Folate biofortification in food plants

Abstract

Folate deficiency is a global health problem affecting many people in the developing and developed world. Current interventions (industrial food fortification and supplementation by folic acid pills) are effective if they can be used but might not be possible in less developed countries. Recent advances demonstrate that folate biofortification of food crops is now a feasible complementary strategy to fight folate deficiency worldwide. The genes and enzymes of folate synthesis are sufficiently understood to enable metabolic engineering of the pathway, and results from pilot engineering studies in plants (and bacteria) are encouraging. Here, we review the current status of investigations in the field of folate enhancement on the eve of a new era in food fortification.

Full text

Folate biofortification in food plants
Samir Bekaert
1
, Sergei Storozhenko
1
, Payam Mehrshahi
2
, Malcolm J. Bennett
2
,
Willy Lambert
3
, Jesse F. Gregory III
4
, Karel Schubert
5
, Jeroen Hugenholtz
6
,
Dominique Van Der Straeten
1
and Andrew D. Hanson
7
1
Unit Plant Hormone Signaling and Bio-imaging, Department of Molecular Genetics, Ghent University, K.L. Ledeganckstraat 35,
B-9000 Gent, Belgium
2
Plant Sciences Division, School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, UK,
LE12 5RD
3
Laboratory of Toxicology, Ghent University, Harelbekestraat 72, B-9000 Gent, Belgium
4
Food Science and Human Nutrition Department, University of Florida, Gainesville, FL 32611, USA
5
Biology Department, Washington University, St. Louis, MO 63130, USA
6
Wageningen Center for Food Sciences, PO Box 557, 6700 AN Wageningen, The Netherlands
7
Horticultural Sciences Department, University of Florida, Gainesville, FL 32611, USA
Folate deficiency is a global health problem affecting
many people in the developing and developed world.
Current interventions (industrial food fortification and
supplementation by folic acid pills) are effective if they
can be used but might not be possible in less developed
countries. Recent advances demonstrate that folate bio-
fortification of food crops is now a feasible complemen-
tary strategy to fight folate deficiency worldwide. The
genes and enzymes of folate synthesis are sufficiently
understood to enable metabolic engineering of the path-
way, and results from pilot engineering studies in plants
(and bacteria) are encouraging. Here, we review the cur-
rent status of investigations in the field of folate enhance-
ment on the eve of a new era in food fortification.
Folates as vitamins and the need for biofortification
Folate is a generic term for tetrahydrofolate (THF) and its
derivatives (Figure 1). Folates are B vitamins, necessary in
almost all organisms as cofactors for one-carbon (C
1
) trans-
fer reactions, generally referred to as C
1
-metabolism.
Vitally important aspects of C
1
-metabolism are nucleotide
biosynthesis, amino acid metabolism and the methylation
cycle, which supplies numerous methylation reactions with
methyl groups (for reviews see Refs [1–3]). Humans and
other animals cannot synthesize folates and, therefore, need
them in the diet, with plants usually being the main dietary
sources [2]. Folate levels vary among food plants; the cereal
staples maize, wheat and, particularly, rice contain extre-
mely low levels (USDA National Nutrient Database for
Standard Reference. Release 19; http://www.nal.usda.gov/
fnic/foodcomp/search/)(Table 1). Reliance on such staples
cannot satisfy recommended dietary allowances (RDA), set
at 400 mg of dietary folate equivalents (DFE) day
1
for
adults National Institutes of Health Office of Dietary
Supplements Dietary Supplement Fact Sheet: Folate;
http://ods.od.nih.gov/factsheets/folate.asp). Clinical and epi-
demiologicalevidence shows that folate intake is suboptimal
for most populations in developing countries – as well as for
surprisingly large population groups in developed countries
[4–6]. Suboptimal folate intake perturbs C
1
-metabolism,
which contributes to megaloblastic anemia, birth defects
[neural tube defects (NTD)], and increased risks for cardi-
ovascular disease and certain cancers (Box 1)[7].
Folate deficiency is, therefore, a global health problem.
