PP62CH04-Hanson ARI 22 January 2011 11:19

V I E W
E
R

S
Review in Advance first posted online
on January 26, 2011. (Changes may
C E
I N

still occur before final publication

online and in print.)
N

A
D V A

Folate Biosynthesis, Turnover,

and Transport in Plants
Andrew D. Hanson1 and Jesse F. Gregory III2
1
Horticultural Sciences Department and 2 Food Science and Human Nutrition Department,
Annu. Rev. Plant Biol. 2011.62. Downloaded from www.annualreviews.org

University of Florida, Gainesville, Florida 32611; email: adha@ufl.edu, jfgy@ufl.edu

by RIJKSUNIVERSITEIT GENT on 02/01/11. For personal use only.

Annu. Rev. Plant Biol. 2011. 62:4.1–4.21 Keywords

The Annual Review of Plant Biology is online at
plant.annualreviews.org biofortification, breakdown, compartmentation, engineering, salvage
This article’s doi: Abstract
10.1146/annurev-arplant-042110-103819
Folates are essential cofactors for one-carbon transfer reactions and are
Copyright  c 2011 by Annual Reviews.
All rights reserved needed in the diets of humans and animals. Because plants are major
sources of dietary folate, plant folate biochemistry has long been of in-
1543-5008/11/0602-0001$20.00
terest but progressed slowly until the genome era. Since then, genome-
enabled approaches have brought rapid advances: We now know
(a) all the plant folate synthesis genes and some genes of folate turnover
and transport, (b) certain mechanisms governing folate synthesis, and
(c) the subcellular locations of folate synthesis enzymes and of folates
themselves. Some of this knowledge has been applied, simply and suc-
cessfully, to engineer folate-enriched food crops (i.e., biofortification).
Much remains to be discovered about folates, however, particularly in
relation to homeostasis, catabolism, membrane transport, and vacuolar
storage. Understanding these processes, which will require both bio-
chemical and -omics research, should lead to improved biofortification
strategies based on transgenic or conventional approaches.

4.1

Changes may still occur before final publication online and in print
 PP62CH04-Hanson ARI 22 January 2011 11:19
Contents
INTRODUCTION . . . . . . . . . . . . . . . . . . 4.2
FOLATE BIOSYNTHESIS . . . . . . . . . . 4.3
Pterin Synthesis . . . . . . . . . . . . . . . . . . . 4.3
p-Aminobenzoate Synthesis. . . . . . . . . 4.4
Folate Assembly, Polyglutamylation,
and Deglutamylation . . . . . . . . . . . . 4.5
Regulation of Folate Synthesis . . . . . . 4.6
FOLATE TURNOVER . . . . . . . . . . . . . . 4.7
Folate Breakdown . . . . . . . . . . . . . . . . . . 4.7
Folate Salvage Processes . . . . . . . . . . . 4.7
Transport of Folates
and Precursors . . . . . . . . . . . . . . . . . . 4.9
Annu. Rev. Plant Biol. 2011.62. Downloaded from www.annualreviews.org
Cloning Plant Folate Transporters
by RIJKSUNIVERSITEIT GENT on 02/01/11. For personal use only.
by Homology . . . . . . . . . . . . . . . . . . . 4.12
ENGINEERING FOLATE
LEVELS . . . . . . . . . . . . . . . . . . . . . . . . . . 4.12
Engineering of Biosynthesis . . . . . . . . 4.12
Engineering of Polyglutamylation . . 4.14
Observations on Biofortification . . . . 4.14
FOLATE FRONTIERS . . . . . . . . . . . . . . 4.15
INTRODUCTION
Tetrahydrofolate (THF) is an essential cofactor
in almost all forms of life, in which it acts as a
carrier for one-carbon (C1 ) units in enzymatic
reactions that form amino acids (methionine,
THF:
tetrahydrofolate glycine, and serine), purines, thymidylate, pan-
tothenate, and formylmethionyl–transfer RNA
C1 : one-carbon
(14, 38). THF and its C1 -substituted deriva-
Biofortification: the
tives are collectively termed folates (vitamin
breeding of crops to
increase their B9). Humans and animals are unable to make
nutritional value by folates de novo and hence depend on dietary
using conventional sources, especially plants (11, 83). The health
crossing and selection impacts of folate deficiency in humans can be
or genetic engineering
severe; they include anemia, spina bifida and
Pterin: a heterocyclic other birth defects, and a higher risk of car-
compound containing
diovascular disease and certain cancers (11, 87).
a pyrazine ring fused to
a pyrimidine ring that Because most plant foods are rather low in fo-
has a carbonyl oxygen lates and folates are lost during processing and
and an amino group cooking, dietary folate deficiency can occur eas-
pABA: ily and is widespread in poorer countries as well
p-aminobenzoate as in some populations within richer ones (11,
DHF: dihydrofolate 83). Folate deficiency is consequently a signifi-
cant worldwide public health problem.
4.2 Hanson · Gregory
Changes may still occur before final publication online and in print
The prevalence of folate deficiency has led
the United States (since 1998) and subsequently
many other Western countries to mandate fo-
late fortification—the addition to cereal grain
products of chemically synthesized folic acid,
which is metabolized to THF (92). An alterna-
tive approach is dietary supplementation with
folic acid, specifically vitamin pills. However,
both fortification and supplementation are dif-
ficult to implement in poorer countries and may
have inherent drawbacks related to the fact that
folic acid is an unnatural compound (11, 43,
92). Over the past decade, these concerns have
spurred research on plant folate biosynthesis
that is directed toward metabolic engineering
of natural folate content (biofortification). This
new research direction has reinforced the drive
to understand plant folate synthesis because of
its fundamental importance in metabolism.
Given that recent research has focused
mainly on folate biosynthesis and its engineer-
ing, most progress has been in these areas,
and reviews have given various aspects of this
progress pride of place (9, 11, 76, 82, 96). In
covering advances in plant folate biosynthesis
and engineering, this review therefore empha-
sizes what is still not known and does likewise
for the much less explored areas of homeostasis,
catabolism, transport, and storage. Through-
out this review, information from studies of mi-
crobes and animals is used to supplement that
available from studies of plants.
THF is a tripartite molecule composed of
pterin, p-aminobenzoate ( pABA), and gluta-
mate moieties (Figure 1). The pterin ring of
folate exists naturally in dihydro or tetrahydro
form; only the latter has cofactor activity. The
ring is fully oxidized in folic acid, which—as
noted above—is not a natural folate, although
it can be reduced via dihydrofolate (DHF) to
THF. C1 units at various levels of oxidation
(formyl, methylene, methyl) can be enzymati-
cally attached to the N5 and/or N10 positions of
THF (Figure 1); the resulting C1 -substituted
folates are enzymatically interconvertible and
serve as C1 donors for various reactions. A key
characteristic of THF, most of its C1 forms,
and DHF is susceptibility to spontaneous
 PP62CH04-Hanson ARI 22 January 2011 11:19

oxidative or photooxidative cleavage of the C9– a O O COOH

H 9 H
N10 bond that links the pterin and pABA moi- N CH2 N C N CH CH2 CH2 COOH
HN 5 H 10 H α γ
eties. This inherent lability underlies the need
of humans and animals for a continual supply H
H2N N N H p-Aminobenzoylglutamate
of folate; what is degraded must be replaced, or H
deficiency ensues. Tetrahydropterin p-Aminobenzoate Glutamate
A short, γ-linked chain of additional
glutamate residues (up to approximately six) THF
is typically attached to the first glutamate
(Figure 1). This polyglutamyl tail is important b O c
to folate function because folate-dependent en- N C1 unit Name
CH2–
HN
zymes generally prefer polyglutamates, whereas N5–CH3 5-methyl-THF
folate transporters prefer monoglutamyl forms H
H2N N N H OHC–N10 10-formyl-THF
(89). The tail thus affects both cofactor activity H
N5–CH–N10 5,10-methenyl-THF
Annu. Rev. Plant Biol. 2011.62. Downloaded from www.annualreviews.org

and membrane transport. Moreover, because Dihydropterin moiety

N5–CH2–N10
by RIJKSUNIVERSITEIT GENT on 02/01/11. For personal use only.

of dihydrofolate 5,10-methylene-THF
the tail promotes enzyme binding and folates
are far more stable to oxidative cleavage when N5–CHO 5-formyl-THF

bound than when free, polyglutamylation can Figure 1

indirectly enhance folate stability (89).
FOLATE BIOSYNTHESIS
Folates are present throughout plant cells—in
the mitochondria, plastids, cytosol, and vac-
uoles (3, 13, 30, 61, 67)—but are synthesized
only in mitochondria. The pterin and pABA
precursors are made in the cytosol and plastids,
respectively (Figure 2).
Pterin Synthesis
Pterin synthesis (Figure 2, steps A–C) be-
gins with the conversion of GTP to dihydro-
neopterin triphosphate, which is mediated by
GTP cyclohydrolase I (GCHI). The plant en-
zyme has an unusual structure. In other organ-
isms, it is a homodecamer of ∼26-kDa subunits,
whereas in plants GCHI is a homodimer of sub-
units with two tandem domains, each of which
is similar to a canonical GCHI monomer (5,
56). Given that neither domain has a full set of
the residues involved in substrate binding and
catalysis in other GCHIs, it is not clear how
plant GCHI functions catalytically (5). Plant
GCHI is thus an interesting target for future
three-dimensional structure-mechanism stud-
ies. Further reasons to pursue such studies are
that GCHI is the committing enzyme of pterin
Structure of folates. (a) Chemical structure of tetrahydrofolate (THF),
monoglutamyl form. The red arrowhead marks the oxidatively labile C9–N10
bond. A polyglutamyl tail can be attached via the γ-carboxyl group of the
glutamate moiety. (b) The 7,8-dihydropterin moiety of dihydrofolate. (c) The
various one-carbon (C1 ) substituents of THF.
synthesis in plants and that, atypically, it shows
substrate inhibition by GTP (5). Plant GCHI
is apparently cytosolic (5, 56).
The dihydroneopterin triphosphate prod-
uct of GCHI is then dephosphorylated to di-
hydroneopterin in two steps. The first step
in plants, as in bacteria, is the removal of
pyrophosphate, which yields dihydroneopterin
monophosphate (46). An Arabidopsis enzyme
from the large Nudix family catalyzes this re-
action in vitro (46), but the same protein may
have other functions (65), and its in vivo role
in folate synthesis awaits genetic confirmation.
As with GCHI, the Nudix hydrolase appears
to be cytosolic (46). Hydrolysis of dihydro-
neopterin monophosphate to dihydroneopterin
may be carried out by a nonspecific phosphatase Polyglutamyl tail: a
in plants, as in Escherichia coli (91), but there is short chain of γ-linked
as yet no biochemical or genetic evidence for glutamate residues
added enzymatically to
this hypothesis for plants, so a specific enzyme
the glutamate moiety
remains a possibility. of folates
The side chain of dihydroneopterin is then
GCHI: GTP
cleaved to 6-hydroxymethyldihydropterin cyclohydrolase I
(HMDHP) and glycolaldehyde by

www.annualreviews.org • Folate Synthesis and Metabolism 4.3

Changes may still occur before final publication online and in print
 PP62CH04-Hanson ARI 22 January 2011 11:19

Mitochondrion
HMDHP 1 HMDHP [Glycosides]
Plastid F C
HMDHP-P2 DHM DHN
pABA pABA pABA
G Pi
K E DHP DHN-P
ADC
pABA-Glc
Glu
H PPi B
D DHF DHN-P3
Chorismate
* 3
I A
THF THF 2 THF GTP
* 4 Glu
Glu J3 J 2
Glu J1
Folate-Glun Folate-Glun Folate-Glun

5 6
Annu. Rev. Plant Biol. 2011.62. Downloaded from www.annualreviews.org

pABA-Glc
L * 7
by RIJKSUNIVERSITEIT GENT on 02/01/11. For personal use only.

