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Journal of Cereal Science 46 (2007) 308– 326
Molecular genetic approaches to increasing mineral availability and
vitamin content of cereals
Henrik Brinch-Pedersen
, Søren Borg, Birgitte Tauris, Preben B. Holm
Department of Genetics and Biotechnology, Faculty of Agricultural Sciences, University of Aarhus, Research Centre Flakkebjerg,
DK-4200 Slagelse, Denmark
Received 17 November 2006; received in revised form 12 February 2007; accepted 12 February 2007
Abstract
The present paper summarizes the current state of knowledge on molecular genetic approaches to increasing iron and zinc availability
and vitamin content in cereals. We have also attempted to integrate the scientific issues into the wider context of human nutrition. In the
cereal grain, iron and zinc are preferentially stored together with phytate in membrane-enclosed globoids in the protein storage vacuole
(PSV) found in the aleurone and the embryo scutellum. The PSV is accordingly central for understanding mineral deposition during
grain filling and mobilization of minerals during germination. Recent studies in Arabidopsis have led to the first identification of iron and
zinc transporters of the PSV and further illustrate some of the dynamics associated with mineral and phytate transport and deposition
into the vacuole. This provides new opportunities for modulating iron and zinc deposition in the cereal grain. Current strategies towards
increasing the iron content of the endosperm are largely based on the expression of legume ferritin genes in an endosperm-specific
manner. However, it is apparent that this approach, at least in rice, only allows a two- to three-fold increase in the iron content of the
grain due to exhaustion of the iron stores in leaves. Further increases thus have to rely on additional uptake and transport of iron from
the root. Phytate is generally considered to be the single most important anti-nutritional factor for iron and zinc availability. In the
current paper we summarize attempts to increase phytase activity in the grain by transformation and evaluate the potential of this
approach as well as the reduction of phytate biosynthesis for improving the bioavailability of iron and zinc. Vitamins constitute the
second important group of micronutrients in grain and we discuss current efforts to increase the amounts of provitamin A, vitamin C and
vitamin E.
r2007 Elsevier Ltd. All rights reserved.
Keywords: Minerals; Bioavailability; Cereals; Vitamins
1. Introduction
Human and animal metabolism, growth and well-being
depend on an appropriate and balanced intake of nutrients.
At present, 49 nutritional components are known to be
essential and indispensable for sustaining human life
(Welch and Graham, 2004). These comprise water and
carbohydrates, 10 essential amino acids, linoleic and
linolenic acids, seven mineral macroelements, 16 mineral
microelements and 13 vitamins (Table 1). In the context of
human nutrition the microelements and the vitamins are
grouped under the common term micronutrients.
Micronutrient deficiencies have plagued the world’s
population from antiquity. Classical examples of vitamin
deficiencies comprise scurvy (vitamin C), beriberi (vitamin
ARTICLE IN PRESS
www.elsevier.com/locate/jcs
0733-5210/$ - see front matter r2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jcs.2007.02.004
Abbreviations: AEA, average energy allowance; CDF, cation diffusion
facilitator; DALY, disability adjusted life yeards; DHAR, dehydroascor-
bate reductase; ER, endoplasmic reticulum; ESADDI, estimated safe and
adequate daily dietary intake; GDP, gross domestic product; HAP, high
available phosphate; HGGT, homogentisic acid geranylgeranyl transfer-
ase; IRT, iron transporter; ITP, iron transport protein; LF, lactoferrin;
lpa, low phytic acid; MIPS, myo-inositol phosphate 3-phosphate synthase;
MR, minimum requirement; MTP, metal tolerance proteins; PSV, protein
storage vacuole; RDA, recommended daily allowance; SCFA, short-chain
fatty acids; YS, yellow stripe
Corresponding author. Tel.: +45 89993651; fax: +45 89993501.
E-mail address: Henrik.BrinchPedersen@agrsci.dk
(H. Brinch-Pedersen).
B1), pellagra (niacin), night blindness and xeropthalmia
(vitamin A), rickets (vitamin D) and pernicious anaemia
(vitamin B12 deficiency) (see McDowell, 2006 for a brief
historic review). Likewise, it has been known for many
years that particular diseases such as goitre result from a
lack of iodine. The last few years have seen increased
interest in the effects of mineral deficiencies and it has
become increasingly apparent that lack of minerals may
have similarly severe negative consequences on human
health and well-being as vitamin deficiencies. Several
human populations have suboptimal intakes of calcium,
manganese, copper and selenium (Bouis et al., 2003;
Combs, 2001;Frossard et al., 2000;Welch, 2002;Welch
and Graham, 2004) and a current estimate suggests that
more than half the world’s population, primarily women of
childbearing age and children, suffer from iron and zinc
deficiencies. These deficiencies have major negative effects
on human health, development, working ability and
quality of life (see Bouis, 2000;Welch and Graham, 2004
for review). They also have severe economic impacts, for
example, iron malnutrition has been calculated to result in
a loss of about 5% of the gross domestic product (GDP) in
Asian regions (Hunt, 2002).
In a few cases such as iodine, zinc, iron and selenium,
micronutrient deficiencies can be attributed to particular
geological conditions where the soils are low in these
minerals. However, in most cases micronutrient deficiencies
result primarily from a too narrow food base due to
poverty, with food intake being almost exclusively based
on staples like rice, wheat, maize, cassava and other crops
that are low in micronutrients. Fortification and supple-
mentation strategies have proved to be unrealistic in
several developing countries for economic reasons, due to
poorly developed education and communication systems
and to lack of general infrastructure. As a consequence
there is increasing interest in breeding for staple crops that
have higher contents of micronutrients: biofortification.
A major driver in this development has been the Harvest-
Plus programme (Welch and Graham, 2004,http://www.
harvestplus.org/), an international alliance of research
laboratories and the CGIAR agricultural research centres,
which has been instrumental in defining, initiating and
integrating initiatives for micronutrient breeding and
nutritional evaluation and has established strategies for
the dissemination and testing of nutritionally improved
cultivars.
Cereals are the primary staple food of humankind and
are accordingly central in strategies aiming at alleviating
micronutrient deficiencies by biofortification. However, it
should be noted that globally 670 million tons of cereals are
used annually as livestock feed which is more than a third
of the total cereal production (Speedy, 2003). Micronu-
trient requirements are often better characterized for
livestock than for humans and developing more nutritious
crops for feed will therefore be an important tool for
increasing agricultural productivity in developing countries
and thereby alleviate poverty. It is also apparent that plant
productivity is highly dependent on the mineral status.
Hence, the application of zinc fertilizers to zinc deficient
soils leads to drastic changes in yields and quality (Cakmak
et al., 1999) as well as to resistance against plant diseases
(Thongbai et al., 1993).
Supplementation and fortification with micronutrients
have met with several difficulties. The initial requirement is
to identify the daily need for micronutrients and this is
complicated by the fact that the uptake of the micro-
nutrients will be highly dependent on the food matrix as
well as on the presence of compounds that may promote or
inhibit the uptake. Furthermore, micronutrients are often
lost during processing and cooking of the food. Bioforti-
fication is faced with numerous challenges but also offers
new solutions. The initial challenge is to use conventional
or molecular breeding to increase the micronutrient
content, preferably in the form of bioavailable minerals
and biologically active vitamins or vitamin precursors.
Supplementary strategies comprise breeding for increased
contents of components that promote nutrient uptake and
reduce amounts of inhibitors of uptake. In this context it
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Table 1
Recommended nutrient intakes for males and females between the ages of
25 and 50 years
Vitamin Assessment Male Female
Energy (kcal) AEA 2900 2200
Protein (g) AEA 63 50
Vitamin A
(mg retinol equivalent) RDA 1000 800
Vitamin D (mg) RDA 5 5
Vitamin E (mg a-tocopherol equivalent) RDA 10 8
Vitamin K (mg) RDA 80 65
Riboflavin (mg) RDA 1.7 1.3
Niacin (mg niacin equivalent) RDA 19 15
Thiamin (mg) RDA 1.5 1.1
Pantothenic acid (mg per day) ESADDI 4–7 4–7
Vitamin B
6
(mg) RDA 2 1.6
Vitamin B
12
(mg) RDA 2 2
Biotin (mg per day) ESADDI 30–100 30–100
Folate (mg) RDA 200 180
Vitamin C (mg) RDA 90 60
Ca (mg) RDA 800 800
P (mg) RDA 800 800
Mg (mg) RDA 350 280
Na (mg) MR 500 500
K (mg) MR 2000 2000
Cl (mg) MR 750 750
Fe (mg) RDA 10 15
Zn (mg) RDA 15 12
Cu (mg) ESADDI 1.5–3.0 1.5–3.0
Se (mg) RDA 70 55
I(mg) RDA 150 150
Mn (mg) ESADDI 2–5 2–5
Mo (mg) ESADDI 75–250 75–250
Cr (mg) ESADDI 50–200 50–200
F (mg) ESADDI 1.5–4.0 1.5–4.0
From Welch and Graham (2004): AEA: average energy allowance, RDA:
recommended daily allowance, ESADDI: estimated safe and adequate
daily dietary intake, MR: minimum requirement.