Although fortification and supplementation (vitamin pills)
are effective ways to improve folate status, they remain far
from accessible to the poor, rural population in developing
countries [8,9]. Hence, there is a compelling case for
the development of folate-enriched food plants as a sustain-
able complement to the existing interventions for fighting
folate deficiency [9–11]. Recently, major progress has been
achieved not only in our understanding of the regulation of
the folate biosynthesis pathway, but also in establishing a
proof of concept for folate biofortification by metabolic engin-
eering of crops plants. Here, we discuss these achievements,
after providing background on the biochemical processes
that affect folate content. Research on bacterial and animal
systems is included where relevant.
Folate biosynthesis and transport
The steps in folate synthesis are the same in plants and
bacteria, and the pathway enzymes and their genes are all
known in both groups [12,13]. In essence, the three parts of
the THF molecule – the pteridine, p-aminobenzoate (p-
ABA) and glutamate moieties (Figure 1) – are produced
separately and then joined together. In bacteria, the whole
process takes place in the cytosol, but in plants three
subcellular compartments are involved: plastids, mito-
chondria and cytosol [14] (Figure 2). The pteridine moiety
is formed from guanosine triphosphate (GTP) in the cytosol
and the p-ABA moiety is formed from chorismate in plas-
tids. Pteridine and p-ABA are then transported to the
mitochondria, where they are coupled together, glutamy-
lated and reduced to produce THF. A short chain of
g-linked glutamates can then be added in mitochondria,
plastids or cytosol, yielding folate polyglutamates (THF-
Glu
n
). Folate molecules exist in vivo mainly as polygluta-
mates and these are preferred by folate-dependent
enzymes involved in C
1
-metabolism [15]. By contrast,
Review
Corresponding authors: Van Der Straeten, D.
(dominique.vanderstraeten@ugent.be); Hanson, A.D. (adha@ufl.edu).
28 1360-1385/$ – see front matter ß2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.tplants.2007.11.001 Available online 20 December 2007

folate transporters typically prefer monoglutamates, thus
glutamylation tends to favor folate retention within cells or
compartments [16]. Folates are found in plant vacuoles, as
well as in the cytosol, mitochondria and plastids [14,17],
and can be taken up by plant cells from the culture medium
[18]. Therefore, the intracellular distribution and localiz-
ation of folates requires various transport steps (Figure 2).
Most of these steps are likely to be carrier-mediated (as in
other organisms), the exceptions being those involving p-
ABA, which is able to cross membranes by diffusion
because it is a hydrophobic weak acid [19]. However, the
only plant folate transporters yet identified are both
plastidial [20,21], thus at least three folate transporters
(mitochondrial, vacuolar and plasmalemmal) and a mito-
chondrial pteridine transporter remain to be found
(Figure 2).
Most folates are labile molecules. Thus, whereas
detailed knowledge of folate biosynthesis in plants enables
engineering of the pathway, the enhancement of the over-
all folate content to a level significantly impacting on
human health also needs an in-depth understanding of
folate degradation and salvage.
Folate breakdown and salvage
Folates undergo spontaneous oxidative or photo-oxidative
cleavage of the C9–N10 bond (Figure 1) to give
dihydropterin-6-aldehyde and p-aminobenzoylglutamate
(p-ABA-Glu) fragments [22]. Folate breakdown can yield
large folate losses in post-harvest fruits and vegetables [2].
For example, in peas 50% of the total folate at harvest was
lost after six days of storage at ambient temperature. In
other plant tissues, breakdown is apparently countered by
salvage reactions that enable re-use of the breakdown
products in folate synthesis [23]. These reactions hydrolyze
p-ABA-Glu to yield p-ABA, and reduce the aldehyde group
of dihydropterin-6-aldehyde to yield the folate synthesis
intermediate hydroxymethyldihydropterin (HMDHP)
(Figure 3)[23]. The aldehyde reduction is mediated by
multiple, non-specific reductases, of which one has been
cloned [24]. If the dihydropteridine ring becomes oxidized
(which can occur spontaneously) before this reaction takes
place, plants cannot reduce the aldehyde reduction product
back to HMDHP and the pteridine is in effect lost [25]; this
is also the case in Escherichia coli [25]. However, a
reductase that catalyzes a nicotinamide adenine dinucleo-
tide phosphate (NADPH)-dependent reduction of oxidized
pteridines is known in trypanosomatids [26]. Lack of such
an enzyme, resulting in failure to reclaim oxidized pter-
idines, might, therefore, be seen as a ‘weak point’ in plant
pteridine and folate metabolism.