Folate-Glun Folates Folates Pterins

Vacuole

8 9

Folates Pterins

Figure 2
The folate biosynthesis pathway and its compartmentation. Red letters denote enzymes that mediate
biosynthetic steps or ancillary reactions: A, GTP cyclohydrolase I (EC 3.5.4.16); B, dihydroneopterin
(DHN) triphosphate diphosphatase (EC 3.6.1.n4); C, DHN aldolase (EC 4.1.2.25); D,
aminodeoxychorismate synthase (EC 2.6.1.85); E, aminodeoxychorismate lyase (EC 4.1.3.38);
F, 6-hydroxymethyldihydropterin (HMDHP) pyrophosphokinase (EC 2.7.6.3); G, dihydropteroate (DHP)
synthase (EC 2.5.1.15); H, dihydrofolate (DHF) synthase (EC 6.3.2.12); I, DHF reductase (EC 1.5.1.3);
J1–J3, isoforms of folylpolyglutamate synthase (EC 6.3.2.17); K, UDP-glucose–p-aminobenzoate ( pABA)
glucosyltransferase; L, γ-glutamyl hydrolase (EC 3.4.19.9). Circled numbers designate known or inferred
transporters; only those marked with an asterisk (transporters 3, 4, and 7) are cloned. Abbreviations: ADC,
aminodeoxychorismate; DHM, dihydromonapterin; Glu, glutamate; Glun , polyglutamate; -P, phosphate;
P2 , diphosphate; P3 , triphosphate; pABA-Glc, p-aminobenzoate β-D-glucopyranosyl ester; THF,
tetrahydrofolate.

dihydroneopterin aldolase; this enzyme p-Aminobenzoate Synthesis

also mediates the epimerization of dihy-
pABA is synthesized from chorismate, the prod-
droneopterin to dihydromonapterin, which
uct of the shikimate pathway, in two steps
it likewise cleaves to yield HMDHP (31).
(Figure 2, steps D, E). Both steps are localized
Arabidopsis and other plants have small, di-
in plastids, as is the shikimate pathway (6, 7).
verged dihydroneopterin aldolase families;
First, chorismate is converted to aminodeoxy-
their members lack obvious targeting signals
chorismate by aminodeoxychorismate synthase
and therefore are presumably cytosolic (31).
(ADCS), a bipartite protein with tandem do-
Dihydroneopterin and dihydromonapterin can
mains that are homologous to the PabA and
be metabolized to β-D-glycosides, at least in
PabB subunits of E. coli ADCS (6, 57). Second,
tomato fruit engineered to overproduce pterins
aminodeoxychorismate lyase converts amin-
(20). Neither the sugar moiety nor the glyco-
ADCS: aminodeoxy- odeoxychorismate to pABA (7). pABA can be
syltransferase(s) involved have been identified,
chorismate esterified to glucose in a reversible reaction me-
synthase nor is it known whether the glycosides serve as
diated by a cytosolic UDP-glucosyltransferase
a mobilizable reserve of pterins.

4.4 Hanson · Gregory

Changes may still occur before final publication online and in print
 PP62CH04-Hanson ARI 22 January 2011 11:19
(Arabidopsis At1g05560, UGT75B1) (Figure 2,
step K) (24, 72). The ester is often more abun-
dant than free pABA (72, 101) and is largely
(≥88%) sequestered in vacuoles in pea leaves
(24). The ester can be reconverted to pABA in
vitro by reversal of the synthesis reaction or via
an esterase activity (72). The relative impor-
tance of these routes in vivo is unknown, and
the protein(s) and gene(s) responsible for the
esterase activity have not been identified.
Folate Assembly, Polyglutamylation,
and Deglutamylation
Annu. Rev. Plant Biol. 2011.62. Downloaded from www.annualreviews.org
The HMDHP and pABA precursors are as-
by RIJKSUNIVERSITEIT GENT on 02/01/11. For personal use only.
sembled into THF in the mitochondrion
(Figure 2, steps F–I). HMDHP is first ac-
tivated by pyrophosphorylation, then coupled
to pABA to yield dihydropteroate. These re-
actions are respectively catalyzed by HMDHP
pyrophosphokinase and dihydropteroate syn-
thase, which are two domains of a single bifunc-
tional protein in plants (57, 77). Aside from the
canonical, mitochondrially targeted HMDHP
pyrophosphokinase–dihydropteroate synthase,
Arabidopsis has a cytosolic form (86), but this
is expressed only in developing seeds and salt-
stressed seedlings and appears to have no coun-
terparts in other higher plants. Next, DHF
synthase couples dihydropteroate to glutamate
to yield DHF (74). DHF synthase is unusual
among plant folate synthesis enzymes in that its
function has been confirmed by mutant stud-
ies; disruption of the Arabidopsis gene encod-
ing DHF synthase (At5g41480, GLA1) results
in folate deficiency and is embryo lethal (42).
Finally, DHF is reduced to THF by DHF re-
ductase, which in plants is fused to thymidylate
synthase (15, 53, 61).
The polyglutamate tail is added to THF and
its C1 -substituted forms, one residue at a time,
via the action of folylpolyglutamate synthase
(FPGS) (41, 74). Arabidopsis has three FPGS
isoforms, and other higher plants appear to have
two or more (57, 94). On the basis of immuno-
logical evidence and green fluorescent protein
fusion experiments, the three Arabidopsis iso-
forms appear to be specifically targeted to the
Changes may still occur before final publication online and in print
cytosol, mitochondria, and plastids (Figure 2,
steps J1–J3) (74). However, data from single
and double FPGS knockouts strongly suggest
FPGS:
that one or more FPGS isoforms are targeted folylpolyglutamate
to multiple organelles (58). Multiple targeting synthase
could account for how, in plants such as tomato, GGH: γ-glutamyl
two FPGS genes suffice (94). In any case, the hydrolase
presence of FPGS in cytosol, mitochondria, and
plastids is consistent with the presence of polyg-
lutamates in these compartments (13, 61, 67)
and with the generalization (89) that monog-
lutamates are the transported forms of folate.
Vacuoles, however, are exceptions; they con-
tain folate polyglutamates (3, 67) but almost
certainly not FPGS or the ATP required for the
FPGS reaction (27, 74). Vacuoles presumably
import polyglutamates, as discussed below.
The folate polyglutamate tail is not a static
entity but can be shortened or removed by
γ-glutamyl hydrolase (GGH), which can have
endo- and exopeptidase activities (2, 14, 67).
GGH is located in vacuoles (Figure 2, step
L) (2, 67). As mentioned above, vacuoles also
contain folate polyglutamates, and vacuolar
GGH activity is sufficiently high to hydrolyze
these polyglutamates within minutes (67). That
polyglutamates survive in the vacuole is there-
fore a mystery. Possible explanations include
the presence of a potent GGH inhibitor, folate-
binding proteins that protect polyglutamates
from hydrolysis, the partitioning of GGH and
folate polyglutamates into distinct vacuolar
subpopulations, or perhaps intravacuolar com-
partmentation similar to that demonstrated for
anthocyanins (54). Although it is not known
how GGH activity is restrained, this activity
plays a role in governing polyglutamyl tail
length in vivo. Thus, in Arabidopsis leaves and
tomato fruit, overexpressing GGH in vacuoles
reduces average folate polyglutamate tail
length (3); total folate content is also reduced,
which accords with the idea that polyglutamy-
lation favors folate binding to proteins and
hence stability (89). Conversely, ablating 99%
of GGH activity in Arabidopsis increases both
tail length and total folate content (3). Taken
together, these results suggest that folates con-
tinuously enter the vacuole as polyglutamates,
www.annualreviews.org • Folate Synthesis and Metabolism 4.5
 PP62CH04-Hanson ARI 22 January 2011 11:19

accumulate there, are eventually hydrolyzed by
GGH, and exit as monoglutamates (Figure 2).
Regulation of Folate Synthesis
The levels of folates and their precursors re-
main within characteristic ranges for different
tissues (44, 66), developmental stages (5, 33,
44), and genotypes (32, 69, 88), which implies
(a) that the synthesis pathway is regulated and
(b) that the regulatory mechanisms vary ge-
netically. Despite their pivotal importance for
biofortification efforts, these mechanisms are
known only in a fragmentary way; a coher-
Annu. Rev. Plant Biol. 2011.62. Downloaded from www.annualreviews.org
ADCS transgenes in tomato fruit causes folate
accumulation and increased expression of the
downstream genes dihydroneopterin aldolase,
aminodeoxychorismate lyase, and mitochon-
drial FPGS (94). The accumulation of dihy-
droneopterin (or its phosphates) apparently
induces the aldolase, and accumulation of amin-
odeoxychorismate induces the lyase; FPGS may
be induced by accumulation of THF or other
monoglutamyl folates (94). Notably, despite
the massive buildup of pterins and pABA asso-
ciated with expression of the GCHI and ADCS
GTP
A

ent overall picture has yet to emerge. Present

by RIJKSUNIVERSITEIT GENT on 02/01/11. For personal use only.