H. Brinch-Pedersen et al. / Journal of Cereal Science 46 (2007) 308–326 309
should also be noted that there appear to be significant
causal relationships between the bioavailabilities of miner-
als, vitamins and amino acids, where vitamins such as
vitamin E, vitamin D, choline, niacine and provitamin A
can promote the uptake of selenium, calcium phosphorus,
methionine, tryptophan, iron and zinc, respectively.
Finally, it is essential that the agronomic performance
and eating quality of the biofortified cultivars are as good,
or preferably better, than those of cultivars currently
grown. The task is formidable but as discussed below, very
significant progress has already been achieved for cereals
and our understanding of the genetic and biochemical
mechanisms for the metabolism of at least some of the
micronutrients is increasing rapidly.
In this review we summarize and address issues that are
particularly relevant to biofortification by genetic trans-
formation with a focus on iron and zinc bioavailability and
vitamin content in cereals. Current efforts to exploit
natural variation in micronutrient content as well as other
health-related aspects of cereals used for human food and
animal feed are presented in separate articles in this issue.
Mineral uptake and transport as well as vitamin biosynth-
esis have been reviewed extensively and will not be repeated
here. Likewise, it is outside our scope to review the highly
complex issue of mineral and vitamin uptake in the human
digestive tract and micronutrient interactions with the food
matrix in general.
2. The distribution and speciation of iron and zinc in the
cereal grain
The cereal grain consists of four major tissues: the
embryo, the aleurone, the starchy endosperm and the outer
layers (testa and pericarp). Elemental microanalyses of
wheat grain sections reveal that phosphate, potassium,
calcium, manganese, iron and zinc appear to be distributed
in a similar way with the highest concentrations being in
the aleurone and the embryo (in particular the scutellum)
and a low concentration in the starchy endosperm. In
contrast sulphur, copper and chloride are quite evenly
distributed between the different tissues (Mazzolini et al.,
1985). Analyses of milled, polished and de-embryonated
grains clearly support the preferential localizations of iron
and zinc in the aleurone and the embryo (Table 2). There is
some variation between the results of individual milling
and polishing experiments which can be attributed to
differences in the processing regimes, with longer milling
and extraction processes increasingly removing the outer
layers and their mineral content. However, the losses of
iron and zinc are typically of the order of 50% or more
with a tendency to greater losses for iron than for zinc
indicating a more peripheral localization of iron. Specific
staining with Prussian Blue for iron (rice, Krishnan et al.,
2003) and dithizone staining for zinc (wheat, Ozturk et al.,
2006) also illustrate that the two minerals are present in the
highest concentrations in the aleurone and the embryo but
with some staining for zinc in the endosperm (Fig. 1).
A few studies have addressed the dynamics of iron and
zinc deposition during grain filling. Zinc accumulates in the
glumes and the testa/pericarp during wheat grain develop-
ment but is predominantly located in the embryo and the
aleurone of the mature grain (Ozturk et al., 2006;Pearson
and Rengel, 1994). During early barley grain development,
90% of the zinc is present in the testa/pericarp, whereas
during mid-development and in the mature grain only 40%
and 5%, respectively, of the zinc is present in this tissue.
Iron accumulates in equal amounts in the testa/pericarp
and the endosperm tissues of barley grains at early to mid
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Table 2
The mineral content in non-milled and milled seeds of rice and wheat
Non-milled (ppm) Milled (ppm) % Loss Reference
Rice
Fe 21.6 3.9 81 Doesthale et al. (1979)
Zn 14.3 13.5 6 Doesthale et al. (1979)
Fe 5.7 4.0 30 Heinemann et al. (2005)
1
Zn 19.8 20.9 5Heinemann et al. (2005)
1
Fe 16 5 (90% extraction) 69 Welch and Graham (1999)
Zn 28 17 (90% extraction) 39 Welch and Graham (1999)
Fe 20 9–11 55 Frossard et al. (2000)
Zn 20 11–12 45 Frossard et al. (2000)
Fe 8.8 4.1 53 Baurenfiend and DeRitter (1991)
Zn 33 18 45 Baurenfiend and DeRitter (1991)
Fe 12–22 1.6–3.8 80 Stenbæk and Østergaard (2006)
Zn 26–40 19–31 20 Stenbæk and Østergaard (2006)
Wheat
Fe 38 22 (70% extraction) 42 Welch and Graham (1999)
Zn 37 12 (70% extraction) 40 Welch and Graham (1999)
Fe 45 13 71 Frossard et al. (2000)
Zn 35 8 77 Frossard et al. (2000)
1
On fresh weight basis.
H. Brinch-Pedersen et al. / Journal of Cereal Science 46 (2007) 308 –326310
developmental stages. However, at maturity only 15–20%
of the total iron is located in the pericarp, with the
endosperm and aleurone together containing about 70% of
the total iron and the embryo 7–8% (Duffus and Rosie,
1976).
Iron and zinc are complexed with various organic
compounds at all stages in the plant, from uptake through
transport to deposition, in order to eliminate their
reactivity. A detailed knowledge of this so-called speciation
is of paramount importance when evaluating their trans-
port and deposition mechanisms as well as their bioavail-
ability. Present evidence indicates that the protein storage
vacuole of the embryo and the aleurone is a primary
depository for iron and zinc in cereals, with the two
minerals being stored together with phytate in so-called
globoids (Fig. 2). In species such as pea 90% of the iron has
been reported to be sequestered by ferritin (Marentes and
Grusak, 1998). There is to our knowledge no information
on the extent of ferritin sequestering of iron in cereals but
we have found significant ferritin gene expression in
different tissues of the wheat grain, as discussed below.
Finally, as iron is a co-factor for a range of enzymes and
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Fig. 1. Iron and zinc detection in rice and wheat. Iron stain with Prussian blue of transverse sections of non-transgenic control (a,b) and transgenic rice
grains (c,d) expressing the soybean ferritin gene. The accumulation of iron in the control material is restricted to the aleurone (al) and embryo (em) layer
while in transgenic seeds iron is present in the entire grain, including the endosperm (endo). E, diphenyl thiocarbazone (DTZ) staining of wheat grain
enriched with Zn by foliar application. A–D adapted from (Krishnan et al., 2003) with permission from the Current Science Association, E adapted from
(Ozturk et al., 2006) with permission from Blackwell Synergy.
Cytoplasm
ZIP YSL
Aleurone cell
InsPn
InsP6
GOLGI
ER
Minerals
VIT1
MTP1
P-type ATP’ase
HMA
ATP
ADP+Pi
Zn2+
NRAMP
Fe
InsP6
GC
Fig. 2. Model for phytate biosynthesis, mineral storage and mineral transport in an aleurone cereal cell. Electron micrographs of protein storage vacuole
(PSV) prepared by high-pressure freezing and freeze substitution (HPF–FS). GC, globoid cavity; SG, soft globoid; In, electron dense inclusion. The
transport of phytate from the endoplasmatic reticulum (ER) to the PSV where it interacts with mineral cations. Plant cells can import minerals using
membrane associated ZIP family and YSL family transporters. To keep homeostasis and avoid toxic effects inside the cytoplasm, minerals e.g., zinc and
iron can be transported into the vacuole through MTP (metal tolerance protein) and VIT (vacuole iron uptake transporter). Export of minerals from the
vacuole is mediated by NRAMP transporters. The cells can export zinc and other metals into the apoplast through HMA (heavy metal ATPase) metal
pumps. See also text for details. The PSV is adapted from (Lonsdale et al., 1999) with permission from Blackwell Synergy and the authors.
H. Brinch-Pedersen et al. / Journal of Cereal Science 46 (2007) 308–326 311
zinc a co-factor for transcription factors as well as
enzymes, it is to be expected that some iron and zinc will
be present bound to these proteins.
2.1. The protein storage vacuole
Protein storage vacuoles are ubiquitous in the aleurone
layer and embryo of seeds and contain stored proteins, a
range of hydrolytic enzymes and phytate. Upon water
imbibition the dormant and dehydrated embryo and
aleurone layer are activated. The protein storage vacuoles
coalesce and acidify triggering the activation of hydrolytic
enzymes. In parallel, the embryos start synthesizing and
secreting the plant hormone gibberellic acid. The aleurone
and the scutellum (cotyledon) of the embryo subsequently
develop into very active secretory tissues with a wide range
of hydrolytic enzymes being synthesized de novo and
secreted into the endosperm to degrade cell walls, starch
granules and storage proteins (Bethke et al., 1998). At the
same time the nutrient uptake mechanisms of the embryo
are activated and upregulated.
The protein storage vacuole therefore contains the
nutrients and enzymes required to kick-start germination,
although little is known about the enzymes that are
present. However, the phytate globoid has been subjected
to energy dispersive X-ray microanalyses in a range of
species (see Lott, 1984 for review). In wheat it was shown
that P, K and Mg are the most universally present minerals
with spatial variation in the distributions of Ca, Fe and Mn
(Lott, 1984). Globoid crystals from the aleurone layer
furthest from the embryo contain Ca, whereas aleurone
globoid crystals near the embryo contain Fe. Manganese
was found in globoid crystals from the pro-vascular tissue
in the base and middle-region of the radicle and not in
globoid crystals elsewhere in the radicle or in other parts of
the grain. Throughout the embryo a few globoid crystals
contain Ca, but no specific patterns of Ca distribution are
evident. Mazzolini et al. (1985) described the elemental
distribution in the various tissues of the mature wheat grain
using an X-ray and nuclear scattered proton analysis and
found that zinc was present in the scutellum in particular
but also in the aleurone. In rice, Wada and Lott (1997)
found that globoids present in most of the embryo tissue
and the zinc were commonly present in globoids in the
scutellum and in the provascular tissues.