Folates are protected from oxidative breakdown by
binding to proteins [22,27], and, because polyglutamyla-
tion generally favors protein-binding [15], there is a
positive correlation between polyglutamylation and folate
stability. Protein-binding also protects polyglutamyl
folates from deglutamylation by g-glutamyl hydrolase
[28]. However, these findings are based entirely on work
in animals. Nothing is yet known about folate-binding
proteins in plants, although the co-occurrence of polyglu-
tamyl folates and high g-glutamyl hydrolase activities in
plant vacuoles [17] suggests that they exist.
Plants have developed mechanisms to cope with folate
instability. Reducing folate degradation rate and salvaging
its degradation products might contribute to folate
Figure 1. Chemical structure of folates. The folate molecule consists of pterin, p-ABA and glutamate moieties marked with square brackets. The folate shown is the
monoglutamyl form of tetrahydrofolate (THF). Plant folates have g-linked polyglutamyl tails of up to approximately six residues attached to the first glutamate. C
1
units at
various levels of oxidation can be attached to N-5 and/or N-10, as indicated by R
1
and R
2
. The list of naturally occurring C
1
units is shown below the structural formula. The
pteridine ring of folates can exist in tetrahydro, dihydro, or fully oxidized forms.
Table 1. Folate content of selected crops
a
Crop Folate content (mg 100 g
S1
)
Rice (white
b
, raw) 6–8
Maize (yellow, seeds, raw) 19
Wheat (hard, white, raw) 38–43
Tomato (fruit, raw) 9–29
Peas (green, raw) 65
Spinach (leaves, raw) 194
Lentils (mature seeds, raw) 433
Beans (pink, mature seeds, raw) 463
a
Values are in mg of folate per 100 g. Data are from the USDA National Nutrient
Database for Standard Reference (http://www.nal.usda.gov/fnic/foodcomp/search/).
b
Polished grains.
Review TRENDS in Plant Science Vol .13 No.1
29

enhancement. However, a greater insight into these mech-
anisms is needed to harness this knowledge into engineer-
ing strategies, which up until now have mainly
concentrated on the engineering of the folate biosynthesis
pathway.
Metabolic engineering of folate synthesis
GTP cyclohydrolase I (GTPCHI) overexpression
To date, all published work on enhancing plant or bacterial
folate content has involved manipulating the activities of
biosynthetic enzymes. Initial studies in tomato fruit and
Arabidopsis thaliana [29,30] overexpressed the first
enzyme of the pteridine branch of the folate pathway,
GTP cyclohydrolase I (GTPCH I; Figure 2). In both cases,
a non-plant gene [based on the mammalian or bacterial
gene (GenBank accessions BE136861 and AE000304),
respectively] was used, because the foreign enzyme was
predicted be free of negative feedback control (inhibition by
pathway products) in planta. Pteridine levels in transgenic
tomatoes and Arabidopsis, respectively, rose to as much as
140- and 1250-fold those in wild-type controls; however,
the rise in folate content was only two- to fourfold,
indicating the need for further engineering of the pathway
[29,30]. Analysis of total p-ABA (p-ABA plus its glucose
ester) in transgenic tomato fruit indicated severe p-ABA
depletion, and, consistent with this depletion, supplying
exogenous p-ABA to GTPCHI-overexpressing transgenic
tomatoes [29] and Arabidopsis [31] increased folate content
by a further 2.5- to 10-fold. This observation points not only
to the need for simultaneous enhancement of both folate
precursors (pterin and p-ABA), but also proves a substan-
tial physiological potential for increasing folate concen-
tration within the plant cell.
Combined overexpression of GTPCHI with
aminodeoxychorismate synthase (ADCS)
These promising findings prompted another round of
engineering in tomato fruit in which the first enzyme of
the p-ABA branch of the folate pathway, aminodeoxychor-
ismate synthase (ADCS; Figure 2), was overexpressed,
using the gene from Arabidopsis (At2g28880; GenBank
accession NP_850127) [32]. The resulting transgenic fruit
contained an average of 19-fold more p-ABA, compared
with wild-type controls, without an increase in folate level.