knowledge is summarized in Figure 3. As in DHN-P3

other biosynthetic pathways, regulatory loops B
appear to operate at both enzyme and gene lev- Plastid DHN-P

els. Thus, in vitro studies have demonstrated +

Chorismate DHN
strong feedback inhibition of dihydropteroate C
D
synthase by dihydropteroate, DHF, and THF
ADC HMDHP
(59). Dihydropteroate and DHF also inhibit +
– E F
the pABA synthesis enzyme ADCS (81), but it
pABA HMDHP-P2
seems unlikely that this effect enables feedback G
control in vivo because dihydropteroate and DHP
DHF pools are, presumably, mainly mitochon- Glu H –
–
drial, whereas ADCS is plastidial. No other DHF
feedback-regulatory properties of plant pABA I –
or pterin synthesis enzymes have been demon-
THF THF
strated (5, 6, 7, 31). However, for human FPGS, J2 +
Glu
–
Glu J1
high folate substrate concentrations curtail the Folate-Glun Folate-Glun
formation of long-chain polyglutamates (93),
and the low average polyglutamyl tail lengths Mitochondrion
in plants engineered to overproduce folates (21,
Figure 3
85) are consistent with a similar negative effect
Potential regulatory loops operating at the enzyme
of high folate levels on plant FPGS enzymes. and gene levels in folate biosynthesis. Broken purple
Transcriptomic analyses have provided lines denote potential enzyme-level feedback
evidence for both feedback and feedforward inhibition mechanisms identified by in vitro studies.
regulation of the expression of folate pathway Broken blue lines denote gene-level feedback
repression (minus signs) or feedforward activation
genes. Specifically, blocking folate synthesis in
( plus signs) mechanisms inferred from transcriptome
Arabidopsis cells with the folate analog data. Folate biosynthesis enzymes are shown by
methotrexate (a DHF reductase inhibitor) letters as in Figure 2. Dihydromonapterin has been
causes folate depletion and an increase in omitted for simplicity. Abbreviations: ADC,
transcript level of a single folate synthesis gene, aminodeoxychorismate; DHF, dihydrofolate;
DHN, dihydroneopterin; DHP, dihydropteroate;
the cytosolic isoform of FPGS (51), which
Glu, glutamate; Glun , polyglutamate; HMDHP,
suggests that folate polyglutamates exercise 6-hydroxymethyldihydropterin; P, phosphate;
feedback control over expression of this gene. P2 , diphosphate; P3 , triphosphate; pABA,
Conversely, overexpression of the GCHI and p-aminobenzoate; THF, tetrahydrofolate.

4.6 Hanson · Gregory

Changes may still occur before final publication online and in print
 PP62CH04-Hanson ARI 22 January 2011 11:19
transgenes, expression of the endogenous
GCHI and ADCS genes is unaffected (94).
Thus, neither gene-level nor enzyme-level
feedback appears to control the committing
reactions of pterin and pABA synthesis, yet
metabolic engineering studies have demon-
strated that these two reactions exert major
control over flux through the folate pathway
(21, 85). Furthermore, folate synthesis in rice
grains is strongly inhibited in lines engineered
to overproduce pABA (85). Such paradoxical
observations nicely illustrate how little the
regulation of folate biosynthesis is understood.
Lastly, mammalian GCHI is regulated by a
Annu. Rev. Plant Biol. 2011.62. Downloaded from www.annualreviews.org
feedback mechanism that involves a regulatory
by RIJKSUNIVERSITEIT GENT on 02/01/11. For personal use only.
protein (99) and also by phosphorylation
(49). Such mechanisms could not have been
detected by the published studies of plant
folate synthesis enzymes because all of them
used single recombinant proteins from a
heterologous host (E. coli ).
FOLATE TURNOVER
Folate breakdown rates in plants can be high.
Thus, postharvest studies of leaves and fruits,
and studies using folate synthesis inhibitors,
point to breakdown rates of ∼10% per day
(66, 70, 83, 88). For comparison, mammalian
whole-body folate breakdown rates are nor-
mally only ∼0.5% per day (35). As steep
postharvest declines in folate levels are of obvi-
ous nutritional significance, understanding the
processes involved in folate breakdown and in
salvage of the breakdown products is as practi-
cally important as understanding de novo folate
biosynthesis.
Folate Breakdown
As stated in the Introduction, most natural
folates are inherently sensitive at physiologi-
cal pH to oxidative or photooxidative scission
of the C9–N10 bond, which yields a pterin
plus p-aminobenzoylglutamate ( pABA-Glu) or
its polyglutamyl forms (Figure 4) (34). Such
nonenzymatic cleavage is thought to be the
main route of folate breakdown in all organisms
(89). However, evidence on this point is lacking
Changes may still occur before final publication online and in print
for plants, so enzyme-mediated cleavage can-
not be excluded; an active cleavage process is
suggested by the unusually high folate break-
Turnover: the
down rates in plants. Folate-cleaving enzymes dynamic balance
from microorganisms have been reported (84), between synthesis and
and mammalian ferritin facilitates folate cleav- degradation of a
age in vitro and in vivo (90). Moreover, the biomolecule
action of the folate-dependent COG0354 pro- pABA-Glu: p-amino-
tein, which participates in synthesis or repair of benzoylglutamate
certain iron-sulfur clusters, may involve folate
oxidation (95).
Folates vary in susceptibility to cleavage:
THF and DHF are the most vulnerable, and
5- and 10-formyltetrahydrofolate are the least
vulnerable (34, 39, 78). For THF and DHF,
the first pterins formed in the reaction are
tetrahydro- and dihydropterin-6-aldehyde, re-
spectively; further oxidation can convert the
tetrahydro to the dihydro form and both to the
fully oxidized, aromatic form pterin-6-aldehyde
(Figure 4a) (38, 78, 97). Additional oxida-
tion converts pterin-6-aldehyde to pterin-6-
carboxylate and perhaps other end products
(52).
Folate Salvage Processes
Like other organisms that synthesize folates,
plants can recycle the pterin and pABA-Glu
cleavage products back to folates (Figure 4b).
The recycling of pABA-Glu moieties appears to
be straightforward (66). First, the polyglutamyl
tail, if present, is removed by GGH, for which
pABA-Glu polyglutamates are good substrates
(2, 67). That GGH is exclusively vacuolar
implies the existence of a tonoplast transport
system for pABA-Glu polyglutamates (which
could be the same as that for folate polyg-
lutamates) (Figure 2). Second, pABA-Glu is
hydrolyzed to yield pABA and glutamate, which
can then be reused for folate synthesis. An
enzyme activity catalyzing this step, pABA-Glu
hydrolase, appears to be ubiquitous in plants, to
be predominantly vacuolar or cytosolic, and to
exist as various isoforms (with native molecular
masses of 90, 200, and 360 kDa in Arabidopsis)
(12). Partial purification and characterization
of the 200-kDa species from Arabidopsis roots
www.annualreviews.org • Folate Synthesis and Metabolism 4.7
 PP62CH04-Hanson ARI 22 January 2011 11:19

showed it to be unstable and probably a in vivo (12). Another possible way to recycle
metalloenzyme (12). The corresponding gene pABA-Glu—direct reincorporation into DHF
has not been cloned, although seven potential via the action of dihydropteroate synthase—was
candidates were tested and ruled out (12). The excluded by a kinetic study of this enzyme (66).
partially purified Arabidopsis root activity also Salvage of the pterin cleavage product is
releases glutamate from folic acid, raising the less well understood, but certain points are
possibility of enzymatic folate degradation fairly clear. First, dihydropterin-6-aldehyde
can be salvaged in vivo by reduction of its
side chain to give the folate synthesis inter-
a THF γ-Glu tail
mediate HMDHP (Figure 4b) (66). In pea
Pterin pABA-Glu leaves, this reaction appears (a) to take place
O H 9 10
O COOH predominantly in the cytosol and (b) to be
N CH2 N C N CH catalyzed by multiple NADPH-dependent
HN H H H
H (CH2)2 COOH pterin aldehyde reductase (PTAR) isoforms
Annu. Rev. Plant Biol. 2011.62. Downloaded from www.annualreviews.org

H2N N N H C N CH that can reduce both dihydropterin-6-aldehyde

H
H
by RIJKSUNIVERSITEIT GENT on 02/01/11. For personal use only.

and pterin-6-aldehyde; similar multiple PTAR

O
(CH2)2 COOH
C N CH isoforms also occur in Arabidopsis seeds (62).
H One Arabidopsis isoform (At1g10310) has been
O
O O n (CH2)2
N CHO N CH2OH cloned; it belongs to the short-chain dehydro-
HN HN COOH
genase/reductase family and attacks diverse
H PTAR H
H2N N N H H2N N N H aromatic and aliphatic aldehydes (62). Like-
H H wise, all the pea isoforms attack other aldehydes
Dihydropterin-6-aldehyde 6-HMDHP
(62). Dihydropterin-6-aldehyde thus seems not
to be salvaged by a single dedicated enzyme but
O O
N CHO N CH2OH rather by a battery of broad-specificity alde-
HN HN hyde reductases. In support of this hypothesis,
PTAR
H2N N N H2N N N
Pterin-6-aldehyde 6-Hydroxymethylpterin ←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−
PTAD Figure 4
O
N Folate breakdown and salvage reactions.
COOH
HN (a) Structures and relationships of folate breakdown
products. Arrowheads mark the oxidatively labile
H 2N N N C9–N10 bond (red ), the bond cleaved by
Pterin-6-carboxylate p-aminobenzoylglutamate hydrolase (black), and
bonds cleaved by γ-glutamyl hydrolase (GGH;
GTP yellow). Dotted arrows show (photo)chemical
b Chorismate oxidations. Solid arrows show enzymatic reactions;
red crosses mark those that appear not to occur in
HMDHP-P2 HMDHP plants (although they occur in other organisms).
pABA The folate synthesis intermediate
6-hydroxymethyldihydropterin (HMDHP) is
PGH DHP
Glu colored blue. (b) Folate salvage reactions ( purple
arrows) in relation to folate biosynthesis.
DHF Abbreviations: DHF, dihydrofolate;
pABA-Glu DHP, dihydropteroate; DHPAld, dihydropterin-
PTAR
THF 6-aldehyde; GGH, γ-glutamyl hydrolase; Glu,
Glu glutamate; Glun , polyglutamate; P2 , diphosphate;
pABA-Glu, p-aminobenzoylglutamate; PGH,
GGH Folate-Glun p-aminobenzoylglutamate hydrolase; PTAD, pterin
aldehyde dehydrogenase; PTAR, pterin aldehyde
pABA-Glun DHPAld
reductase; THF, tetrahydrofolate.