It is generally assumed that the minerals form salts with
phytate and precipitate. However, Casaravilla et al. (2006)
have argued that the stoichiometry has not yet been
established for a plant phytate. They analysed the parasitic
cestode Echinococcus granulosus that secretes globoids into
an extracellular matrix and found that the globoids were
largely composed of Ca
5
H
2
L, 16H
2
O (L representing the
fully deprotonated phytate) with Mg as a minor compo-
nent. It is clear, therefore, that we still have limited
knowledge of the formation of salts between the minerals
and phytate in plants. In the present paper we will refer to
phytate as a mineral salt, phytate.
Ultrastructural analyses including freeze etch studies
(Fig. 2)(Buttrose, 1971;Lonsdale et al., 1999;Swift and
Buttrose, 1972) have revealed the presence of a membrane
surrounding the globoids. Jiang et al. (2001) suggested that
the globoid compartment was a lytic vacuole internalized
in the protein storage vacuole and provided evidence that
the membrane possessed H
+
-pyrophosphatase and the
tonoplast intrinsic protein g-TIPp. However, we currently
have no further information on the nature of this
membrane or the origin of the globoid compartment.
Recent studies in Arabidopsis have shed additional light
on mineral and phytate deposition in the globoids (Otegui
et al., 2002). Ultrastructural analyses and energy dispersive
X-ray spectroscopy revealed that Mg, Ca, K, Mn and also
Zn are stored together with phytate in three different
compartments in the developing seed. In the embryo,
typical globoids of Mg, Ca, K and phytate were deposited
in protein storage vacuoles. However, the chalazal en-
dosperm had high concentrations of Mn and phytate in the
endoplasmic reticulum (ER) while the vacuoles of the
endosperm appeared to preferentially contain Zn–phytate
salts. Zn and Mn were thereafter gradually transferred to
the developing embryo. It was proposed that lower
amounts of myo-inositol phosphates or phytate were
transported into the lumen of the ER with the transient
formation of Mn–phytate while there was transport of
phytate from the ER to the vacuoles where Zn-rich
globoids were formed. A phytate transport route from
the ER via the Golgi to the protein storage vacuole with
subsequent formation of Ca, Mg and K phytate globoids
was proposed for the embryos. The uptake of Mn into the
embryo also coincided with the synthesis of the two Mn-
binding enzymes namely Mn-super oxide dismutase and
the Mn-binding protein of the oxygen-evolving complex.
The influx and efflux mechanisms of minerals into the
protein storage vacuole have been further explored in
Arabidopsis. Two probably functionally redundant trans-
porters of the NRAMP family, termed AtNRAMP3 and
AtNRAMP4, were able to retrieve iron from the vacuolar
globoids of germinating seeds. However, in lines with
mutations in the two genes encoding these transporters,
iron was stored properly but could not be retrieved
(Lanquar et al., 2005). An iron vacuolar uptake transpor-
ter, termed VIT1, has also recently been identified in
Arabidopsis protein storage vacuoles (Kim et al., 2006).
VIT1 is a plant homologue of the yeast CCC1 transporter
that mediates iron storage by transporting iron from the
cytosol to the vacuole (Li et al., 2001). Abolishment of the
VIT1 function resulted in a more diffuse distribution of
iron in the germinating seed while in wild-type seeds the
iron was mainly localized in the provascular strands of the
hypocotyl, radicle and cotyledons (Kim et al., 2006).
There is to our knowledge no information on the
dynamics of phytate transport and deposition or on the
transport mechanisms that facilitate the influx and
potential efflux of iron and zinc into the protein storage
vacuoles of the aleurone. However, work on Arabidopsis
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H. Brinch-Pedersen et al. / Journal of Cereal Science 46 (2007) 308 –326312
shows that the transport of iron and zinc into the globoids
may be complex, involving the ER and transfer across
protein storage vacuoles as well as across the globoid
membrane. It is also possible that additional transporters
are required. Members of the cation diffusion facilitator
(CDF) family, also called metal tolerance proteins (MTP),
catalyse the efflux of metal ions from the cytoplasm to the
outside of the cell or into sub-cellular compartments. In
Arabidopsis MTP1 and MTP3 are involved in cellular zinc
detoxification by transporting the metal ions into the
vacuole. AtMTP1 is localized in the vacuolar membrane of
leaf and root cells while expression of AtMTP3 is primarily
observed in the roots of seedlings. Consequently, AtMTP1
seems to play a role in storage of zinc in the vacuoles of the
leaves whereas AtMTP3 immobilize zinc in vacuoles of
root cells, thereby restricting its mobilization to the shoot
(Arrivault et al., 2006;Drager et al., 2004). Another
transporter that may be involved in zinc sequestration in
the vacuole is the tonoplast-located MHX transporter from
Arabidopsis, which exchanges protons with Mg, Zn and Fe
ions. The affinity of AtMHX is in the micromolar range for
Zn and Fe and in the millimolar range for Mn, which is in
accordance with the concentrations of these minerals found
in the cytosol (Elbaz et al., 2006). Future work will reveal
whether homologues of these genes are involved in the
transport across the membrane of the protein storage
vacuole and the globoid.
3. Phytate biosynthesis and deposition
Phytate (InsP
6
, phytic acid, myo-inositolphosphate
1,2,3,4,5,6-hexakisphosphate), the primary storage form
of phosphate and inositol in cereal seeds, is considered to
be the single most important anti-nutritional factor for the
bioavailability of minerals (Bouis, 2000). It has a high P
content (28.2%), with six phosphate groups and 12
hydrogens titratable in water. During germination phytate
is degraded by a specific group of enzymes, the phytases
(Brinch-Pedersen and Hatzack, 2006).
Phytate accumulates rapidly during seed development
and can account for up to several percent of the seed dry
weight (Lott, 1984). An analysis of 30 cereals revealed that
the phytate content ranged from 0.06% of the dry weight in
basmati rice to 2.22% in durum wheat (Reddy, 2002). In
small grained cereals such as rice and wheat, the starchy
endosperm is almost devoid of phytate, with 80% or
more being in the aleurone and pericarp and 10% in the
scutellum. In contrast, maize stores 88% of the phytate in
the embryo, 3% in the endosperm and about 10% in the
outer layers (O’Dell et al., 1972).
Our knowledge of the biosynthetic genes and of the
genetic regulation and localization of phytate biosynthesis
is poor. There appear to be two separate pathways, one
being a phosphoinisitide pathway with the second invol-
ving the sequential phosporylation of the myo-inositol
backbone (Fig. 3, see Brinch-Pedersen and Hatzack, 2006
for recent review). As described above, the phytate is
deposited as globoids within storage protein vacuoles in the
aleurone and the scutellum and is digested by de novo
synthesized phytases during germination.
4. Phytate and mineral bioavailability
Phytates, and to some extent the lower isomers of
inositol phosphate InsP
5
,InsP
4
and InsP
3,
are strong
chelators and bind positively charged proteins, amino
acids and minerals in insoluble complexes in the digestive
tract (Brinch-Pedersen and Hatzack, 2006;Kies et al.,
2006;Sandberg and Andlid, 2002). When moved from the
stomach (pH 2–5) to the small intestine (pH 6.5–7.5) , the
phytate becomes more ionized and begins to bind cations
(for a review of pH effects on phytate–mineral interactions
see Champagne and Phillippy, 1989). The decreasing order
of stability of mineral phytate complexes in vitro is Zn
2+
,
Cu
2+
,Ni
2+
,Co
2+
,Mn
2+
,Ca
2+
and Fe
2+
(Cheryan,
1980).
Phytate must be hydrolysed by 60–70% in order to make
the phytate-bound minerals biologically available, (Lon-
nerdal, 2000;Sandberg et al., 1996;Sandberg et al., 1999;
Sandstrom and Sandberg, 1992). The stepwise hydrolysis
of phytate is catalysed by phytase (myo-inositol hexaki-
sphosphate 3- and 6-phosphohydrolase) [EC 3.1.3.26 and
EC 3.1.3.8], with minerals and orthophosphate being
released and a series of lower isomers of myo-inositol
phosphates and eventually myo-inositol being formed
(Brinch-Pedersen et al., 2002). Several human nutritional
studies have shown that phytase-mediated reduction of
phytate via fermentation of food with yeast, germination of
seeds or treatment of food with phytase, significantly
improves the absorption of minerals (see Weaver and
Kannan, 2002 for review). Hydrolysis of phytate with acid
plus salt has also been shown to improve the absorption of
iron (Hurrell et al., 1992).