When this trait was combined with the pteridine-over-
production trait by crossing, the double transgenic fruit
accumulated an average of 19-fold more folate than con-
trols; the folate levels achieved (840 mg per 100 g edible
portion) provide the complete adult daily requirement in
less than one standard serving (1/2 cup). However, this
engineering strategy also resulted in a 20-times higher
accumulation of pteridines and p-ABA as compared with
the wild-type control. Although the level reached for p-ABA
in the transgenic tomatoes is harmless for human health,
the situation for pteridines is unclear and needs investi-
gation [32]. Humans and animals synthesize an important
pterin, tetrahydrobiopterin (H4B), which participates in
the synthesis of nitric oxide and neurotransmitters, such
as dopamine (reviewed in [33,34]). In addition, dihydro-
neopterin and its oxidized form, neopterin, are well known
markers of immune system stimulation and are widely
used in diagnostics of numerous diseases [33]. They
possibly also participate in the stress response [33,35].
Therefore, perturbing pteridine status might have con-
sequences for human health, but relative roles and inter-
actions of endogenously synthesized versus dietary
pteridines in mammalian metabolism are unclear.
In an independent approach, folate biofortification of rice
was recently achieved [36]. In this case, Arabidopsis genes
encoding GTPCHI (Gene Bank accession AF489530) and
ADCS were overexpressed under the control of strong endo-
sperm-specific promoters on a single genetic locus. The
presumably negatively feedback-regulated plant GTPCHI
[37] was chosen to avoid an undesirably high accumulation
of intermediates. Transgenic rice seeds overexpressing both
Arabidopsis genes contained up to 100-fold higher folate
levels as compared with the wild type (1723 versus 17 mg/
100 g fresh weight). Cooking experiments have demon-
strated that it is probable that 100 g of the biofortified rice
grains can satisfy the daily folate requirement for an aver-
age adult person or at least supply most of it [36]. Moreover,
the levels of the biosynthesis intermediates, pterins and
Box 1. Folate deficiency and health
Humans cannot synthesize folates (vitamin B9) and thus have to rely
on plant food supplying these essential vitamins. The recommended
dietary allowance (RDA) for an adult person is 400 mg and 600 mg for
pregnant women (http://ods.od.nih.gov/factsheets/folate.asp).
Folate deficiency results in serious health problems, including
megaloblastic anemia and neural tube defects (NTD), such as spina
bifida and anencephaly. Adequate dietary folate intake can prevent
these conditions [62]. Given that the neural tube is formed between
days 21 and 27 after conception (before most women realize they
are pregnant), NTD risk can only be minimized if women take high
amounts of folate from the peri-conceptional phase until week 12 of
gestation. Recent studies indicate that NTD incidence in the poorest
regions in India and China can be up to 10 times higher than that in
Western surveillance systems [63,64]. Low folate status is also
associated with the occurrence of several neurodegenerative
disorders (such as Alzheimer’s disease) [65], a higher risk of
cardiovascular disease [66] and development of a range of cancers
[67], although no causal relationship has been established so far.
Folate deficiency first becomes visible in high-turnover cells, such
as erythrocytes, resulting in megaloblastic anemia because of a
deficit in DNA, necessary for normal cell division [2].Other
consequences are the induction of hyper-homocysteinemia, a risk
factor for cardiovascular disease [66], misincorporation of uracil in
DNA, and ultimately chromosomal breakage [68], resulting in
cellular degeneration. Finally, folate deficiency leads to aberrant
DNA-methylation patterns associated with carcinogenesis [67,69].
To reduce the risk of NTD, mandatory fortification of cereal-
derived foods with synthetic folic acid has been implemented in the
USA and other countries [70]. The amount of added folic acid is such
that the predicted average intake resulting from consumption of
fortified food products equals 100 mg/day, corresponding to
170 mg dietary folate equivalents (DFE) because folic acid is
assumed to be 1.7 times more bioavailable than natural folates
[71]. Evaluation of efficacy showed that reality surpasses this
prediction, and that the RDA was met or exceeded in most adults
[72,73]. However, as excessive intakes of folic acid (>1 mg/day)
might mask the diagnosis of vitamin B
12
deficiency [74,75],
fortification remains a controversial issue in the EU.