4.8 Hanson · Gregory

Changes may still occur before final publication online and in print
 PP62CH04-Hanson ARI 22 January 2011 11:19
At1g10310 knockout plants have only a subtle
reduction in total PTAR activity (62) but
display an altered fatty acid desaturation
phenotype that may be related to the aliphatic
aldehyde reductase activity of At1g10310 (1).
The combined activity of PTAR isoforms is rel-
atively high. For instance, in pea leaves, PTAR
activities are at least 13- to 1,500-fold higher
than those of de novo folate synthesis enzymes
(62). This high PTAR activity may favor rapid
conversion of dihydropterin-6-aldehyde to
HMDHP, thereby minimizing the tendency
for further oxidation to pterin-6-aldehyde and
pterin-6-carboxylate (Figure 4a).
Annu. Rev. Plant Biol. 2011.62. Downloaded from www.annualreviews.org
Second, if PTAR activity fails to intercept
by RIJKSUNIVERSITEIT GENT on 02/01/11. For personal use only.
dihydropterin-6-aldehyde before it is oxidized
to pterin-6-aldehyde, then the pterin moiety
can no longer be salvaged and can only be fur-
ther oxidized (63). Such a situation arises be-
cause plants lack the capacity to reduce the
fully oxidized pterin ring to the dihydro state,
a deficit shared with E. coli (63). The lack of
pterin-reducing capacity is surprising inasmuch
as enzymes with this activity occur in other or-
ganisms; pteridine reductase 1 of Leishmania is
one example (10). Further oxidation of pterin-
6-aldehyde to pterin-6-carboxylate can occur
spontaneously or enzymatically; plant extracts
have both NAD-dependent and -independent
activities (63). Pterin-6-carboxylate seems to be
a dead-end product; its relative scarcity in most
plant tissues suggests that folate salvage is nor-
mally efficient (66).
The following points about folate salvage
are still obscure. First, the oxidative cleavage
product of THF is tetrahydropterin-6-
aldehyde (89), and it is not clear how oxidation
to the dihydro form occurs or even whether
it is necessary (given that the tetrahydro form
could conceivably be recycled directly to
THF). Second, due to the resistance of 5-
methyl- and 5- and 10-formyltetrahydrofolates
to oxidative cleavage (55), it is likely that THF
and DHF are the primary folates that undergo
nonenzymatic oxidative cleavage. However,
in the case of 5-methyltetrahydrofolate,
for example, facile and reversible oxidation
to 5-methyl-5,6-DHF can occur, and this
Changes may still occur before final publication online and in print
compound can undergo rapid cleavage at
acidic pH to pABA-Glu and a methylated
pterin (55). Presumably the levels of ascorbate,
glutathione, and other reductants are sufficient
in plants to keep 5-methyl-5,6-DHF to a
minimum, but its cleavage may nonetheless
represent a secondary pathway for nonenzy-
matic folate degradation. In any case, it is not
known how pterin moieties produced by ox-
idative cleavage of 5-methyl- and, presumably,
5-formyltetrahydrofolate are metabolized.
These pterins presumably still carry a C1
substituent at the 5-position; in principle, this
C1 unit might be enzymatically removed, or
removal might not be a necessary precondi-
tion for recycling. Lastly, the predominantly
cytosolic location of PTAR activity seems
anomalous, given that 30–50% of the folates
in metabolically active plant cells are in mito-
chondria (28, 44, 61) and that mitochondrial
folates are almost certainly at a high risk
of oxidative cleavage due to the prevalence
of reactive oxygen species in mitochondria
(50). The implication is that pterin cleavage
products cannot be recycled until they are
exported from the mitochondria, which seems
to be at odds with the need for PTAR to
intervene before further oxidation puts the
pterins beyond rescue.
Transport of Folates and Precursors
The compartmentation of the enzymes of
folate biosynthesis and metabolism, and of
folates themselves, means that there must be
substantial transmembrane traffic in folates
and their precursors. Of this traffic, only that in
pABA (a hydrophobic weak acid) appears to be
by simple diffusion (72). On the basis of com-
partmentation data, experimental evidence,
and comparative biochemistry, a minimal set of
nine probable carrier-mediated transport steps
can be defined (Figure 2, steps 1–9). These
steps, and the evidence for them, are as follows.
1. Mitochondrial pterin import. If
HMDHP is synthesized in the cy-
tosol, it must enter mitochondria
to support folate synthesis. Also,
www.annualreviews.org • Folate Synthesis and Metabolism 4.9
 PP62CH04-Hanson ARI 22 January 2011
Annu. Rev. Plant Biol. 2011.62. Downloaded from www.annualreviews.org
by RIJKSUNIVERSITEIT GENT on 02/01/11. For personal use only.
4.10
11:19
mitochondria probably export pterins
resulting from folate degradation (see
above). Pterin transport has not been
studied in plant mitochondria, but it
seems likely to be carrier mediated given
that the only well-studied pterin uptake
process, in the protistan parasite Leishma-
nia, involves a specific transporter (BT1)
that belongs to the folate-biopterin
transporter (FBT) family (48). The
Arabidopsis genome encodes nine FBT
family proteins, only one of which has a
known function (in folate transport; see
below). It is therefore possible that the
other eight include a pterin transporter.
However, complementation tests with
five of these eight proteins in a Leishma-
nia BT1 knockout strain yielded negative
results (26).
2. Mitochondrial export of THF and/or
other folates. Because THF is made in
mitochondria, and mitochondria can
convert THF to most of its C1 derivatives
(36), THF and/or C1 folates must be
exported from mitochondria to account
for the folates found elsewhere in the
cell. Mitochondria can almost certainly
also import folates, because supplied
5-formyltetrahydrofolate restores folate-
dependent C1 fluxes in Arabidopsis when
folate synthesis is blocked (70); this
effect requires 5-formyltetrahydrofolate
to access the mitochondrial enzyme 5-
formyltetrahydrofolate cycloligase (79).
In view of its centrality, mitochondrial fo-
late transport is one of the most important
folate-related processes to understand in
plants, but nothing is known about it in
terms of either biochemical activity or
genes. In contrast, the mammalian mito-
chondrial folate transporter (MFT) has
been cloned and extensively characterized
(68); it is a member of the mitochondrial
carrier family. However, the closest ho-
molog of MFT in Arabidopsis (At5g66380,
AtFOLT1), although it demonstrates fo-
late transport activity in two heterologous
systems, is targeted to the chloroplast
Hanson · Gregory
Changes may still occur before final publication online and in print
envelope, not to mitochondria (8). Two
other, less close Arabidopsis homologs
of MFT appear to lack folate transport
activity (8).
3,4. Plastidial folate import. Two chloro-
plast envelope proteins that can medi-
ate folate transport have been identi-
fied in Arabidopsis. Both are homologs
of known folate transporters. The first,
AtFOLT1 (8), is described above. At-
FOLT1 complements the mft mutation
in Chinese hamster ovary cells and con-
fers folate uptake when expressed in E. coli
cells. However, its significance in planta
is uncertain, given that ablation of At-
FOLT1 affects neither growth, nor leaf
or chloroplast folate content (8). This
lack of a mutant phenotype may re-
flect redundancy of function with the
second chloroplastic folate transporter,
At2g32040 (45). At2g32040 and its Syne-
chocystis ortholog Slr0642 belong to the
FBT family. When expressed in E. coli,
At2g32040 and Synechocystis Slr0642 en-
able uptake of monoglutamyl folates and
folate analogs (45), whereas other Ara-
bidopsis FBT family proteins do not (26).
Ablation of At2g32040 increases chloro-
plast folate content and reduces the pro-
portion of 5-methyltetrahydrofolate (45),
which provides evidence for a folate
transport role in planta, although growth
is unaffected. A mutational analysis of
the Slr0642 protein identified 22 residues
essential to folate transport activity, of
which seven are conserved in all known
FBT family folate transporters (26). The
majority of the eight Arabidopsis FBT pro-
teins other than At2g32040 lack at least
one of these residues, which suggests that
they may transport other substrates. In-
terestingly, the FBT family includes a
member, from Leishmania, that is a spe-
cific, high-affinity S-adenosylmethionine
transporter (22).
5. Vacuolar pABA glucose ester import.
It is necessary to invoke a tonoplast
transporter for pABA glucose ester
 PP62CH04-Hanson ARI 22 January 2011 11:19
because the ester is made in the cytosol
but is almost entirely located in vac-
uoles and, being hydrophilic, almost cer-
tainly cannot diffuse readily across mem-
branes (24, 72). No biochemical evidence
on the transport process is available, and
no gene has been cloned. In both these
respects pABA glucose transport is not
alone: Although many glucose conju-
gates of natural products undergo carrier-
mediated uptake into the vacuole, little
is known about the mechanisms or genes
(17).
6. Vacuolar folate polyglutamate import.
Annu. Rev. Plant Biol. 2011.62. Downloaded from www.annualreviews.org
The argument for a tonoplast transport
by RIJKSUNIVERSITEIT GENT on 02/01/11. For personal use only.
system for folate polyglutamates is
outlined above (see the section entitled
Folate Assembly, Polyglutamylation, and
Deglutamylation); there is no biochem-
ical information on this system, and no
gene is known. Although such a system
would be unusual in that most folate
transporters strongly prefer monoglu-
tamyl folates (25, 45, 89), a precedent
exists in mammalian lysosomes, which,
like plant vacuoles, import polyglu-
tamates and contain GGH (4). The
mammalian polyglutamate transport sys-
tem has unfortunately not been cloned.
7. Vacuolar folate monoglutamate import.
Folates and their analogs are substrates
for certain mammalian multidrug
resistance–associated protein (MRP)
subfamily ATP-binding cassette trans-
porters (100), and there is evidence that
MRP proteins import folates into plant
vacuoles. Thus, the Arabidopsis vacuolar
MRP protein AtMRP1 (At1g30400)
expressed in yeast, and its functional
equivalent(s) in vacuolar membrane vesi-
cles from red beet root, are competent in
the MgATP-dependent transport of folic
acid and methotrexate (73). Polyglutamyl
folates are poor substrates, so AtMRP1
is unlikely to correspond to the polyg-
lutamate transporter discussed above.
Ablation of AtMRP1 results in increased
sensitivity to, and impaired vacuolar
Changes may still occur before final publication online and in print
accumulation of, methotrexate (73),
indicating that this protein functions in
planta, at least in folate analog transport.
Whether it plays a significant role in
folate transport is less certain. First, the
Km values for folate for both AtMRP1
and the beet vacuole system are very high
(0.19 mM) compared with the intracel-
lular concentrations of free (i.e., non–
protein bound) folate monoglutamates,
which are probably submicromolar (73).
Second, like other MRPs, AtMRP1 trans-
ports glutathione conjugates and thus is
not folate specific (73). Third, ablation of
AtMRP1 eliminates only approximately
half of vacuolar methotrexate uptake ac-
tivity, which indicates the presence of at
least one other transport system. Finally,
because ATP-binding cassette trans-
porters work in only one direction (75),
AtMRP1 is unlikely to mediate the ex-
port from vacuoles of the monoglutamyl
folate products of GGH action.
8. Cellular folate import. There is strong
evidence that plant cells or tissues can
take up intact folates from experiments
in which supplied folates are metabolized
(70, 80) or reverse the effects of folate syn-
thesis inhibitors (16, 51). There is like-
wise direct evidence for uptake of the
close folate analog methotrexate (16, 51,
70), including a 1993 study of Datura in-
noxia cells that determined a Km value of
66 nM and showed that uptake is pH and
energy dependent (98). The same study
also showed that selection for methotrex-
ate resistance resulted in an increase in the
Km value for methotrexate uptake. This
pioneering work invites extension to the
gene level via the molecular and genetic
tools now available.
9. Cellular pterin import. There is good
evidence that plant tissues take up and
metabolize supplied pterins (62, 66), as
do E. coli, other bacteria, and yeast (63).
Pterin transport is, however, a gener-
ally neglected research area and has been
studied only in Leishmania (48).
www.annualreviews.org • Folate Synthesis and Metabolism 4.11
 PP62CH04-Hanson ARI 22 January 2011
Annu. Rev. Plant Biol. 2011.62. Downloaded from www.annualreviews.org
by RIJKSUNIVERSITEIT GENT on 02/01/11. For personal use only.
4.12
11:19
Cloning Plant Folate Transporters
by Homology
The cloning-by-homology approach that
yielded AtFOLT1, At2g32040, and AtMRP1
may have ceased to be fruitful. Taking all
organisms together, seven classes of folate
transporters have so far been cloned (26). Five
are from mammals: the mitochondrial trans-
porter, the reduced folate carrier, the intestinal
proton–coupled folate transporter, various
MRPs, and glycosylphosphatidylinisotol-
linked folate receptors, which mediate uptake
by an endocytic mechanism. Of these, only
the mitochondrial and MRP transporters have
reasonably close plant homologs (AtFOLT1
and AtMRP1, respectively), which are known
to transport folates, as described above. The
sixth class is the FBT family, one of whose
plant homologs (At2g32040) also transports
folates. The seventh class, members of the
bacterial energy–coupling factor family, do not
have homologs in plants.
ENGINEERING FOLATE LEVELS
As summarized in Table 1, the biosynthesis
pathway and the control of polyglutamylation
have been the targets for almost all plant folate
engineering studies so far. The sole exception
is an Arabidopsis model study (30) designed to
enhance folate content by blocking metabolism
of 5-formyltetrahydrofolate, as proposed by
Scott et al. (83). This strategy indeed raises
total folate level, but only by twofold, and
leads to slowed growth and delayed flowering.
It is consequently not promising and has not
been pursued. In contrast, several of the other
studies achieved a far-greater-than-twofold
enrichment, and none reported negative effects
on growth or development.
Engineering of Biosynthesis
As is common in pathway engineering studies,
various species, organs, promoters, and ana-
lytical methods have been used by different
groups, which precludes direct comparisons.
Hanson · Gregory
Changes may still occur before final publication online and in print
Some useful generalizations can nonetheless be
made. Thus, of seven studies that manipulated
the biosynthetic pathway, six inserted genes
coding for GCHI, the first enzyme of pterin
synthesis (Figure 2, step A), alone or together
with ADCS, the first enzyme of pABA synthesis
(Figure 2, step D). In most cases, the GCHI
transgene was from a nonplant source
(E. coli, mouse, or chicken), on the basis of the
reasonable but undocumented premise that
plant GCHI enzymes are subject to feedback
inhibition, which would limit their potential
to increase flux. Expressed alone, the nonplant
transgenes generally led to substantial pterin
overproduction, but so did a plant transgene
(Table 1). It is therefore not clear whether
plant or nonplant GCHIs are preferable, but
it can at least be concluded that the former
need not be avoided on principle. Whatever
the case, excessive overproduction of pterins
may be undesirable, not least because their
health effects and potential roles in human
metabolism are unknown (21).
Despite massively boosting pterin levels,
the introduction of a GCHI transgene alone
generally raises folate content only modestly,
typically by approximately twofold (Table 1).
When total pABA levels (i.e., pABA plus
its glucose ester) were analyzed in GCHI-
overexpressing tomato fruit (20), severe pABA
depletion was observed, indicating that the
pABA supply limits further folate accumulation.
Consistent with this view, adding an Arabidopsis
ADCS transgene greatly increases the total
pABA pool and raises folate content to as
much as 25 times the content in wild-type fruit
(Table 1). Expression of the ADCS transgene
alone had no effect on fruit folate content.
The results of expressing GCHI and ADCS
transgenes separately and together in rice
grains are very similar, except that folate con-
tent is substantially (and unexpectedly) reduced
by expression of ADCS alone (as discussed in
the section entitled Regulation of Folate Syn-
thesis). Taken together, the tomato and rice
data indicate that expression of ADCS in com-
bination with GCHI is far more effective than
expression of GCHI alone, and that expression
 Annu. Rev. Plant Biol. 2011.62. Downloaded from www.annualreviews.org
by RIJKSUNIVERSITEIT GENT on 02/01/11. For personal use only.