Phytate is almost indigestible by monogastric animals
because they have little or no phytase activity in their
digestive tract. This implies that the digestion of phytate
and the effect of phytate on mineral bioavailability in
animals including humans are affected by a range of other
factors. As discussed above there are substantial differences
in the phytate content of different cereals. Furthermore,
the phytate content also depends on the processing regimes
where milling and polishing remove the phytate and
minerals. A major factor is the amount of preformed
phytase in the mature seed which differs widely between
different species. Hence, Eeckhout and Depaepe (1994)
determined the phytase activity of rye, triticale, wheat,
barley, oats, maize, rapeseed meal and soybean meal to be
5130, 1688, 1193, 582, 41, 15, 16 and 40 U/kg, respectively
(1U, the amount of enzyme that liberates 1 mmol orthopho-
sphate/min from phytin). The final parameter is the
cooking regime. Endogenous phytases are inactivated at
about 65 1C, implying that the phytase activities are lost in
boiled cereals. On the other hand, substantial phytate
degradation occurs during leavening where phytase from
ARTICLE IN PRESS
H. Brinch-Pedersen et al. / Journal of Cereal Science 46 (2007) 308–326 313
ARTICLE IN PRESS
5-PP-Ins(1,2,3,4,6)P5
(InsP7)
5,6-PP-Ins(1,2,3,4)P4
(InsP8)
Glucose 6-phosphate
Ins(3)P1 Synthase
(MIPS)
PtdIns PtdIns(4)P1
PtdIns
synthase
PtdIns
4-kinase
InsP6 kinase
DiphosphoIns polyP
Diphosphatase
DiphosphoIns polyP
Diphosphatase
PP-InsP5 kinase?
myo-inositol
H1
H2
H3
H4
H5
OH
OH
OH
OH
HO
HO
H6
Ins(1,4,5)P3
PtdIns(4,5)P2
PtdIns(4)P1
5-kinase
Phospholipase C
Inositol phosphate multikinase
(IPMK)
PP
PP
P
H1
H2
H3
H4
H5
OH
OH
OH
H6
Ins(1,4,5,6)P4
Inositol phosphate multikinase
(IPMK)
Ins(3,4,5,6)P4
1-kinase
PP
PP
PP
P
H1
H2
H3
H4
H5
OH
OH
H6
Ins(3,4,5,6)P4
PP
PP
PP
P
H1
H2
H3
H4
H5
OH
OH
H6
PP
H4 OH
Ins(1,3,4,5,6)P5
PhytaseIns(1,3,4,5,6)P5
2-kinase
Ins(1,3,4,5,6)P5
2-kinase
Multi Ins-polyP phosphatase
(MINPP)
PP
PP
PP
P
H1
H2
H3
H5
H6
phytate
Ins(1,2,3,4,5,6)P6
PP
PP
PP
PP
PP
P
H1
H2
H3
H4
H5
H6
P
PP
PP
PP
PP
PP
P
H1
H2
H3
H4
H5
P
H6
PP
PP
PP
PP
P
H1
H2
H3
H4
H5
P
H6
PP
Ins(3)P1
Ins(3,6)P2 Ins(3,4)P2
Ins(3,4,5,6)P4Ins(1,3,4,6)P4
Ins(3)P1 4-kinaseIns(3)P1 6-kinase
Ins(3,4/6)P2
4/6-kinase
Ins(3,4,6)P3
1/5-kinase
Ins(1,3,4,6)P4
1-phosphatase
Ins(1,3,4,6)P4
5-kinase
Ins(3,4,5,6)P4
1-kinase
PP
H1
H2
H3
H4
H5
OH
OH
OH
HO
PP
H6
PP
H1
H2
H3
H4
H5
OH
OH
HO
HO
PP
H6
Ins(1,3,4,5,6)P5
H1
H5
PP PP
PP H2
H3
H4 OH
PP
PP
H6
PP
PP
H1
H2
H3
H4
H5
OH
OH
PP
PP
H6
PP
PP
H1
H2
H3
H4
H5
OH
HO
PP
PP
H6
Ins(3,4,6)P3
PP
H1
H2
H3
H4
H5
OH
OH
HO
PP
PP
H6
H1
H2
H3
H4
H5
OH
OH
OH
HO
HO
PP
H6
Ins mono-
phosphatase
Ins 3-kinase
Fig. 3. The biosynthetic pathway from glucose 6-phosphate to phytate with indications of critical step enzymes and the most abundant isomers. On the
left the stepwise phosphorylation of Ins(3)P
1
; on the right the phosphatidyl inositol phosphate pathway proceeding via phospholipase cleavage of
PtdIns(4,5)P
2
and phosphorylation of Ins(1,4,5)P
3
by inositol phosphate multikinase (IPMK). P represents a phosphate moiety; PP represents a
pyrophosphate moiety.
H. Brinch-Pedersen et al. / Journal of Cereal Science 46 (2007) 308 –326314
the yeast and cereal hydrolyse the phytate (for a review see
Gibson, 2006).
5. Strategies to increase the iron content of the cereal grain
Several studies have attempted to increase the iron
content of the endosperm by expressing iron-binding
proteins such as lactoferrin and, in particular, ferritin.
Lactoferrin (LF) is an 80 kDa iron-binding glycoprotein
related to transferrin (Kanyshkova et al., 2001) which is
present in high concentrations (1–2 g/l) in human milk. In
order to increase the iron content of rice, human LF was
expressed in the rice grain under the control of the rice
glutelin 1 promoter (Nandi et al., 2002). The heterologous
protein accounted for up to 0.5% of the grain weight in the
dehusked rice and evaluation in human Caco-2 cells
indicated that the heterologous LF was bioavailable.
Ferritin is a major storage form for iron in plants being
localized in the plastids and essential for iron homoeos-
tasis. Twenty-four ferritin polypeptides form a nanocage
that can accommodate some 4500 iron atoms and about
600 molecules of phosphate. In plants the synthesis of
phytoferritin is transcriptional controlled and induced by
excess iron in the cells (Briat et al., 1999). In dicotyledo-
nous plants such as pea up to 90% of the iron stored in
embryo cells may be sequestered by ferritin (Marentes and
Grusak, 1998). Information on the localization of ferritin
in the cereal grain is limited to expression studies. In wheat,
as in all the major cereals, ferritin is encoded by two genes
(with three homoeoalleles for each in wheat). Quantitative
RT-PCR has revealed significant expression in all grain
tissues. In the embryo and the testa/pericarp the expression
levels amounted to 20% of that found in the leaves and in
the endosperm to 5% (Borg, unpublished results). The
bioavailability of iron in ferritin has been evaluated in iron
deficient rats, showing that purified ferritin and transgenic
ferritin rice are as bioavailable as FeSO
4
when supplemen-
ted into feed (Beard et al., 1996;Murray-Kolb et al., 2002).
Likewise, iron as ferritin and iron in the form of FeSO
4
have been shown to be equally bioavailable in non-anaemic
women (Davila-Hicks et al., 2004).
Ferritin is therefore an ideal sink for iron and a number
of studies have focused on increasing the ferritin levels and
thereby the iron content of the endosperm. Expression of a
soybean ferritin cDNA in wheat and rice using the
constitutive maize ubiquitin promoter resulted only in
increases in the iron content of the leaves while the seeds
had a lower iron content (Drakakaki et al., 2000). This
shows that excess ferritin sequesters the iron in the leaves
reducing the iron mobilisation to the seeds. The over-
expression of ferritin in rice is summarized in Table 3. In all
cases a legume (soybean or bean) ferritin gene was used
under the control of an endosperm-specific promoter. The
results consistently showed two- to three-fold increases in
the iron content. Furthermore, these increases were in some
cases accompanied by increases in the amounts of zinc
(Goto et al., 1999;Lucca et al., 2001;Vasconcelos et al.,
2003). Rice lines expressing high levels of soybean ferritin
were generated using the endosperm-specific rice globulin
promoter that drives 10-fold higher expression than the rice
glutelin promoter. The ferritin levels were increased up to
13-fold compared to those previously achieved using the
glutelin promoter (Qu et al., 2005). However, there was
only a moderate increase (30%) in iron content. Moreover,
all of the over-expressing lines had decreased levels of iron
in their leaves (10% of the non-transformed control) with
low levels of iron in the rest of the plant and the plants also
showed signs of chlorosis in the leaves during seed
development. The contents of Mg and Zn in the seed
tended to be higher while other minerals (Ca, Cd, Cu, Mn)
were present at the same concentrations as in wild-type
plants (Qu et al., 2005).
It can therefore be suggested that over-expression of the
soybean ferritin gene in the rice endosperm exhausts the
iron reserves in the leaves. Consequently, it will be
necessary to improve the iron transport from the roots
and also possibly increase iron uptake if higher levels of
iron are required in the endosperm.
A number of routes can be taken to alter iron uptake and
transport (see Colangelo and Guerinot, 2006;Curie and
Briat, 2003;Hell and Stephan, 2003;Reid, 2001 for reviews
of iron uptake and transport). Iron is generally abundant
ARTICLE IN PRESS
Table 3
Cereals transformed with soybean ferritin genes
Plant Promoter Gene Iron seed mg Fe/g DW
(ppm)
Reference
Control Transgenic
Rice Kitaake Rice glutelin 1.3kb GluB-1 Soybean ferritin Soyfer H-1 8.6–14.3 13.3–38.1 Goto et al. (1999)
Rice Taipei 309 Rice glutelin 1.8 kb Gt-1 Phaseolus ferritin 9.99–10.65 11.53–22.07 Lucca et al. (2001)
Rice Maize ubiquitin-1 Soybean ferritin 10 10
1
Drakakaki et al. (2000)
Wheat Maize ubiquitin-1 Soybean ferritin 40 40
1
Drakakaki et al. (2000)
Rice IR68144 Rice glutelin GluB-1 Soybean ferritin 17.0 16.2–34.7
2
Vasconcelos et al. (2003)
Rice Kitaake Rice glutelin 1.3 kb GluB-1, globulin Glb-1 (10X) Soyfer H-1 11.2 15.1
2
Qu et al. (2005)
Rice Basmati Rice glutelin GluB-1 Soybean ferritin nd nd Sivaprakash et al. (2006)
1
Only increase in vegetative tissues.