In third world countries, a solid infrastructural platform for
effective population-based prevention in the form of fortification,
supplementation or educational campaigns is lacking. Therefore,
biofortification of staple crops used in concert with conventional
public health practices will help in attaining the recommended
dietary intake of folates, especially in developing countries.
Review TRENDS in Plant Science Vol .13 No.1
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p-ABA, were substantially lower than in biofortified tomato
fruit. The molar ratios of folates:p-ABA:pterins in folate-
enhanced tomatoes are 1:2.5:0.75 [32], whereas they are
1:0.5:0.013 in biofortified rice [36]. It is therefore tempting to
speculate that the use of plant GTPCHI, which probably
retains its intrinsic negative feedback regulation, in combi-
nation with plant ADCS, results in a balanced tuning of both
enzyme activities, enabling a more optimal flux of pteridine
precursors and p-ABA through the pathway.
The accumulated folates in double transgenic tomato
fruit and rice grains showed normal proportions of C
1
forms but were not as extensively polyglutamylated as in
controls [32,36]. A reduction in glutamylation has no
negative impact on the nutritional value of folates and
can even be beneficial by enhancing bioavailability,
because a negative correlation with polyglutamate tail
length has been demonstrated [38,39].Theaccumulation
of folate precursors in double transgenic tomato fruit, and
to a lesser extent in transgenic rice grains, indicated a flux
constraint at the downstream HMDHP-pyrophosphoki-
nase (HPPK) step, suggesting the utility of a further
round of engineering to boost the activity of this enzyme
[32].
These two successful attempts at folate biofortification
in a dicot and a monocot plant species demonstrate that the
simultaneous enhancement of pterin and p-ABA branches
can be used as a universal approach applicable to other
plants. Optimization of metabolite fluxes can probably be
achieved by engineering other pathway enzymes.
Examples of such engineering in bacteria show a potential
for application to plants.
Enhancing folates in bacteria
Increasing GTPCHI and HPPK activity by overexpression of
folKE (a gene that encodes a bifunctional protein, displaying
both aforementioned activities; GenBank accession number
YP_809225) increased folate production threefold in Lacto-
coccus lactis and, as in plants, reduced the extent of poly-
glutamylation [40,41]. Co-expressing folylpolyglutamyl
synthase (GenBank accession YP_809228), which is respon-
sible for adding a glutamyl-chain to THF, restored normal
polyglutamylation. Overexpressing the downstream
enzymes dihydropteroate synthase (DHPS) (GenBank
accession YP_809226) and dihydrofolate reductase (DHFR)
(GenBank accession YP_001033455) along with FolKE gave
no additional increase in folate production, or even
Figure 2. The folate biosynthesis pathway, its compartmentalization in plant cells and carrier-mediated transport steps. The two known folate carriers (both plastidial) are
shown in black. Hypothetical carriers are shown in gray with dotted lines indicating hypothetical transport steps (the movement of p-ABA is most probably by diffusion).
The hypothetical vacuolar folate carrier might transport polyglutamyl forms, unlike most other folate carriers. p-ABA occurs mainly as its glucose ester, which is formed in
the cytosol via a reversible reaction with UDP-glucose [19]. Compound abbreviations: ADC, aminodeoxychorismate; DHF, dihydrofolate; DHM, dihydromonapterin; DHN,
dihydroneopterin; DHP, dihydropteroate; –Glc, glucose ester; –Glu
n
, polyglutamate; HMDHP, hydroxymethyldihydropterin; –P, phosphate; –P
2
, diphosphate; –P
3
,
triphosphate; THF, tetrahydrofolate. Enzymes: 1, GTP cyclohydrolase I; 2, DHN-P
3
pyrophosphatase; 3, non-specific phosphatase; 4, dihydroneopterin aldolase (which
mediates the epimerization of DHN to DHM, and the aldol cleavage of both); 5, aminodeoxychorismate synthase; 6, aminodeoxychorismate lyase; 7,
hydroxymethyldihydropterin pyrophosphokinase; 8, dihydropteroate synthase; 9, dihydrofolate synthase; 10, dihydrofolate reductase; 11, folylpolyglutamate synthase;
12, p-ABA glucosyltransferase.