PP62CH04-Hanson
ARI

Table 1 Projects aiming to metabolically engineer folate content

Year Species Organ(s) tested Gene(s) added or ablated Change in folate Comments Reference
2004 Arabidopsis Leaves + Escherichia coli GCHI +2- to +4-fold 1,250-fold increase in pterins 40
2004 Tomato Fruit + Mouse GCHIa +2-fold Up-to-140-fold increase in pterins 20
2005 Arabidopsis Leaves 5-FCL ablated +2-fold Growth slowed 20%; flowering delayed 30
22 January 2011

2007 Tomato Fruit + Arabidopsis ADCS None Up-to-40-fold increase in pABA 21

+ Mouse GCHIa / Up to +25-fold >20-fold increases in pterins and pABA
11:19

+ Arabidopsis ADCS
2007 Rice Grain + Arabidopsis GCHI None Up-to-29-fold increase in pterins 85
+ Arabidopsis ADCS Up to –6-fold Up-to-89-fold increase in pABA
+ Arabidopsis GCHI/ADCS Up to +100-fold Average 4-fold increase in pterins and
25-fold increase in pABA
2008 Rice Leaves + Wheat HPPK/DHPS +75% Precursors not analyzed 29
Grain +40%
2009 Maize Grain + E. coli GCHI +2-fold Precursors not analyzed; β-carotene and 60
ascorbate pathways also engineered
2009 Lettuce Leaves + Chicken GCHIa +2- to +9-fold Precursors not analyzed 64
2010 Arabidopsis Leaves + AtGGH2 –39% Polyglutamate tail length decreased 3
2010 Tomato Fruit + LeGGH2 –46% Polyglutamate tail length decreased 3
2010 Arabidopsis Leaves + GGH RNAi +30% Polyglutamate tail length increased 3

a
Codon usage modified to improve plant expression.
b
Abbreviations: 5-FCL, 5-formyltetrahydrofolate cycloligase; ADCS, aminodeoxychorismate synthase; GCHI, GTP cyclohydrolase I; HHPK/DHPS, 6-hydroxymethyldihydropterin
pyrophosphokinase–dihydropteroate synthase; pABA, p-aminobenzoate; RNAi, RNA interference.

Changes may still occur before final publication online and in print
www.annualreviews.org • Folate Synthesis and Metabolism
4.13
 PP62CH04-Hanson ARI 22 January 2011
Annu. Rev. Plant Biol. 2011.62. Downloaded from www.annualreviews.org
by RIJKSUNIVERSITEIT GENT on 02/01/11. For personal use only.
4.14
11:19
of ADCS alone is useless at best. The pABA
accumulations caused by ADCS transgenes are
of less potential concern than pterin accumu-
lations because pABA is among the compounds
that are generally recognized as safe (GRAS)
in terms of regulatory status (21, 85).
The only synthesis engineering study that
did not manipulate GCHI or ADCS ex-
pressed a wheat HMDHP pyrophosphokinase–
dihydropteroate synthase gene in rice by using
a constitutive promoter (29). Small (≤75%) in-
creases in leaf and grain folate content were
reported, which suggests that one or another
of the activities of this bifunctional enzyme ex-
erts significant control of flux through the fo-
late synthesis pathway, a possibility also raised
by analysis of engineered tomato fruit (21).
A rational engineering strategy might there-
fore be to insert HMDHP pyrophosphokinase–
dihydropteroate synthase into plants that ex-
press both GCHI and ADCS transgenes.
Engineering of Polyglutamylation
The generally accepted hypothesis that polyg-
lutamylation indirectly stabilizes folates by
promoting enzyme binding (89) predicts that
folate levels can be (a) reduced by shorten-
ing polyglutamyl tails and (b) increased by
lengthening them. Studies in which GGH
was over- or underexpressed in Arabidopsis and
tomato (3) support both predictions (Table 1).
Thus, threefold overexpression of GGH
reduces average tail length and cuts folate
content by ∼40%, whereas reducing GGH
activity by 99% increases tail length and raises
folate content by 30%. Because the folates that
accumulate in engineered tomato fruit and
rice grains are largely unglutamylated (21, 85),
another rational engineering strategy might be
to increase polyglutamylation by suppressing
GGH activity in these systems.
Observations on Biofortification
Four further observations may be made about
folate engineering and biofortification. The
first is that all engineering studies to date have
Hanson · Gregory
Changes may still occur before final publication online and in print
relied on simple one- or two-gene strategies,
and almost all have overexpressed enzymes at
the head of the pathway, which drives flux down
the pathway by increasing precursor supply.
This type of “push” strategy inevitably entails
a buildup of precursors, which is undesirable,
as mentioned above. More desirable would be
a “pull” strategy in which the folate end prod-
uct is sequestered in vacuoles, thereby relieving
the (little understood) feedback controls over
pathway activity and so enhancing flux without
precursor accumulation. Although we do not
yet know enough about vacuolar folate trans-
port and storage to implement such a strategy,
its intrinsic advantages provide a sound ratio-
nale for research to make it possible.
The second observation concerns serendip-
ity and arises from a retrospective study of folate
pathway gene expression in engineered tomato
fruit (mentioned in the section entitled Regu-
lation of Folate Synthesis) (94). Basically, a cer-
tain amount of luck was involved in the success
of the two-gene (GCHI-plus-ADCS) strategy
in tomato fruit: Overexpression of these genes
induced expression of three downstream path-
way genes, which presumably enhanced path-
way flux capacity. Such feedforward effects are
neither predictable nor understood and may not
occur in other systems, meaning that the GCHI-
plus-ADCS strategy may not necessarily work
as well as in tomato fruit. That it did so in rice
(85) is, however, an encouraging sign.
The third observation is that in microorgan-
isms there exist many variants of the canonical
biosynthesis pathway that include alternatives
to almost all its enzymes (18). For instance,
certain prokaryotes have a radically different
type of GCHI (23), whereas others have a novel
enzyme that replaces both dihydroneopterin
triphosphate diphosphatase and dihydro-
neopterin aldolase (Figure 2, steps B, C) (71).
Such evolutionary novelties expand the parts
list for engineering projects and in principle
enable construction of hybrid folate synthesis
routes not found in nature. These potential
alternative routes could have the advantage of
escaping endogenous constraints on pathway
flux (whatever those constraints may be).
 PP62CH04-Hanson ARI 22 January 2011 11:19

