(II) for absorption: gene sources and expression strategies can have a profound strategy II (graminaceous plants, i.e. grasses and cereals) effect on achievable b-carotene levels. Although the original involves the secretion of chelating chemicals, called phy- Golden Rice line was criticized because of the large amounts tosiderophores, that bind to Fe(III) before absorption, of rice that would need to be consumed, the latest fortified whereas strategy I (all other plants) involves the expres- potato [36] contains enough b-carotene to provide 50% of the sion of Fe(III) reductases and the subsequent absorption of recommended daily allowance in one 250 g serving, assum- Fe(II). Specific transport proteins are then used to absorb ing a realistic b-carotene-to-retinol conversion rate of 6:1. the minerals into the roots, and they are transported Whereas vitamin A is a single molecule, vitamin E is a through the phloem to sink tissues, such as leaves in the group of eight hydrophobic compounds (known as vita- form of complexes with nicotianamine, which specifically mers), the most potent of which is a-tocopherol. Dietary chelate Fe(II). The overexpression of several of these trans- vitamin E is obtained mainly from seeds, and its function port and chelating proteins promotes metal accumulation. in the body is to prevent the oxidation and polymerization For example, efforts to increase iron uptake in roots by of unsaturated fatty acids. Vitamin E deficiency leads to genetic modification have focused on strategy I plants, for general wasting, kidney degeneration and infertility. In instance, through the expression of iron transport proteins plants, tocopherol synthesis requires input from two meta- [41]. For strategy II plants, iron accumulation can be bolic pathways (Figure 1c). enhanced by the production of higher levels of phytosider- Work in Arabidopsis has shown that the levels of vita- ophores; for instance, the expression in rice of the barley min E activity can be increased either by increasing the naat-A and naat-B genes, encoding nicotianamine amino- total amount of vitamin E or by shifting the metabolic flux (involved in phytosiderophore biosynthesis) towards a-tocopherol. Shintani and Della-Penna [37] resulted in increased iron uptake [42]. There seems to be expressed the Synechocystis PCC6803 and Arabidopsis some cross-talk between the iron and zinc transport path- genes encoding g-tocopherol methyltransferase (g-TMT) ways because transgenic plants and mutants with over- in Arabidopsis seeds, resulting in a fundamental shift from expressed Fe(III) reductases and iron transporters also g/d-toa/b-tocopherol; this showed that nutritional show increased zinc accumulation. This could reflect the enhancement in plants was possible without altering total enhanced synthesis of nicotianamine, which increases the vitamin E levels. By contrast, the expression of Arabidop- mobilization of both metals in the vascular tissue. Thus, sis homogentisic acid prenyltransferase (HPT) produced the overexpression of nicotianamine synthase also leads to twice the level of vitamin E found in normal seeds [38], iron and zinc accumulation; for example, the expression of whereas expression of the Escherichia coli tyrA gene, which barley HvNAS1 in tobacco (Nicotiana tabacum) doubled encodes a dual-function enzyme (chorismate mutase and the iron and zinc concentrations in leaves [43]. prephenate dehydrogenase) resulted in up to three times The second approach to mineral biofortification is to the normal level of vitamin E [39]. A more recent study was express recombinant proteins that enable minerals to be based on the simultaneous expression of multiple genes in stored in a bioavailable form, for instance, overexpression soybean; in this case, the Arabidopsis genes encoding of soybean ferritin in rice using an endosperm-specific 2-methyl-6-phytylbenzoquinol (MPBQ) methyltransferase promoter [44,45]. Ferritin is an iron-storage protein. This and g-TMT were used [40]. Transgenic soybeans showed a produced rice grains with three [44] and 4.4 [45] times significant elevation in the total amount of vitamin E the wild-type iron levels. Iron concentration was also activity (fivefold greater than that of wild-type plants), measured in polished grains, because minerals are lost which was attributable mainly to an eightfold increase during polishing, but the levels of iron (and zinc) were still in the levels of a-tocopherol, from its normal 10% of total higher than in non-polished wild-type grains [45]. By con- vitamin E to over 95%. Recently, plants have been engin- trast, the use of a constitutive (see Glossary) promoter to eered to accumulate several other vitamins, including drive ferritin expression resulted in elevated iron levels in folate and ascorbate (vitamin C) (Box 3). the leaves of transgenic rice plants, but not in grains, owing to higher expression levels of ferritin in vegetative tissues Iron and zinc [46]. Although metabolic engineering is the most suitable Although the total amount of iron and other minerals in approach to fortify plants with organic nutrients, a different plants is an important determinant of nutritional quality, www.sciencedirect.com Review TRENDS in Plant Science Vol.12 No.12 553