Review TRENDS in Plant Science Vol .13 No.1
31

decreased production, as in the case of DHFR [40]. Taken
together, these results warrant trials for engineering HPPK
activity in plants becausethe effects of engineering HPPK as
well as GTPCHI were not separated in L. lactis and might
have been additive. A more theoretical approach to folate
pathway engineering in Bacillus subtilis achieved an eight-
fold increase in folate production by amplifying expression of
the genes in a folate operon, combined with genetic manip-
ulations designed to increase p-ABA formation [42].
Other metabolic engineering strategies
Several ways to engineer folate content besides overex-
pressing biosynthesis genes potentially exist. Leaf
vacuoles contain folates that are probably protein-bound
[17], so if vacuolar transporter and folate-binding proteins
(FBPs) had been identified (which is not yet the case),
either or both might be manipulated to enhance storage
in vacuoles, in which folates are able to accumulate to high
levels [17]. For grains or other storage organs, a plant FBP
(if these exist) or the well characterized bovine milk FBP
[27] might be targeted to protein-storage vacuoles. Tightly
sequestering the end-products of the pathway in this man-
ner could bring about a folate-sink effect, increasing bio-
synthetic flux by releasing negative-feedback controls and
causing precursor pools to shrink rather than expand.
Another strategy for fruits and vegetables would be to
cut post-harvest losses [2,23]. To do this, we would first
need to know (which we do not) whether the net folate
breakdown reflects increased degradation, or decreased
salvage or synthesis. When both p-ABA and pteridines
were overproduced in tomato, it was observed that pter-
idines were largely oxidized after ripening [29], in which
state they can no longer be used for folate synthesis.
Introducing a foreign (and potentially feedback-free)
pteridine reductase [26] might thus be helpful to boost
folate salvage capacity.
A more readily implemented strategy is to reduce
expression of the enzyme that returns 5-formyl-THF (a
stable folate that is not a C
1
donor) to the active C
1
folate
pool [2]. This should cause build-up of 5-formyl-THF, and
probably of total folate, and indeed the first experiments
exploring this path showed 5-formyl-THF accumulation
and a doubling of total folate levels [43]. However,
5-formyl-THF accumulation is a double-edged sword:
although this folate is not a C
1
donor, it inhibits folate-
dependent enzymes and hence can disrupt C
1
metabolism
[43]. Despite observations that some over-accumulation of
5-formyl-THF (using 10mM 5-formyl-THF in feeding trials
on Arabidopsis plants) is well tolerated [43], these inhibi-
tory effects most probably limit the extent to which
5-formyl-THF can accumulate in the cytosol, mitochondria
or plastids (but not vacuoles, which contain no folate-
dependent enzymes to inhibit).
Parallel to using transgenic technology to enhance
folate levels in plants, it is important that the pursuit of
classical breeding strategies is continued, because pro-
ducts emerging from this approach are readily accepted
by consumers.
Natural variation in folate levels
Exploiting natural genetic variation within species and
between related species is a paradigm for crop improvement,
and combining genomics with conventional breeding
methods has an enormous potential for nutritional improve-
ment of crops [9–11]. Varieties can be phenotypically ana-
lyzed on a large scale as soon as high-throughput procedures
for folate determination are available [44]. This should
enable mapping of quantitative trait loci (QTLs), ultimately
to be integrated in molecular marker-assisted selection.
Crops such as rice, maize and wheat are appealing targets
for enrichment, because they contain low levels of folates
and are staple foods indeveloping countries. Given their low
intrinsic folate concentrations, it might be the case that only
limited enhancement can be achieved through breeding
strategies [45]. However, this has yet to be established, so
it is important to explore this approach. Significant oppor-
tunities exist to enhance folate in tomato where wild genetic
resources are being harnessed through conventional breed-
ing. This goal is now a realistic possibility because of the
availability of well characterized interspecific introgression
lines (ILs) and novel populations. Introgression populations
are available, which are comprised of marker-defined
regions of wild-species genomes introgressed into a Sola-
num lycopersicum background [46]. Preliminary studies
employing the microbiological assay to screen ILs have
observed several-fold variation in folate abundance within
the population (G. Tucker, personal communication). These
near isogenic lines are a powerful tool for fine mapping of
QTLs [47]. Moreover,mapped QTLs are potentially valuable
in identifying and eventually cloning new target genes
involved in folate metabolism and its regulation. The avail-
ability of complete genome sequence information and a large
array of molecular markers make a QTL approach particu-
larly feasible in rice [48].