Finally, conventional breeding based on nat-
ural variation may be a good alternative to
metabolic engineering and such breeding could
be informed by the outcomes of engineering
experiments (9). Surveys of tomato (9), potato
(32), wheat (69), and strawberry (88) found
roughly twofold ranges in folate content among
cultivars or breeding lines. Such natural varia-
tion, if heritable, could form the basis for breed-
ing programs. Given the engineering evidence
that both pterin and pABA supplies limit fo-
late synthesis (Table 1), such programs might
profitably analyze precursors as well as folates
in parents and progenies.
Annu. Rev. Plant Biol. 2011.62. Downloaded from www.annualreviews.org
Other frontiers are at least implicit in the
foregoing sections on biosynthesis regulation,
turnover, and transport but bear reempha-
sizing. Regarding biosynthesis regulation, the
general lack of biochemical studies of enzymes
isolated from plants (as opposed to recombinant
proteins) means that if phosphorylation or reg-
ulatory proteins were involved, we would prob-
ably not know. Similarly, at the gene level, if fo-
late regulons exist, published studies would not
necessarily have detected them. Future tran-
scriptomics studies have the potential to do so.
For turnover, there are fascinating hints
from postharvest and inhibitor studies that fo-

late breakdown in plants may be too fast to ex-

by RIJKSUNIVERSITEIT GENT on 02/01/11. For personal use only.

plain by spontaneous chemical processes alone,

FOLATE FRONTIERS
The frontiers include all the topics in this re-
view, including—despite the effort invested in
it—the biosynthesis pathway itself. Specifically,
only one step in this pathway (DHF synthase)
has been validated by mutant studies (42); all the
others rest on biochemical data, comparative
genomics, and functional complementation of
microbial mutants. Thus, if alternatives to the
classical folate synthesis steps exist in plants, we
would not know about them. Moreover, it is
not clear whether all cells and organs are au-
tonomous for folate synthesis or whether some
depend on intercellular or interorgan traffic in
folates or their precursors. The presence of a
high-affinity cellular folate uptake system (98)
makes such traffic seem probable, as does ev-
idence that maternal tissues supply folates to
early embryos (42). A first step to address this
issue would be metabolic profiling of folates and
folate precursors in xylem and phloem sap.
and other hints—particularly from compara-
tive biochemistry—that folate degradation can
be enzyme mediated. Biochemistry, compara-
tive genomics, transcriptomics, and proteomics
could be applied and integrated to search for
candidate folate-degrading proteins (18, 19, 37,
47). Such work has yet to begin.
Folate transport and vacuolar storage are
perhaps the frontiers with the greatest poten-
tial short-term payoffs in engineering terms.
Few folate transporters have been discovered
in plants, and they are probably the least
interesting ones for engineering. Far more
crucial are the transporters that mediate mito-
chondrial folate export, vacuolar folate polyg-
lutamate import, and folate uptake into the cell.
Although homology to known folate trans-
porters is nearly if not completely mined out as a
way to identify such transporters, more creative
approaches, including comparative genomics
and other -omics technologies, hold consider-
able potential but, again, have yet to be applied.

SUMMARY POINTS
1. Enzymes for each step of the folate synthesis and polyglutamylation pathways have been
cloned and partially characterized as recombinant proteins. The subcellular compart-
mentation of the pathway is broadly understood, as is that of folates themselves.
2. Certain mechanisms that regulate folate biosynthesis at the enzyme and gene levels have
been identified, and engineering studies have identified enzymes that exert significant
control over flux through the biosynthetic pathway.

www.annualreviews.org • Folate Synthesis and Metabolism 4.15

Changes may still occur before final publication online and in print
 PP62CH04-Hanson ARI 22 January 2011 11:19

3. There is evidence that plants exhibit relatively high folate breakdown rates and can salvage
the breakdown products for reuse in folate synthesis. Salvage enzyme activities have been
identified, and one salvage enzyme has been cloned.
4. Three plant folate transporters—two chloroplastic and one vacuolar—have been cloned.
Enzyme and folate compartmentation data point to the existence of at least five other
folate transport systems in plant cells.
5. Metabolic engineering efforts that overexpressed two folate synthesis genes in combi-
nation have increased folate levels by up to 25-fold in tomato fruit and 100-fold in rice
grains. Lesser increases have been obtained in these and other species through the over-
expression of single genes.
Annu. Rev. Plant Biol. 2011.62. Downloaded from www.annualreviews.org

FUTURE ISSUES
by RIJKSUNIVERSITEIT GENT on 02/01/11. For personal use only.

1. Most of the steps in plant folate synthesis lack genetic confirmation, so it is not clear
whether the known enzymes are the only, or even the major, significant ones in vivo. It
is also unclear whether the pathway is active in all cells or whether some cells depend on
folate import.
2. Knowledge of folate biosynthesis regulation is fragmentary, and no coherent overall
picture has emerged. Comparative biochemistry suggests the possibility of enzyme-level
regulation by phosphorylation and specific regulatory proteins, but no study carried out
so far could have detected such mechanisms.
3. The high rates of folate breakdown in plants suggest that this process may have enzyme-
mediated as well as purely chemical components, but this possibility remains wholly
unexplored. Most folate salvage enzymes remain to be cloned and characterized.
4. The crucial transporters that export folates from their site of synthesis in mitochondria,
that import polyglutamyl folates into vacuoles, and that import folates into the cell have,
for the most part, not been biochemically characterized, and none of them have been
cloned.
5. The folate engineering strategies used to date have resulted in buildup of precursors to
high, perhaps undesirable, levels and may have succeeded in part due to luck. Alternative
rational strategies based on increasing the flux capacity of later as well as early pathway
steps, or on manipulating vacuolar sequestration of folates, have not yet been explored,
in part due to lack of basic knowledge.

DISCLOSURE STATEMENT
The authors are not aware of any affiliations, memberships, funding, or financial holdings that
might be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS
Folate metabolism research in the authors’ laboratories has been funded by the U.S. National
Science Foundation (current grant MCB-0839926). We thank Linda Jeanguenin, Océane Frelin,
and Kenneth Ellens for comments on the manuscript.

4.16 Hanson · Gregory

Changes may still occur before final publication online and in print
 PP62CH04-Hanson ARI 22 January 2011 11:19

LITERATURE CITED
1. Ajjawi I, Lu Y, Savage LJ, Bell SM, Last RL. 2010. Large-scale reverse genetics in Arabidopsis: case studies
from the Chloroplast 2010 Project. Plant Physiol. 152:529–40
2. Akhtar TA, McQuinn RP, Naponelli V, Gregory JF 3rd, Giovannoni JJ, Hanson AD. 2008. Tomato γ-
glutamylhydrolases: expression, characterization, and evidence for heterodimer formation. Plant Physiol.
148:775–85
3. Akhtar TA, Orsomando G, Mehrshahi P, Lara-Núñez A, Bennett MJ, et al. 2010. A central role for
γ-glutamyl hydrolases in plant folate homeostasis. Plant J. 64:256–66
4. Barrueco JR, Sirotnak FM. 1991. Evidence for the facilitated transport of methotrexate polyglutamates
into lysosomes derived from S180 cells. Basic properties and specificity for polyglutamate chain length.
J. Biol. Chem. 266:11732–37
5. Basset G, Quinlivan EP, Ziemak MJ, Dı́az de la Garza R, Fischer M, et al. 2002. Folate synthesis in
plants: The first step of the pterin branch is mediated by a unique bimodular GTP cyclohydrolase I.
Proc. Natl. Acad. Sci. USA 99:12489–94
6. Basset GJ, Quinlivan EP, Ravanel S, Rébeillé F, Nichols BP, et al. 2004. Folate synthesis in plants: The
Annu. Rev. Plant Biol. 2011.62. Downloaded from www.annualreviews.org

p-aminobenzoate branch is initiated by a bifunctional PabA-PabB protein that is targeted to plastids.

by RIJKSUNIVERSITEIT GENT on 02/01/11. For personal use only.

Proc. Natl. Acad. Sci. USA 101:1496–501

7. Basset GJ, Ravanel S, Quinlivan EP, White R, Giovannoni JJ, et al. 2004. Folate synthesis in plants: The
last step of the p-aminobenzoate branch is catalyzed by a plastidial aminodeoxychorismate lyase. Plant J.
40:453–61
8. Bedhomme M, Hoffmann M, McCarthy EA, Gambonnet B, Moran RG, et al. 2005. Folate metabolism
in plants: An Arabidopsis homolog of the mammalian mitochondrial folate transporter mediates folate
import into chloroplasts. J. Biol. Chem. 280:34823–31
9. Bekaert S, Storozhenko S, Mehrshahi P, Bennett MJ, Lambert W, et al. 2008. Folate biofortification in
food plants. Trends Plant Sci. 13:28–35
10. Bello AR, Nare B, Freedman D, Hardy L, Beverley SM. 1994. PTR1: a reductase mediating salvage of
oxidized pteridines and methotrexate resistance in the protozoan parasite Leishmania major. Proc. Natl.
Acad. Sci. USA 91:11442–46
11. Blancquaert D, Storozhenko S, Loizeau K, De Steur H, De Brouwer V, et al. 2010. Folates and folic
acid: from fundamental research toward sustainable health. Crit. Rev. Plant Sci. 29:14–35
12. Bozzo GG, Basset GJ, Naponelli V, Noiriel A, Gregory JF 3rd, Hanson AD. 2008. Characterization of
the folate salvage enzyme p-aminobenzoylglutamate hydrolase in plants. Phytochemistry 69:29–37
13. Chen L, Chan SY, Cossins EA. 1997. Distribution of folate derivatives and enzymes for synthesis of 10-
formyltetrahydrofolate in cytosolic and mitochondrial fractions of pea leaves. Plant Physiol. 115:299–309
14. Cossins EA. 2000. The fascinating world of folate and one-carbon metabolism. Can. J. Bot. 78:691–708
15. Cox K, Robertson D, Fites R. 1999. Mapping and expression of a bifunctional thymidylate synthase,
dihydrofolate reductase gene from maize. Plant Mol. Biol. 41:733–39
16. Crosti P, Malerba M, Bianchetti R. 1993. Growth-dependent changes of folate metabolism and biosyn-
thesis in cultured Daucus carota cells. Plant Sci. 88:97–106
17. Dean JV, Mills JD. 2004. Uptake of salicylic acid 2-O-β-D-glucose into soybean tonoplast vesicles by
an ATP-binding cassette transporter–type mechanism. Physiol. Plant. 120:603–12
18. de Crécy-Lagard V, El Yacoubi B, Dı́az de la Garza R, Noiriel A, Hanson AD. 2007. Comparative
genomics of bacterial and plant folate synthesis and salvage: predictions and validations. BMC Genomics
8:245
19. de Crécy-Lagard V, Hanson AD. 2007. Finding novel metabolic genes through plant-prokaryote phy-
logenomics. Trends Microbiol. 15:563–70
20. Dı́az de la Garza R, Quinlivan EP, Klaus SM, Basset GJ, Gregory JF 3rd, Hanson AD. 2004. Folate
biofortification in tomatoes by engineering the pteridine branch of folate synthesis. Proc. Natl. Acad. Sci.
USA 101:13720–25
21. Dı́az de la Garza R, Gregory JF 3rd, Hanson AD. 2007. Folate biofortification of tomato fruit. Proc.
Natl. Acad. Sci. USA 104:4218–22