Box 3. Recent work on other vitamins

Although vitamin enhancement in plants has focused predomi- nantly on vitamins A and E, there has been some recent progress with other vitamins. is associated with several diseases but the most important is dietary folate deficiency during early pregnancy, which is linked to the occurrence of neural tube defects, such as spina bifida [59]. In the West, the incidence of these birth defects is reduced by the availability of fresh green vegetables, government-sponsored fortification programs and the provision of folate supplements during pregnancy [59]. In developing countries, these measures are largely unavailable, resulting in an estimated 200 000 severe birth defects every year [see the UNICEF report: The Micronutrient Initiative (2004), Vitamin and : A global progress report. http://www.micronutrient.org/reports/]. Be- cause cereals are a poor source of folate, various strategies have been used in an attempt to fortify cereal grains with this vitamin [60]. Folates are tripartite molecules consisting of pteridine, p-amino- benzoate (PABA) and glutamate moieties, with pteridines synthesized in the cytosol and PABA in the plastids. These moieties are then transported to the mitochondria, where they condense to form dihydropteroate and are conjugated to glutamate (Figure I). Attempts to increase folate levels in tomato (Lycopersicon esculentum) and Arabidopsis initially involved the overexpression of GTP cyclohydro- Figure I. (a) Structure of the monoglutamyl form of tetrahydrofolate, although lase I (GCHI) [61,62] in the pteridine branch of the pathway, which plant folates can have additional polyglutamyl tails. (b) Simplified plant folate increased the amount of pteridine 140-fold but only increased overall biosynthesis. Pteridines are synthesized in the cytosol (outer area): DHN; DHM; folate levels by twofold owing to the limited free PABA pool in the HMDHP (additional Ps indicate additional phosphate groups). PABA is synthesized from chorismate in the plastids via ADC. HMDHP and PABA are imported plastids. This was addressed by generating plants in which GCHI and into the mitochondria for conversion into tetrahydrofolate. The enzymes aminodeoxychorismate synthase (ADCS) (a key enzyme in the PABA GCHI and ADCS, as discussed in the main text, are shown. Abbreviations: branch) were overexpressed simultaneously, thus increasing the ADC, aminodeoxychorismate; ADCS, aminodeoxychorismate synthase; DHN, levels of both precursors [63]. Fruits from these tomato plants dihydroneopterin; DHM, dihydromonapterin; GCHI, GTP cyclohydrolase I; contained up to 20 times the wild-type amount of folate, such that HMDHP, hydroxymethyldihydropterin; PABA, p-aminobenzoate. one serving (100 g) contains the full adult recommended daily allowance (400 mg). Progress has also been made in the enhancement of ascorbate (vitamin C), which is an essential metabolic substrate, electron donor, enzyme and antioxidant, whose absence leads to scurvy. At least three separate metabolic pathways converge on ascorbate in plants, which means several strategies can be used to enhance its synthesis (Figure II). The most successful include the overexpression of a rat L-gulonolactone oxidase gene in tobacco, which resulted in a sevenfold increase in the ascorbate pool [64], and the expression of myoinositol oxidase in Arabidopsis, resulting in a threefold enhancement [65]. Encouraging results have also been achieved by reducing the rate at which ascorbate is recycled, which can be up to 40% per day in some plants. For example, the overexpression of wheat dehydroascorbate reductase in tobacco (targeted to chloroplasts) increased the ascorbate pool fourfold [66]. Modulation of ascorbate oxidase activity has also led to moderate increases in the ascorbate pool [67]. In tomato, an unexpected five- to sixfold increase in ascorbate levels was recently achieved through the suppression of mitochondrial malate dehydrogenase [68]. Figure II. Simplified plant ascorbate biosynthesis and recycling. The enzymes gulonolactone oxidase, myoinositol oxidase, dehydroascorbate reductase and ascorbate oxidase, as discussed in the main text, are shown. Abbreviations: Gal, galactose; GalL, galactonolactone; GDP, guanosine diphosphate; Gul, gulose; GulL, gulonolactone; Man, mannose; P, phosphate; UDP, uridine diphosphate.

what really matters is the amount of bioavailable iron and increased. The combined use of multiple strategies for iron how well it is absorbed by the human gut. Phytic acid (also fortification therefore provides the maximum levels of known as phytate) is an antinutritional compound that bioavailable iron. chelates minerals and reduces their bioavailability in the gut. Therefore, a combined approach has been developed Concluding remarks and future perspectives that involves the expression of both ferritin and phytase (a Malnutrition is a significant challenge, particularly in the fungal enzyme that breaks down phytate); this has been developing world, where measures that are commonplace achieved in rice [47] and maize [48]. The rice grains con- in developed countries (varied diet, fortification schemes tained twice the wild-type amount of iron, and simulations and dietary supplements) are largely absent. Transgenic of digestion and absorption using the maize kernels biofortification strategies could help to alleviate malnu- showed that the amount of bioavailable iron had also trition although further work is required to identify and www.sciencedirect.com 554 Review TRENDS in Plant Science Vol.12 No.12 manipulate relevant metabolic pathways and to improve 12 Hagan, N.D. et al. (2003) The redistribution of protein sulfur in the degree of nutritional improvement that can be achieved. transgenic rice expressing a gene for a foreign, sulfur-rich protein. Plant J. 34, 1–11 Other nutrients could also be targeted, such as , 13 Chiaiese, P. et al. (2004) Sulphur and nitrogen nutrition influence the and minerals, such as magnesium and calcium. response of chickpea seeds to an added, transgenic sink for organic Future goals include the combination of multiple nutri- sulphur. J. Exp. Bot. 55, 1889–1901 tional improvements into elite crop varieties without dis- 14 Tabe, L. et al. 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Plant Science Conferences in 2008

Keystone joined meetings: ‘Plant innate immunity’ and ‘Plant hormone signalling’ 10–15 February 2008 Keystone, USA http://www.keystonesymposia.org

50th Maize Genetics Conference 27 February – 2 March 2008 Washington, DC, USA http://www.maizegdb.org/maize_meeting/2008/

4th EPSO meeting ‘Plants for Life’ 22–26 June 2008 Toulon, France http://www.epsoweb.org/catalog/conf2008.htm

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