Figure 3. Folate salvage reactions (blue arrows) in relation to folate biosynthesis.
Oxidative or photo-oxidative cleavage of folates gives rise to p-
aminobenzoylglutamate (p-ABA-Glu) or its polyglutamyl forms (p-ABA-Glu
n
) and
a pterin aldehyde. In the case of tetrahydrofolate (THF), the aldehyde is
tetrahydropterin-6-aldehyde, which can oxidize spontaneously to dihydropterin-
6-aldehyde (DHPA). DHPA can also be formed directly from cleavage of
dihydrofolate (DHF). Enzymes: GGH, g-glutamyl hydrolase (which removes the
polyglutamyl tail of p-ABA-Glu
n
); PGH, p-ABA-Glu hydrolase; PTAR, pterin
aldehyde reductase.
Review TRENDS in Plant Science Vol .13 No.1
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Enhancing folate bioavailability
Bioavailability issues will have to be addressed regardless
of what method is being applied, because poor bioavail-
ability could annihilate the effect of enhanced folate con-
tent; conversely, improving bioavailability could increase
the nutritional value of a crop without the need to enhance
nutrient levels as such. Natural folates exhibit reduced
bioavailability compared with synthetic forms. It is known
that polyglutamylation can reduce folate bioavailability,
because dietary folates need to be deglutamylated by the
intestinal conjugase, an enzyme that hydrolyses the poly-
glutamate tail, before efficient intestinal uptake can take
place [45]. Recently, however, it has been shown that the
ratio of monoglutamate to polyglutamate in natural food
folate-derivatives had no apparent influence on the intes-
tinal absorption [38], suggesting that the amount of the
intestinal conjugase is more than adequate to remove the
polyglutamate tail without affecting the absorption rate.
This suggests that other factors have an impact on bioa-
vailability, such as entrapment of folates in the food
matrix, rendering them inaccessible to the conjugase that
is tethered to the intestinal cell membranes. Therefore, one
potential strategy to increase bioavailability could be to
enhance levels of the plant conjugase activity of gamma-
glutamyl hydrolase (GGH), which would be released from
the vacuole following maceration and facilitate folate
release within the food matrix before digestion. An alterna-
tive strategy is the use of FBPs. A hitherto unidentified
component of cow’s milk, possibly FBP, has been reported
to improve the bioavailability of food folates [49], but the
relevance to human nutrition of the study on which this
conclusion was based has been questioned. One must also
consider the fact that the presence of FBPs in the food
matrix, although mechanistically unclear, can lead to a
reduction in folate absorption [50]. Nutritional studies
might in this way present new approaches that could be
implemented in the creation of a health beneficial crop.
Determination of folate bioavailability and bioefficacy in
such a crop will ultimately require data from controlled
absorption tests in human volunteers, and data from larger
feeding trials in folate-deficient test populations will have
to be acquired to demonstrate the efficacy of enriched crop
varieties. In addition, there will be a need to assess
whether high pterin or p-ABA concentrations accumulated
as a result of engineering have any effect on folate uptake,
transport or metabolism [51].
Finally, it should be mentioned that the water-soluble
nature of the folate cofactor removes a major criticism [52]
that has been leveled at provitamin A-enriched rice grains
[53]. Because provitamin A is fat-soluble, adequate intake
of fats is essential for its absorption, which is not guaran-
teed in the diet of the poor. This is not a problem for folates.
The feasibility of folate-biofortified crops
Direct advantages of biofortification
Using a folate-enriched staple crop to help combat
folate deficiency has two immediate advantages. First,
biofortification offers sustainability when compared with
industrial fortification (addition of synthetic folic acid to
cereal-derived foods) and pharmaceutical supplementation
(Box 1). Development of a biofortified crop is largely a
one-time investment that can benefit the health of millions
and therefore establishes a multiplier effect [54]. Second,
biofortified crops can improve folate intake by malnour-
ished rural populations that are unlikely to benefit from
commercially fortified foods or supplements, which are
often only available in cities. Therefore, biofortification
is complementary to these other intervention strategies.