www.annualreviews.org • Folate Synthesis and Metabolism 4.17

Changes may still occur before final publication online and in print
 PP62CH04-Hanson ARI 22 January 2011 11:19

22. Dridi L, Ahmed Ouameur A, Ouellette M. 2010. High affinity S-adenosylmethionine plasma membrane
transporter of Leishmania is a member of the folate biopterin transporter (FBT) family. J. Biol. Chem.
285:19767–75
23. El Yacoubi B, Bonnett S, Anderson JN, Swairjo MA, Iwata-Reuyl D, de Crécy-Lagard V. 2006. Discovery
of a new prokaryotic type I GTP cyclohydrolase family. J. Biol. Chem. 281:37586–93
24. Eudes A, Bozzo GG, Waller JC, Naponelli V, Lim EK, et al. 2008. Metabolism of the folate precursor
p-aminobenzoate in plants: glucose ester formation and vacuolar storage. J. Biol. Chem. 283:15451–59
25. Eudes A, Erkens GB, Slotboom DJ, Rodionov DA, Naponelli V, Hanson AD. 2008. Identification
of genes encoding the folate- and thiamine-binding membrane proteins in Firmicutes. J. Bacteriol.
190:7591–94
26. Eudes A, Kunji ER, Noiriel A, Klaus SM, Vickers TJ, et al. 2010. Identification of transport-critical
residues in a folate transporter from the folate-biopterin transporter (FBT) family. J. Biol. Chem.
285:2867–75
27. Farré EM, Tiessen A, Roessner U, Geigenberger P, Trethewey RN, Willmitzer L. 2001. Analysis of the
compartmentation of glycolytic intermediates, nucleotides, sugars, organic acids, amino acids, and sugar
alcohols in potato tubers using a nonaqueous fractionation method. Plant Physiol. 127:685–700
Annu. Rev. Plant Biol. 2011.62. Downloaded from www.annualreviews.org
by RIJKSUNIVERSITEIT GENT on 02/01/11. For personal use only.

28. Gambonnet B, Jabrin S, Ravanel S, Karan M, Douce R, Rébeillé F. 2001. Folate distribution during
higher plant development. J. Sci. Food Agric. 81:835–41
29. Gillies SA, McIntosh SR, Henry RJ. 2008. A cereal crop with enhanced folate: rice transgenic for wheat
HPPK/DHPS. Presented at ComBio 2008, Canberra, Aust.
30. Goyer A, Collakova E, Dı́az de la Garza R, Quinlivan EP, Williamson J, et al. 2005. 5-
Formyltetrahydrofolate is an inhibitory but well tolerated metabolite in Arabidopsis leaves. J. Biol. Chem.
280:26137–42
31. Goyer A, Illarionova V, Roje S, Fischer M, Bacher A, Hanson AD. 2004. Folate biosynthesis in higher
plants. cDNA cloning, heterologous expression, and characterization of dihydroneopterin aldolases.
Plant Physiol. 135:103–11
32. Goyer A, Navarre DA. 2007. Determination of folate concentrations in diverse potato germplasm using
a trienzyme extraction and a microbiological assay. J. Agric. Food Chem. 55:3523–28
33. Goyer A, Navarre DA. 2009. Folate is higher in developmentally younger potato tubers. J. Sci. Food
Agric. 89:579–83
34. Gregory JF 3rd. 1989. Chemical and nutritional aspects of folate research: analytical procedures, methods
of folate synthesis, stability, and bioavailability of dietary folates. Adv. Food Nutr. Res. 33:1–101
35. Gregory JF 3rd, Quinlivan EP. 2002. In vivo kinetics of folate metabolism. Annu. Rev. Nutr. 22:199–220
36. Hanson AD, Gage DA, Shachar-Hill Y. 2000. Plant one-carbon metabolism and its engineering. Trends
Plant Sci. 5:206–13
37. Hanson AD, Pribat A, Waller JC, de Crécy–Lagard V. 2009. ‘Unknown’ proteins and ‘orphan’ enzymes:
the missing half of the engineering parts list—and how to find it. Biochem. J. 425:1–11
38. Hanson AD, Roje S. 2001. One-carbon metabolism in higher plants. Annu. Rev. Plant Physiol. Plant Mol.
Biol. 52:119–37
39. Hillcoat BL, Nixon PF, Blakley RL. 1967. Effect of substrate decomposition on the spectrophotometric
assay of dihydrofolate reductase. Anal. Biochem. 21:178–89
40. Hossain T, Rosenberg I, Selhub J, Kishore G, Beachy R, Schubert K. 2004. Enhancement of folates in
plants through metabolic engineering. Proc. Natl. Acad. Sci. USA 101:5158–63
41. Imeson HC, Cossins EA. 1997. Folylpolyglutamate synthase from higher plants. Methods Enzymol.
281:141–45
42. Ishikawa T, Machida C, Yoshioka Y, Kitano H, Machida Y. 2003. The GLOBULAR ARREST1 gene,
which is involved in the biosynthesis of folates, is essential for embryogenesis in Arabidopsis thaliana.
Plant J. 33:235–44
43. Iyer R, Tomar SK. 2009. Folate: a functional food constituent. J. Food Sci. 74:R114–22
44. Jabrin S, Ravanel S, Gambonnet B, Douce R, Rébeillé F. 2003. One-carbon metabolism in plants.
Regulation of tetrahydrofolate synthesis during germination and seedling development. Plant Physiol.
131:1431–39

4.18 Hanson · Gregory

Changes may still occur before final publication online and in print
 PP62CH04-Hanson ARI 22 January 2011 11:19

45. Klaus SM, Kunji ER, Bozzo GG, Noiriel A, Dı́az de la Garza R, et al. 2005. Higher plant plastids and
cyanobacteria have folate carriers related to those of trypanosomatids. J. Biol. Chem. 280:38457–63
46. Klaus SM, Wegkamp A, Sybesma W, Hugenholtz J, Gregory JF 3rd, Hanson AD. 2005. A nudix enzyme
removes pyrophosphate from dihydroneopterin triphosphate in the folate synthesis pathway of bacteria
and plants. J. Biol. Chem. 280:5274–80
47. Lee I, Ambaru B, Thakkar P, Marcotte EM, Rhee SY. 2010. Rational association of genes with traits
using a genome-scale gene network for Arabidopsis thaliana. Nat. Biotechnol. 28:149–56
48. Lemley C, Yan S, Dole VS, Madhubala R, Cunningham ML, et al. 1999. The Leishmania donovani LD1
locus gene ORFG encodes a biopterin transporter (BT1). Mol. Biochem. Parasitol. 104:93–105
49. Li L, Rezvan A, Salerno JC, Husain A, Kwon K, et al. 2010. GTP cyclohydrolase I phosphorylation and
interaction with GTP cyclohydrolase feedback regulatory protein provide novel regulation of endothelial
tetrahydrobiopterin and nitric oxide. Circ. Res. 106:328–36
50. Logan DC. 2006. The mitochondrial compartment. J. Exp. Bot. 57:1225–43
51. Loizeau K, De Brouwer V, Gambonnet B, Yu A, Renou JP, et al. 2008. A genome-wide and metabolic
analysis determined the adaptive response of Arabidopsis cells to folate depletion induced by methotrexate.
Annu. Rev. Plant Biol. 2011.62. Downloaded from www.annualreviews.org

Plant Physiol. 148:2083–95

by RIJKSUNIVERSITEIT GENT on 02/01/11. For personal use only.