2
Did also show increase in seed zinc levels.
H. Brinch-Pedersen et al. / Journal of Cereal Science 46 (2007) 308–326 315
in most soils, but exists mainly in the oxidized ferric form
(Fe
3+
), which is insoluble and not readily available to
plants. Plants use two strategies to acquire iron from iron-
deficient soils. Dicots and non-graminaceous monocots are
‘‘strategy I’’ plants that reduce ferric to ferrous ions (Fe
2+
)
using Fe (III) reductase. The ferrous ions are then taken up
by the iron transporter IRT, a member of the ZIP-like
transporters (Hell and Stephan, 2003). The graminaceous
monocots are so-called ‘‘strategy II’’ plants that release
chelating non-proteinogenic amino acids (phytosidero-
phores) from the roots to acquire iron from the rhizosphere
(Takahashi et al., 1999). Fe
3+
complexed to the phytosi-
derophore is then transported into the root by specific
transporters belonging to the yellow stripe-1 (YS1)-like
protein family (Curie et al., 2001). Interestingly, it has
recently been demonstrated that rice, like the strategy I
plants, has the ability to take up Fe
2+
using IRT
transporters (Ishimaru et al., 2006). After entering the
plant, the iron is bound to chelators in order to neutralize
this very reactive metal. Nicotianamine, which is also a
precursor in the formation of phytosiderophores, is
ubiquitous in plants and can bind Fe
2+
,Fe
3+
and other
ions such as Zn
2+
and Cu
2+
(Schaaf et al., 2004, 2005;von
Wiren et al., 1999). The iron is translocated to the xylem,
probably bound to nicotianamine and is subsequently
transported as a Fe
3+
citric acid complex within the
transpiration stream (Hell and Stephan, 2003). During
senescence iron appears to be transported in the phloem
from leaves to seed as Fe
3+
bound to iron transport
proteins (ITP) (Kruger et al., 2002) and as Fe
2+
bound to
nicotianamine (Stephan and Scholz, 1993). YS1-like
proteins are considered to be important for the loading
and unloading of nicotianamine-chelated Fe
2+
in the
phloem (Koike et al., 2004).
Iron uptake, transport and mobilization to the seed can
accordingly be manipulated in a range of different ways.
Enhanced iron uptake and transport have been reported
for rice and barley. Rice is especially susceptible to low iron
supply as it has low secretion of phytorosiderophores. In
rice the biosynthetic pathway of phytosiderophores was
enhanced by transformation with a barley genomic
fragment containing two nicotianamide aminotranferase
genes. The transgenic plants were more tolerant to growth
to iron-limiting calcareous soils and gave approximately
four times higher grain yields than the non-transgenic
controls (Takahashi et al., 2001).
In another experiment, the Arabidopsis AtFRO2 gene
was introduced into rice in order to determine the ability of
a strategy I root reductase to enhance the iron acquisition
in a strategy II plant. In Arabidopsis the FRO2 reductase is
responsible for the conversion of Fe
3+
to Fe
2+
at the root
surface with subsequent IRT transport of Fe
2+
across the
root membrane (Hell and Stephan, 2003). However, when
introduced into rice, the activity was very limited and no
functionality was found. However, this strategy may be
pursued further as two rice FRO candidate genes coding
for Fe
3+
-chelate reductases (Gross et al., 2003) and
functional Fe
2+
transporters of the ZIP family have
recently been identified (Ishimaru et al., 2006).
6. Strategies to increase the zinc content of the cereal grain
Manipulation of the zinc content of the cereal grain may
be less straightforward than for iron. Proteins are a major
sink for zinc as about 300 enzymes and more than 1000
transcription factors require zinc as a co-factor (Bao et al.,
2003). Furthermore, studies of related Triticum species
showed strong correlations between protein content and
zinc content (Ozturk et al., 2006). Introduction of the high
grain protein content (Gpc-B1) locus from the wild
tetraploid wheat Triticum turgidum ssp. dicoccoides into
different recombinant chromosome substitution lines
resulted in 10–34% higher concentrations of zinc, iron,
manganese and protein in the grain compared to lines
carrying the allele from cultivated wheat and the authors
proposed that the Gpc-B1 locus promoted remobilization
of protein, Zn, Fe and Mn from the leaves to the grains
(Distelfeld et al., 2006).
Another way to increase the zinc content of the cereal
grain may be to manipulate the transporters involved in
zinc translocation. A large number of cation transporters
have been identified in rice, but few have been character-
ized with respect to substrate specificity, expression pattern
and cellular localization. The cation transporters can be
categorized into several distinct families and include the
family of heavy metal P
1B
-type ATPases (HMA) as well as
the family of ZIP, CDF, NRAMP and YSL transporters as
discussed above. Members of the ZIP and CDF families
appear to play a predominant role in zinc uptake,
translocation and deposition.
Further studies of zinc transport mechanisms should
lead to a strategy to improve the zinc content of the cereal
grain. For example, zinc uptake from the soil, transport in
the plant and remobilization could be improved and the
zinc transporters in the aleurone and the embryos
manipulated to increase the zinc content of the globoids.
Alternatively, strategies may be developed to bypass the
sequestering in the globoids, rendering more free zinc
available for entry into the endosperm. However, in this
context the ionic selectivity of plant zinc transporters is of
particular relevance. It is well known that most zinc
transporters are not entirely specific but may also facilitate
the uptake of metals such as cadmium. For example, it was
demonstrated that OsZIP1 is capable of transporting zinc,
magnesium and cadmium whereas the primary substrates
for OsZIP3 are zinc, magnesium and to some extent
calcium (Ramesh et al., 2003). Further research should
therefore be devoted to improving the ion selectivity of the
transporters in order to prevent the long-term exposure to
even low levels of heavy metals which could have severe
impacts on public health (see: http://www.phime.org for
further description). The feasibility of this approach has
been documented by Rogers et al. (2000) who found that a
single amino acid substitution in the IRT1 transporter of
ARTICLE IN PRESS
H. Brinch-Pedersen et al. / Journal of Cereal Science 46 (2007) 308 –326316
Arabidopsis abolished its affinity for cadmium while
preserving its ability to take up zinc.
7. Strategies to reduce inhibitors of iron and zinc
bioavailability
A number of studies in a range of species (oilseed rape,
soybean, alfalfa, barley, maize, wheat and rice) have been
devoted to introducing microbial genes encoding phytase
(see Brinch-Pedersen et al., 2002 for review). The primary
objective of these studies has been to determine the effects
of expressing heterologous phytases on the bioavailability
of phosphate and has included animal feeding trials.
However, only one study has assessed the effects of this
strategy on mineral bioavailability. In this case in vitro
studies showed that the heterologous expression of phytase
in maize promoted iron uptake in Caco-2 cells (Drakakaki
et al., 2005), indicating that a transgenic approach is indeed
feasible. Furthermore, as discussed below, decreasing
phytate by supplementation or by mutation clearly resulted
in positive effects on mineral bioavailability.
It was initially demonstrated that expression of the
Aspergillus niger phyA (phytase) gene in wheat, driven by
the constitutive maize ubiquitin-1 promoter, resulted in
stable accumulation and significantly increased seed
phytase activity (Brinch-Pedersen et al., 2000). Subsequent
studies in rice used alternative phytase genes from
Escherichia coli (Hong et al., 2004), the ruminal bacterium
Selenimonas ruminatium (Hong et al., 2004), A. fumigatus
(Lucca et al., 2001) and the yeast Schwanniomyces
occidentalis (Hamada et al., 2005). In order to maximize
accumulation, seed-specific promoters were used, such as
the rice glutelin (Lucca et al., 2001) and the wheat high
molecular weight subunit 1D 5 gene promoters (Brinch-
Pedersen et al., 2006). Germination-specific expression
using the a-amylase aamy8 promoter has also been
reported (Hong et al., 2004).
Most microbial phytases are secreted and glycosylated.
Signal peptides from barley and rice a-amylases and from
barley b-glucanase have therefore been used to target the
proteins to the ER and the secretory pathway to ensure
secretion into the apoplast. However, a series of papers
showed that while such proteins are targeted to the
apoplast in leaves, they were transported to the protein
storage vacuole in the grain (Brinch-Pedersen et al., 2006;
Drakakaki et al., 2006). Immuno-localization in transgenic
wheat grains similarly showed that the phytase was not
targeted to the apoplast but to the protein storage vacuoles
of the endosperm (Brinch-Pedersen et al., 2006). This is
probably due to the fact that the secretory machinery of the
cereal grain primarily addresses the need for synthesis of
storage proteins with the vacuole acting as the default
destination. We can therefore conclude that the phytase is
deposited in a compartment of the endosperm that is
separate from that of its substrate, phytate.