Regional adaptation
To ensure the adoption of biofortification by farmers, it is
important that crop productivity and/or profits are
increased simultaneously. This can be accomplished by
introducing the biofortification trait into high-yielding
(elite) genotypes [55]. Therefore, biofortified lines will have
to enter breeding programs to be crossed with varieties
that are locally adapted to a given agro-ecosystem. This
will evidently require a certain level of national research
capacity [56]. However, in regions such as South-East Asia,
effective systems for disseminating improved crop
varieties are already in place, making such implementa-
tion costs minimal [57].
Cost-effectiveness of biofortification
Biofortification is expected to be cost-effective based on
studies quantifying the potential health benefits for vita-
min A, iron and zinc-enhanced crops using the disability-
adjusted life years (DALY) approach [57,58]. This model
calculates the reduction of the current disease burden
associated with vitamin deficiency (i.e. the mortality and
morbidity burden quantified as the number of DALYs lost)
resulting from biofortification, taking into account current
intake of the staple crop, the expected intake of micronu-
trient(s) after crop biofortification, and the percentage of
the population expected to consume the biofortified crop.
Application of the DALY methodology to the impact of
golden rice 2 (an improved version of b-carotene bioforti-
fied golden rice) in India gave an estimated reduction of the
burden of vitamin A deficiency (VAD) of between 9% (low-
impact scenario) and 59% (high-impact scenario) [59].
Even the 9% reduction translates into saving many thou-
sands of people from blindness or death from infectious
disease. Moreover, golden rice 2 promises to be cost effec-
tive, because it was estimated that even under a low impact
scenario the cost of saving one DALY is less than US$20 as
compared with a cost of at least US$134 of doing it by
vitamin A supplementation [59].
Towards consumer acceptance
It is unlikely that folate-enrichment of food plants will
result in any sensory change, as is the case for golden rice,
so from this standpoint consumer acceptance should not be
compromised. However, concerns about potentially harm-
ful environmental (e.g. loss of biodiversity) and health (e.g.
allergy) impacts are inevitable, especially in the European
Union, particularly if biofortification is achieved by meta-
bolic engineering. This does not mean that engineering
approaches should be abandoned, because acceptance of
‘genetically modified’ crops is likely to grow as their
benefits become clear to consumers [60], as has happened
for other scientific and medical breakthroughs of the past
two centuries (e.g. the widespread controversy around the
Review TRENDS in Plant Science Vol .13 No.1
33

first mass-vaccination, an intervention that ended up
eradicating smallpox, at that time a deadly disease). The
health risks and benefits of genetic manipulation of food
crops depend almost exclusively on the chemical compo-
sition (profiles of nutrients, metabolites, antinutritional
factors, etc.) of the resulting product, not on the technology
used to achieve the modification [61]. Indeed, it can be
argued that genetic engineering provides more targeted,
specific and predictable alterations of food crop composition
than occurs with the various ‘conventional’ approaches
(including conventional breeding, mutation breeding, soma-
clonal variation, somatic hybridization, etc.) [61]. Moreover,
much can be learned from metabolic engineering studies
about how folate content is controlled in plants, and this
knowledge suggests how folate levels can be increased with-
out using transgenic technology.
Conclusion
Folate-biofortified rice, tomatoes and Arabidopsis plants
have already been developed using simple metabolic
engineering stratagems, and enough is known about plant
folate biochemistry to envisage other biofortified crops,
such as wheat, banana and potato. Currently, folate
deficiency persists, and will continue to do so until we
deploy agriculture-based strategies to help reduce this
global burden. Folate biofortification of staple crops
should be a valuable, complementary and cost-effective
intervention in fighting folate deficiency worldwide, above
all in poor countries.
Acknowledgements
Our work was supported by Ghent University (Bijzonder
Onderzoeksfonds, GOA 1251204) (S.B., S.S., W.L. and D.V.D.S.), by the
Home Grown Cereals Association (P.M. and M.J.B.), by National Science
Foundation Grant MCB0443709 (A.D.H. and J.F.G.), and by the C.V.
Griffin Sr Foundation (A.D.H.). We thank Greg Tucker (University of
Nottingham) for sharing unpublished information on natural variation of
folates in tomato.
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