52. Lowry OH, Bessey OA, Crawford EJ. 1949. Photolytic and enzymatic transformations of pteroylglutamic
acid. J. Biol. Chem. 180:389–98
53. Luo M, Piffanelli P, Rastelli L, Cella R. 1993. Molecular cloning and analysis of a cDNA coding for the
bifunctional dihydrofolate reductase–thymidylate synthase of Daucus carota. Plant Mol. Biol. 22:427–35
54. Markham KR, Gould KS, Winefield CS, Mitchell KA, Bloor SJ, Boase MR. 2000. Anthocyanic vacuolar
inclusions—their nature and significance in flower colouration. Phytochemistry 55:327–36
55. Maruyama T, Shiota T, Krumdieck CL. 1978. The oxidative cleavage of folates. A critical study. Anal.
Biochem. 84:277–95
56. McIntosh SR, Brushett D, Henry RJ. 2008. GTP cyclohydrolase 1 expression and folate accumulation
in the developing wheat seed. J. Cereal Sci. 48:503–12
57. McIntosh SR, Henry RJ. 2008. Genes of folate biosynthesis in wheat. J. Cereal Sci. 48:632–38
58. Mehrshahi P, Gonzalez-Jorge S, Akhtar TA, Ward JL, Santoyo-Castelazo A, et al. 2010. Functional
analysis of folate polyglutamylation and its essential role in plant metabolism and development. Plant J.
64:267–79
59. Mouillon JM, Ravanel S, Douce R, Rébeillé F. 2002. Folate synthesis in higher-plant mitochondria:
coupling between the dihydropterin pyrophosphokinase and the dihydropteroate synthase activities.
Biochem. J. 363:313–19
60. Naqvi S, Zhu C, Farre G, Ramessar K, Bassie L, et al. 2009. Transgenic multivitamin corn through
biofortification of endosperm with three vitamins representing three distinct metabolic pathways. Proc.
Natl. Acad. Sci. USA 106:7762–67
61. Neuburger M, Rébeillé F, Jourdain A, Nakamura S, Douce R. 1996. Mitochondria are a major site for
folate and thymidylate synthesis in plants. J. Biol. Chem. 271:9466–72
62. Noiriel A, Naponelli V, Bozzo GG, Gregory JF 3rd, Hanson AD. 2007. Folate salvage in plants: Pterin
aldehyde reduction is mediated by multiple nonspecific aldehyde reductases. Plant J. 51:378–89
63. Noiriel A, Naponelli V, Gregory JF 3rd, Hanson AD. 2007. Pterin and folate salvage. Plants and Es-
cherichia coli lack capacity to reduce oxidized pterins. Plant Physiol. 143:1101–9
64. Nunes AC, Kalkmann DC, Aragao FJ. 2009. Folate biofortification of lettuce by expression of a codon
optimized chicken GTP cyclohydrolase I gene. Transgenic Res. 18:661–67
65. Ogawa T, Ueda Y, Yoshimura K, Shigeoka S. 2005. Comprehensive analysis of cytosolic Nudix hydrolases
in Arabidopsis thaliana. J. Biol. Chem. 280:25277–83
66. Orsomando G, Bozzo GG, Dı́az de la Garza R, Basset GJ, Quinlivan EP, et al. 2006. Evidence for
folate-salvage reactions in plants. Plant J. 46:426–35
67. Orsomando G, Dı́az de la Garza R, Green BJ, Peng M, Rea PA, et al. 2005. Plant γ-glutamyl hydrolases
and folate polyglutamates: characterization, compartmentation, and co-occurrence in vacuoles. J. Biol.
Chem. 280:28877–84

www.annualreviews.org • Folate Synthesis and Metabolism 4.19

Changes may still occur before final publication online and in print
 PP62CH04-Hanson ARI 22 January 2011 11:19

68. Perchiniak E, Lawrence SA, Kasten S, Woodard BA, Taylor SM, Moran RG. 2007. Probing the
mechanism of the hamster mitochondrial folate transporter by mutagenesis and homology modeling.
Biochemistry 46:1557–67
69. Piironen V, Edelmann M, Kariluoto S, Bedo Z. 2008. Folate in wheat genotypes in the HEALTHGRAIN
diversity screen. J. Agric. Food Chem. 56:9726–31
70. Prabhu V, Chatson KB, Lui H, Abrams GD, King J. 1998. Effects of sulfanilamide and methotrexate
on 13 C fluxes through the glycine decarboxylase/serine hydroxymethyltransferase enzyme system in
Arabidopsis. Plant Physiol. 116:137–44
71. Pribat A, Jeanguenin L, Lara-Núñez A, Ziemak MJ, Hyde JE, et al. 2009. 6-Pyruvoyltetrahydropterin
synthase paralogs replace the folate synthesis enzyme dihydroneopterin aldolase in diverse bacteria.
J. Bacteriol. 191:4158–65
72. Quinlivan EP, Roje S, Basset G, Shachar-Hill Y, Gregory JF 3rd, Hanson AD. 2003. The folate precursor
p-aminobenzoate is reversibly converted to its glucose ester in the plant cytosol. J. Biol. Chem. 278:20731–
37
73. Raichaudhuri A, Peng M, Naponelli V, Chen S, Sánchez-Fernández R, et al. 2009. Plant vacuolar ATP-
binding cassette transporters that translocate folates and antifolates in vitro and contribute to antifolate
Annu. Rev. Plant Biol. 2011.62. Downloaded from www.annualreviews.org

tolerance in vivo. J. Biol. Chem. 284:8449–60

by RIJKSUNIVERSITEIT GENT on 02/01/11. For personal use only.

74. Ravanel S, Cherest H, Jabrin S, Grunwald D, Surdin-Kerjan Y, et al. 2001. Tetrahydrofolate biosynthesis
in plants: molecular and functional characterization of dihydrofolate synthetase and three isoforms of
folylpolyglutamate synthetase in Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA 98:15360–65
75. Rea PA. 2007. Plant ATP-binding cassette transporters. Annu. Rev. Plant Biol. 58:347–75
76. Rébeillé F, Alban C, Bourguignon J, Ravanel S, Douce R. 2007. The role of plant mitochondria in the
biosynthesis of coenzymes. Photosynth. Res. 92:149–62
77. Rébeillé F, Macherel D, Mouillon JM, Garin J, Douce R. 1997. Folate biosynthesis in higher
plants: purification and molecular cloning of a bifunctional 6-hydroxymethyl-7,8-dihydropterin
pyrophosphokinase/7,8-dihydropteroate synthase localized in mitochondria. EMBO J. 16:947–57
78. Reed LS, Archer MC. 1980. Oxidation of tetrahydrofolic acid by air. J. Agric. Food Chem. 28:801–5
79. Roje S, Janave MT, Ziemak MJ, Hanson AD. 2002. Cloning and characterization of mitochondrial
5-formyltetrahydrofolate cycloligase from higher plants. J. Biol. Chem. 277:42748–54
80. Roje S, Wang H, McNeil SD, Raymond RK, Appling DR, et al. 1999. Isolation, characterization, and
functional expression of cDNAs encoding NADH-dependent methylenetetrahydrofolate reductase from
higher plants. J. Biol. Chem. 274:36089–96
81. Sahr T, Ravanel S, Basset G, Nichols BP, Hanson AD, Rébeillé F. 2006. Folate synthesis in plants:
purification, kinetic properties, and inhibition of aminodeoxychorismate synthase. Biochem. J. 396:157–
62
82. Sahr T, Ravanel S, Rébeillé F. 2005. Tetrahydrofolate biosynthesis and distribution in higher plants.
Biochem. Soc. Trans. 33:758–62
83. Scott J, Rébeillé F, Fletcher J. 2000. Folic acid and folates: the feasibility for nutritional enhancement in
plant foods. J. Sci. Food Agric. 80:795–824
84. Scott JM. 1984. Catabolism of folates. In Folates and Pterins, Vol. 1, ed. RL Blakley, SJ Benkovic,
pp. 307–27. New York: Wiley
85. Storozhenko S, De Brouwer V, Volckaert M, Navarrete O, Blancquaert D, et al. 2007. Folate fortification
of rice by metabolic engineering. Nat. Biotechnol. 25:1277–79
86. Storozhenko S, Navarrete O, Ravanel S, De Brouwer V, Chaerle P, et al. 2007. Cytosolic hydrox-
ymethyldihydropterin pyrophosphokinase/dihydropteroate synthase from Arabidopsis thaliana: a specific
role in early development and stress response. J. Biol. Chem. 282:10749–61
87. Stover PJ. 2004. Physiology of folate and vitamin B12 in health and disease. Nutr. Rev. 62:S3–12
88. Stralsjo LM, Witthoft CM, Sjoholm IM, Jagerstad MI. 2003. Folate content in strawberries
(Fragaria × ananassa): effects of cultivar, ripeness, year of harvest, storage, and commercial process-
ing. J. Agric. Food. Chem. 51:128–33
89. Suh JR, Herbig AK, Stover PJ. 2001. New perspectives on folate catabolism. Annu. Rev. Nutr. 21:255–82
90. Suh JR, Oppenheim EW, Girgis S, Stover PJ. 2000. Purification and properties of a folate-catabolizing
enzyme. J. Biol. Chem. 275:35646–55

4.20 Hanson · Gregory

Changes may still occur before final publication online and in print
 PP62CH04-Hanson ARI 22 January 2011 11:19

91. Suzuki Y, Brown GM. 1974. The biosynthesis of folic acid. XII. Purification and properties of dihydro-
neopterin triphosphate pyrophosphohydrolase. J. Biol. Chem. 249:2405–10
92. Sweeney MR, McPartlin J, Scott J. 2007. Folic acid fortification and public health: report on threshold
doses above which unmetabolised folic acid appear in serum. BMC Public Health 7:41
93. Tomsho JW, Moran RG, Coward JK. 2008. Concentration-dependent processivity of multiple glutamate
ligations catalyzed by folylpoly-γ-glutamate synthetase. Biochemistry 47:9040–50
94. Waller JC, Akhtar TA, Lara-Núñez A, Gregory JF 3rd, McQuinn RP, et al. 2010. Developmental and
feedforward control of the expression of folate biosynthesis genes in tomato fruit. Mol. Plant. 3:66–77
95. Waller JC, Alvarez S, Naponelli V, Lara-Nuñez A, Blaby IK, et al. 2010. A role for tetrahydrofolates in
the metabolism of iron-sulfur clusters in all domains of life. Proc. Natl. Acad. Sci. USA 107:10412–17
96. Webb ME, Marquet A, Mendel RR, Rébeillé F, Smith AG. 2007. Elucidating biosynthetic pathways for
vitamins and cofactors. Nat. Prod. Rep. 24:988–1008
97. Whiteley JM, Drais J, Kirchner J, Huennekens FM. 1968. Synthesis of 2-amino-4-hydroxy-6-formyl-
7,8-dihydropteridine and its identification as a degradation product of dihydrofolate. Arch. Biochem.
Biophys. 126:955–57
98. Wu K, Atkinson IJ, Cossins EA, King J. 1993. Methotrexate resistance in Datura innoxia (uptake and
Annu. Rev. Plant Biol. 2011.62. Downloaded from www.annualreviews.org
by RIJKSUNIVERSITEIT GENT on 02/01/11. For personal use only.

metabolism of methotrexate in wild-type and resistant cell lines). Plant Physiol. 101:477–83
99. Yoneyama T, Hatakeyama K. 1998. Decameric GTP cyclohydrolase I forms complexes with two pen-
tameric GTP cyclohydrolase I feedback regulatory proteins in the presence of phenylalanine or of a
combination of tetrahydrobiopterin and GTP. J. Biol. Chem. 273:20102–8
100. Zeng H, Chen ZS, Belinsky MG, Rea PA, Kruh GD. 2001. Transport of methotrexate (MTX) and folates
by multidrug resistance protein (MRP) 3 and MRP1: effect of polyglutamylation on MTX transport.
Cancer Res. 61:7225–32
101. Zhang GF, Mortier KA, Storozhenko S, Van De Steene J, Van Der Straeten D, Lambert WE. 2005.
Free and total para-aminobenzoic acid analysis in plants with high-performance liquid chromatography/
tandem mass spectrometry. Rapid Commun. Mass Spectrom. 19:963–69

www.annualreviews.org • Folate Synthesis and Metabolism 4.21

Changes may still occur before final publication online and in print