Phytate and the lower myo-inositols have been
assigned a number of functions in cellular metabolism
(Brinch-Pedersen et al., 2002). We therefore determined the
hydrolysis profile of phytate in incubated flour of
transgenic wheat lines using metal dye detection HPLC
analysis. In this case the phytase activity comprises the
combined action of in planta synthesized A. niger phytase
(a 3-phytase) and endogenous wheat phytase (a 6-phytase)
(Brinch-Pedersen et al., 2003). After 50 min incubation of
the flour, phytate was reduced by 45% in the non-
transgenic material and 86% in the transgenic flour. In
the non-transgenic material, breakdown products accumu-
lated in the form of inositol di- to pentakisphosphates over
the 50 min. In transgenic material the breakdown products
were present only transiently and after 50 min of
incubation virtually all lower inositol phosphates were
completely hydrolysed. A similar breakdown pattern was
revealed when native A. niger phytase was added to wild-
type seeds.
Because most cereals are processed for human food by
boiling or baking the thermotolerance of phytase will be of
great importance. For example, rice is cooked by boiling
for several minutes while processing of wheat may include
leavening that allows for phytate degradation before
baking. Most phytases are not particular thermotolerant
and start to loose activity around 60 1C(Ullah and
Mullaney, 1996). For example, the T
m
of the A. niger
phyA phytase is 63.3 1C and the denaturation is associated
with an irreversible conformational change and the loss of
70–80% of the activity. Various strategies have been
adopted in order to obtain transgenic seeds with heat
stable phytase. The PhyA phytase from A. fumigatus is
known to possess a strong capacity for refolding into the
active conformation after thermal denaturation. When
transgenic rice and wheat seeds expressing the A. fumigatus
phytase were boiled for 20 min, only 8% of the initial
phytase activities were retained, which was much lower
than the 59% residual activity obtained after boiling a
commercial preparation of the fungal enzyme for the same
period of time (Brinch-Pedersen et al., 2006;Lucca et al.,
2001). This indicates that in planta expression may affect
the refolding of the enzyme or provide an environment
unfavourable to refolding after heating. However, when
the engineered phytase 10-thermo-[3]-Q50T-K91A, which
was designed to have a high unfolding temperature
(89.3 1C), was expressed in wheat the residual activity after
boiling for 20 min was 12% of the initial activity of
4777 FTU/kg. This residual activity of 573 FTU/kg re-
duced the phytate level by 42%. Current evidence from pig
feeding experiments indicates that at low-to-moderate zinc
levels, supplementation with 500 FTU/kg of feed leads to
20–50% increases in plasma zinc levels (Jondreville et al.,
2005). It thus appears that cereals expressing a heat stable
phytase such as the consensus phytase do indeed have the
potential to improve the bioavailability of iron and zinc.
Rice is an extreme example but with many other cereal
products the flour is mixed with water before heat
treatment, allowing for the action of the endogenous and
the introduced phytases. In addition, the heterologous
ARTICLE IN PRESS
H. Brinch-Pedersen et al. / Journal of Cereal Science 46 (2007) 308–326 317
phytase activity can be readily increased and a barley
line with 10.000 FTU/kg was recently obtained (Brinch-
Pedersen et al., 2006).
A number of issues have to be addressed before
implementing a transgenic strategy for improving iron
and zinc uptake in humans. Transgenic wheat, expressing
high amounts of a codon optimized A. fumigatus phytase,
was therefore compared for substantial equivalence to non-
transgenic wheat by microarray analyses using a 9 K
unigene cDNA array. Most genes present on the array
were derived from cDNA libraries generated from wheat
grain indicating that it has good coverage of the genes
expressed during grain filling. No significant differences in
the overall gene expression pattern were found when
compared to wild-type grain (Gregersen et al., 2005).
However, current evidence suggests that it will be
important to maintain a minimum level of inositol
phosphates in the cereal product as these also have
potential health benefits (anti-oxidative and anti-neoplastic
properties, ability to reduce serum lipids and cholesterol
levels, prevention of renal calculi via mineral-binding and
hypoglycemic effects of relevance in diabetes, Burgess and
Gao, 2002;Jenab and Thompson, 2002). It is important to
establish whether phytate and some of the lower myo-
inositols do indeed possess significant health benefits.
If so, phytate could be reduced to a level that can maintain
these benefits as well as improve the iron and zinc
bioavailability.
7.1. The low phytate mutants
Substantial efforts have also been made to identify
mutants with impaired phytate biosynthesis. Low phytate
mutants have been reported for several plant species,
including wheat, barley, rice and maize (see Guttieri et al.,
2003;Raboy, 2001 for review). For example in barley, the
low phytate mutations lpa1-1,lpa2-1,lpa3-1 and M955
result in 50%, 40%, 65% and 95% reductions of the
phytate levels, respectively. These reductions are accom-
panied by equivalent molar increases in inorganic phos-
phate or lower isomers of inositol phosphate, leaving the
total P content almost unaffected. Interestingly, there are
no major changes in the mineral content of these mutants
(Hatzack et al., 2000;Liu et al., 2004). The lpa-1 mutants
have been used to breed the so-called HAP (high available
phosphate) cultivars of soybean, barley and maize. How-
ever, unfortunately the lpa mutations often have reduced
yields while the germination rates of lpa soybean and maize
were reduced by 30% and 23%, respectively (Oltmans
et al., 2005;Pilu et al., 2003).
The reduction of phytate biosynthesis in rice has also
recently been addressed by transformation with the
Ins(3)P1 synthase (MIPS) gene (RINO1) in the antisense
orientation (Kuwano et al., 2006). The MIPS-catalysed
conversion of glucose 6-phosphate to myo-inositol-3-
phosphate (Fig. 3) is regarded as the first step in the
biosynthesis of phytate, and there appears to be a close
relationship between the biosynthesis of phytate and
the formation of myo-inositol-3-phosphate by MIPS
(Brinch-Pedersen et al., 2002). In the rice study, the
phytate-P content was reduced considerably and this was
associated with a similar increase in the level of Pi (up to
48%). In comparison, the Pi fraction in lpa rice represented
32% of the total seed P (Larson et al., 2000). There were no
differences in germination between the transgenic and wild-
type seeds.
The effects of reduced phytate content on mineral
bioavailability, including zinc absorption, have been
evaluated in human feeding trials, where the subjects were
fed simple diets of tortillas or polenta made from lpa maize
flour. In both cases there was a positive correlation
between a reduced phytate content and increased zinc
absorption (Adams et al., 2002;Hambidge et al., 2004).
Similarly, increased availability of iron and calcium has
been reported from human feeding trials using tortilla
based diets (Hambidge et al., 2005;Mendoza et al., 1998).
8. Other inhibitors of iron and zinc bioavailability
8.1. Phenolic compounds
The tannins, also known as polyphenols, are present
throughout the plant kingdom including in foods such as
tea, coffee, cereals, grapes and vegetables. They are
particularly abundant in millets and sorghum (Klopfen-
stein and Hoseney, 1995) and are located primarily in the
pericarp and testa tissues. Accordingly, milling and
polishing remove large amounts of the tannins. The tannins
form insoluble complexes with iron (III) via the orthodihy-
droxyl groups in the phenolic structure making the iron
unavailable for absorption in the gastrointestinal tract
(Gillooly et al., 1984). The polyphenol levels of cereal seeds
can be reduced via incubation with polyphenol oxidase
which, when combined with a phytase-mediated phytate
reduction, has been shown to significantly increase the
availability of iron (Matuschek et al., 2001).
8.2. Fibre
Dietary fibre (non-starch polysaccharides) is resistant to
digestion by humans. The fibre contents of cereals can be
very high (up to 16%) and the majority of fibre consists
of insoluble polysaccharides and lignin (Charalampopou-
los et al., 2002). Dietary fibre can bind a range of minerals
and reduce their bioavailability. Moreover, the insoluble
components act as bulking agents and speed up the passage
through the digestive tract, reducing the time available for
absorption of nutrients. Fibre is also known to interact
with other food components such as phytate, tannins and
oxalates and these interactions may be more important in
hindering mineral absorption than binding by the fibre
itself (Harland, 2006). Consequently, the addition of pure
cellulose, lignin, mucilages and gums to the diet has little or
no effect on iron absorption (Cook et al., 1983).
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9. Enhancers
9.1. Inulin
Most plants store starch as a reserve carbohydrate, but
about 15% of all flowering plant species, including cereals,
also store carbohydrates in the form of fructans. In the US,
wheat provides 70% of the dietary fructans, oligofructose
and inulin (Moshfegh et al., 1999). When non-digestible
oligosaccharides such as inulin and oligofructose reach the
gastrointestinal tract, they are fermented by the local
microflora and stimulate the growth of bifidobacteria and
lactobacilli, which are considered to have health-promoting
effects (Gibson et al., 1995). Several studies in humans and
animals have shown that inulin and oligofructose can
increase the intestinal absorption of minerals (see Scholz-
Ahrens and Schrezenmeir, 2002 for review). Short-chain
fatty acids (SCFA) produced during fermentation lower
the pH in the intestinal lumen which improves mineral
solubility and absorption. Moreover, SCFA can form
complexes with the minerals and thereby increase their
uptake. Bacterial metabolites may also stimulate the
intestinal epithelium and increase the absorption.
The fructan biosynthetic pathway has been genetically
modified in several plant species. In cereals, the fructan
level in maize kernels could be increased by 9-fold via
expression of the gene SacB from Bacillus amyloliquefa-
ciens which encodes levansucrase (Caimi et al., 1996), but
no information on the impact on mineral bioavailability is
available.
9.2. Vitamins and other enhancers
A diverse range of other food components can counteract
the adverse effects of inhibitors on mineral absorption,
although in many cases their precise mechanism is not clear.
They are mainly organic compounds and include vitamins
(ascorbic acid, vitamin C; b-carotene, pro-vitamin A;
riboflavin, vitamin B
2
; calciferol, vitamin D), cysteine in
proteins, a variety of amino and organic acids and
chemically similar ions competing for the same transport
carriers (see Lopez et al., 2002;White and Broadley, 2005
for review). A classic example is ascorbic acid which reduces
ferric iron to ferrous iron which is then bound in an
absorbable chelate that does not react with the inhibitors
(Ballot et al., 1987;Gillooly et al., 1983). Similarly, b-
carotene probably improves the bio-availability of iron by
increasing the solubility (Garcia-Casal et al., 1998). An
interesting example is vitamin D, which is altered metabo-
lically in the liver and kidneys to the hormone 1,25-
dihydroxyvitamin D which in turn controls the absorption
of calcium ions from the intestine (Lopez et al., 2002).
10. Increasing the vitamin content of cereals
Vitamins constitute the second major group of micro-
nutrients required for the growth and health of humans
and animals. In developed countries a balanced vitamin
supply is easily achieved by supplementation but the poor
in developing countries primarily depend on the vitamin
sources in the staple foods they consume. Hence, the
vitamin content of cereals is of great importance. The
problems encountered are very similar to those encoun-
tered for the minerals. The contents of several vitamins are
low in cereals. They are also primarily present in the
embryo, the aleurone and the outer parts of grain (see
Anonymous, 2002, 2003, 2004 for review) and are
accordingly lost during milling and polishing. In addition,
the remaining vitamins are often extracted during cooking.
Vitamins are classified into two major groups, the lipid-
soluble and the water-soluble. Lipid-soluble vitamins
comprise provitamin A (b-carotene) and vitamins D
(calciol), E (tocopherols and tocotrienols) and K (phyllo-
quinone) while vitamins B1 (thiamine), B2 (riboflavin,
nicotianamide, folate, pantothenate), B3 (niacin), B6
(pyridoxal), B9 (folic acid), B12 (cobalamine), C (ascor-
bate) and H (biotin) are water-soluble. Among the three
major cereals (wheat, rice and maize), whole grain wheat is
considered to be a good source of B vitamins (thiamine,
riboflavin, niacin, pyridoxine and folic acid) and vitamin E.
On the other hand, the wheat grain has low contents of the
other lipid soluble vitamins (provitamin A, vitamin D and
vitamin K) which are only present in significant amounts in
the embryo. However, durum wheats do contain yellow
coloured carotenoids (carotene, xanthophylls and xantho-
fyll ester) in the endosperm and the embryo. Maize is the
only one of the three major cereals that contains significant
amounts of vitamin A precursors in the endosperm and
yellow corn is the main source of provitamin A in pig feed
(Egesel et al., 2003). Rice is similar to wheat in that it
contains low amounts of vitamin A precursors and milled
rice has low contents of lipid-soluble as well as water-
soluble vitamins.
Vitamin supplements to food and feed are today
primarily produced by chemical synthesis or by fermenta-
tion using microorganisms, and to a lesser extent by
isolation from natural sources. The value of the global
market for vitamin supplements amounts to $2.75 billion
annually (Herbers, 2003). Considerable research effort has
been devoted to characterizing the biosynthetic pathways
for vitamins in plants, to cloning the genes encoding the
individual steps and to understanding the regulation of the
pathways. Herbers (2003) lists several advantages of
vitamin production in natural hosts rather than by
synthetic chemistry. Firstly, the active stereoisomers are
produced exclusively. Secondly, vitamins made in and
derived from plants are considered ‘‘natural’’ and might
therefore have positive public acceptance and thirdly,
plant-based production systems may be cheaper.
It is clear that there is considerable potential to increase
the vitamin contents of cereals to alleviate vitamin
deficiencies in humans and livestock. The task is challen-
ging, as it will require the transfer or re-establishment of
whole biosynthetic pathways and transport systems in the
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H. Brinch-Pedersen et al. / Journal of Cereal Science 46 (2007) 308–326 319
endosperm. Furthermore, the endosperm is a highly
specialized tissue, designed for the production and storage
of hydrophilic compounds such as starch, cell walls and
proteins.
So far there have been attempts to manipulate the
contents of provitamin A, vitamin E and vitamin C in
plants with work in cereals being primarily focused on
increasing the amounts of precursors for vitamin A. One
recent review (Herbers, 2003) also refers to a patent
application describing increasing the vitamin H content.
We will therefore focus on the current efforts to increase
the provitamin A content and briefly describe the
possibilities for increasing the amounts of vitamins C and
E. Several excellent recent reviews are available on the
biosynthesis of these three vitamins and current effort to
increase their contents (provitamin Al Babili and Beyer,
2005;DellaPenna and Pogson, 2006; vitamin C: Agius
et al., 2003;Agius et al., 2006;Hancock and Viola, 2002;
vitamin E: DellaPenna and Pogson, 2006;Hofius et al.,
2006;Schneider, 2005). Finally, we will describe a novel
approach taken for vitamin B12, the plant-based produc-
tion of protein factors that ensure the recycling of the
vitamin.
10.1. Provitamin A (b-carotene)
Vitamin A deficiency affects hundreds of millions of
people in the developing countries, particularly in South
and Southeast Asia. The estimates vary but are of the same
order of magnitude. A recent analysis estimated that about
127 million preschool children were vitamin A deficient of
which 4.4 million suffered from xerophthalmia (West,
2002). About 10 million pregnant women have low vitamin
A status and some develop night blindness. In addition, a
number of diseases are being recognized as relating to
vitamin A deficiencies, which include effects on embryo
development as well as increased morbidity and mortality
during pregnancy (Calgett-Dame and DeLuca, 2002).
The genetic engineering of rice for high production of
b-carotene, the precursor for vitamin A, constitutes an
outstanding example of the potential for engineering
biosynthetic pathways in cereals. In a series of studies
Peter Beyer, Ingo Potrykus and their co-workers demon-
strated that a biosynthetic pathway for b-carotene could be
established in the amyloplasts of the rice endosperm. In the
initial study, the first step in the pathway (the formation of
phytoene from geranylgeranyl phosphate) was achieved in
endosperm of japonica rice by the introduction of a
phytoene synthase gene from daffodil, regulated by a rice
endosperm-specific glutelin promoter (Burkhardt et al.,
1997). Subsequently, a gene was introduced encoding a
bifunctional Erwinia uredovora phytoene desaturase–x-
carotene desaturase catalyzing the conversion of phytoene
to x-carotene and then to lycopene together with a daffodil
gene encoding a lycopene b-cyclase that converts lycopene
to b-carotene (Ye et al., 2000). All constructs encoded
targeting sequences to ensure entry of the enzymes into the
amyloplasts. Interestingly, b-carotene synthesis was also
achieved in lines without a lycopene b-cyclase indicating
that rice contains a functional gene encoding this enzyme
that may be expressed normally or activated by the
intermediates of the pathway.
Subsequent effort has been directed towards the
introduction of the pathway into indica rice (Datta et al.,
2003), using the phosphomannose isomerase selection
system instead of antibiotic resistance, and replacement
of the daffodil phytoene synthase gene with a gene from
maize (Paine et al., 2005) (see Al Babili and Beyer, 2005 for
review). The best lines contained up to 37 mg/g carotinoids
of which 31 mg/g was b-carotene. Assuming a b-carotene:-
vitamin A equivalence ratio of 12:1, it is estimated that the
recommended daily allowance of 300 mg vitamin A for
young children can be met by a daily consumption of
100–200 g of rice per day. The most promising lines are
currently undergoing field trials and propagation for
nutritional evaluation. The potential impact and cost
effectiveness of Golden Rice have recently been evaluated
using a disability-adjusted life years approach (DALYs). It
was estimated that the burden of vitamin A deficiency
amounts to 2.328.000 DALYs lost annually. In a high
impact scenario the number of DALYs was reduced by
59% and in a low impact scenario by 9%. The cost of
saving one DALY through Golden Rice was estimated to
be o$20 compared to $134–599 if using vitamin A
supplementation (Stein et al., 2006).
10.2. Vitamin E
Vitamin E is generally assigned a function as a radical
scavenger in lipophilic environments and hence as a
protectant of the polyunsaturated fatty acids in membrane
lipid (Schneider, 2005). Vitamin E is generic term that
combines the a,b,dand gforms of tocotrienols and
tocopherols, also referred to collectively as tocochroma-
nols. a-Tocopherol is the predominant form of vitamin E in
human and animal tissues and has the highest vitamin E
activity, hence it has been the main focus of attention.
However, the tocotrienols have also been ascribed im-
portant functions with neuroprotective, anti-cancer and
cholesterol-lowering properties (Sen et al., 2006). It is
generally assumed that in developed countries the food
intake ensures an adequate supply of these components but
it is possible that further long-term health benefits may be
achieved by dietary supplementation. Vitamin E has been
used in high doses in livestock production to improve the
quality and shelf life of meat. Tocopherols are primarily
synthesized from isophytol and trimethylhydroquinone
and 10% of the annual production of 40,000 ton is
extracted from soybean oil (see Valentin and Qi, 2005 for
review). In plants, the only well documented effect of
vitamin E is the protection of seed storage lipids from
oxidation during dormancy and germination and toco-
pherols are known to improve the flavour stability of corn
oil during storage and cooking.
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All of the genes involved in the biosynthesis of these
compounds have been cloned but there is no information
on how their accumulation, storage and degradation are
regulated (Hofius et al., 2006). Metabolic pathway
engineering has so far primarily been performed on seeds
of oilseed crops and Arabidopsis using two different types
of strategy to improve the conversion of the tocochroma-
nols into a-tocopherol and to increase the total amount of
tocochromanols. In the former approach Shintani and
DellaPenna (1998) showed that it was possible to convert
g-tocopherols to a-tocopherols by seed-specific over-ex-
pression of the VTE4 gene (encoding a metyltransferase),
resulting in a nine-fold increase in vitamin E activity.
Co-expression of the VTE3 and VTE4 genes resulted in the
almost complete conversion of b-, d-,andg-tocopherols
to a-tocopherol (Van Eenennaam et al., 2003). The
second approach included the introduction of a feed-back
insensitive gene encoding a bifunctional prephenate
dehydratase (TYRA) into tobacco, together with the
Arabidopsis HPPD gene (p-hydroxyphenylpyruvate dioxy-
genase), HPP (p-hydroxyphenylpyruvate) production being
regulated by feedback inhibition of arogenate dehydrogen-
ase by tyrosine. The result was an eight-fold increase in
tocochromanols in tobacco leaves (Rippert et al., 2004).
Similarly, two- to three-fold increases in total seed
tocochromanols were reported in Arabidopsis, canola
and soybean, which were almost entirely due to increases
in tocotrienols (Karunanandaa et al., 2005). However, the
seeds were black due to oxidative polymerization of HGA
and germination was impaired.
In general tocotrienols are the major form of vitamin E
in cereals and in monocots. To our knowledge there has
only been one attempt to increase the amount of vitamin E
in the seed of a cereal. This showed that the introduction of
a barley cDNA encoding homogentisic acid geranylgeranyl
transferase (HGGT) resulted in a 20-fold increase in
tocotrienols in the embryo with the total content of
tocochomanols being increased four- to six-fold (Cahoon
et al., 2003).
It is thus apparent that most if not all of the tools are
available for increasing the vitamin E content of cereals
and for modulating the relative proportions of the eight
different forms. It is also relevant that tocopherols and
tocotrienols are distributed in a tissue-specific manner, with
the tocopherols in maize, barley, oat and wheat being
mainly present in the embryo while the tocotrienols are
primarily confined to the pericarp and the endosperm. Falk
et al. (2004) therefore monitored the formation of the
different forms of vitamin E in different tissues of the
barley grain during grain filling. According to them, only
13% of the tocochromanols, mainly tocopherols with very
little tocotrienols, were present in the embryo while the
pericarp and the endosperm contained 50% and 37% of
these components, respectively. Eighty-five percent of the
total tocochromanols were tocotrienols. In the endosperm
fraction a-tocotrienol was dominant followed by b-andg-
tocotrienols.
10.3. Vitamin C
Vitamin C (L-ascorbic acid, 2-oxo-L-threo-hexono-1,
4-lactone 2,3-endiol, C
6
H
8
O
6
) is abundant in many fresh
fruits and leafy vegetables, often being present in the
millimolar range, and an adequate supply can readily be
obtained by ingesting a varied diet. While most animals are
able to synthesize vitamin C in the liver from glucose,
primates lack this ability due to mutations in the gene
encoding the enzyme L-gulono-1,4-lactone oxidase which
catalyses the last step in the pathway (Woodall and Ames,
1997). The annual world production of vitamin C amounts
to 80,000 ton and is based on chemical synthesis. About
50% of this is used for vitamin supplements and
pharmaceuticals, 25% as a food additive, 15% in beverage
production and 10% for animal feed (Hancock and Viola,
2002).
In plants, vitamin C is synthesized from D-glucose-6-P
via a 10 step pathway known as the Wheeler and Smirnoff
pathway (Wheeler et al., 1998) or from D-fructose-6-P.
Several of the genes in the pathways are now cloned (see
Agius et al., 2006 for review). The last step in the pathway
is mediated by 8-gulono-1,4-lactone dehydrogenase or
L-galactono-1,4-lactone dehydrogenase and takes place on
the inner membrane of the mitochondrion, from where it is
transported to all other subcellular compartments (Bartoli
et al., 2000). The primary function of vitamin C appears to
be as an enzyme co-factor, anti-oxidant and donor/
acceptor in electron transfer. Enzymes that require vitamin
C as a co-factor include 2-oxoglutarate- and Fe(II)-
dependent oxygenases such as prolyl hydroxylases required
for post-translational hydroxylation of prolyl residues in
hydroxyproline-rich cell wall glycoproteins and enzymes
involved in the biosynthesis of flavanoids, gibberellins and
ethylene. In animals, vitamin C is essential for collagen
synthesis, the transformation of cholesterol into bile, as a
co-factor for the synthesis of neurotransmitters, as an anti-
oxidant and for proper immune function.
Until now few attempts have been made to increase the
vitamin C content of plants and none in cereals.
Transformation of tobacco and lettuce with a rat cDNA
encoding L-gulono-g-lactone oxidase resulted in an increase
in the vitamin C content of up to seven-fold (Jain and
Nessler, 2000). Similarly, expression of a D-galacturonic
acid reductase from strawberry in Arabidopsis resulted in
two- to three-fold increases in L-ascorbic acid (Agius et al.,
2003). An alternative strategy, to over-express a wheat
dehydroascorbate reductase (DHAR) in maize and tobac-
co, was taken by Chen et al. (2003). DHAR is the enzyme
responsible for regenerating ascorbic acid from dehydroas-
corbate and the L-ascobic acid levels were increased by two-
to four-fold.
10.4. Vitamin B12
Vitamin B12 (cobalamin) is exclusively synthesized in
microorganisms. It is required by animals including
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H. Brinch-Pedersen et al. / Journal of Cereal Science 46 (2007) 308–326 321
humans while plants and fungi are thought to neither
synthesize it nor require it. In animals it is indispensable for
the functions of two essential enzymes, methionine
synthase and malonyl-CoA mutase. If untreated, deficien-
cies in vitamin B12 result in the fatal condition of
pernicious anemia and partial deficiencies have been
correlated with a range of neurological symptoms (Martens
et al., 2002). It has been estimated that 10–15% of people
over the age of 60 are affected by inadequate access to
vitamin B12.
About 30 genes and 70 enzymatic steps are required for
the synthesis of vitamin B12 (Roth et al., 1993). This
implies that the transfer of the pathway to plants is far
beyond current technological possibilities. However, an
alternative approach has been taken which is to improve
vitamin B12 uptake. Vitamin B12 metabolism in humans is
very complicated, with three transport proteins (haptocor-
rin, intrinsic factor and transcobalamin) and several
receptors being required to ensure efficient uptake.
Intrinsic factor is synthesized and excreted by the cells of
the gastric mucosa and binds vitamin B12 in the
duodenum, acting as a chaperone delivering the vitamin
to receptors on the enterocytes of the terminal ileum for
subsequent uptake.
Pernicious anemia is now thought to result from a gastric
autoimmune disease where antibodies are generated
towards the intrinsic factor. Because intrinsic factor can
at present only be isolated from gastric juice, Fedosov et al.
(2003) explored the possibility of plant-based synthesis of
this protein. Constitutive expression of a cDNA encoding
intrinsic factor resulted in the production of 70 mg/kg fresh
weight, with the key properties of the recombinant protein
being similar to those of the endogenous form. The plant-
derived intrinsic factor has now passed clinical trials and is
scheduled for commercialization (http://www.cobento.
com/).
11. Concluding remarks
Biofortification of the staple cereals by exploiting
molecular tools is a challenging task. However, given the
severity of the problem of micronutrient deficiencies among
the poor in developing countries and the absence of
feasible supplementation and fortification-based alterna-
tives, the task is justified. In a wider context, biofortifica-
tion reflects a new and rapidly advancing paradigm in
agricultural production, namely to breed for nutritional
quality rather than only yield. Biofortification initiatives
have led to greatly increased scientific interest in micro-
nutrients, resulting in a rapidly developing field of
research comprising and integrating agronomy, breeding,
molecular biology, genetics, plant physiology and nutri-
tion. In the current paper we have reviewed the present
state of the art on molecular genetic approaches to improve
mineral bioavailability and vitamin content in the most
important plants for human and livestock nutrition, the
cereals.
Acknowledgement
We would like to acknowledge the economic support
from HarvestPlus, International Food Policy Research
Institute 2033 K Street, Washington DC, USA.
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