folate biosynthesis_hanson
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Folate Biosynthesis, Turnover,and Transport in Plants
Andrew D. Hanson1 and Jesse F. Gregory III2
1Horticultural Sciences Department and 2Food Science and Human Nutrition Department,University of Florida, Gainesville, Florida 32611; email: [email protected], [email protected]
Annu. Rev. Plant Biol. 2011. 62:4.14.21
The Annual Review of Plant Biology is online atplant.annualreviews.org
This articles doi:10.1146/annurev-arplant-042110-103819
Copyright c 2011 by Annual Reviews.All rights reserved
1543-5008/11/0602-0001$20.00
Keywords
biofortification, breakdown, compartmentation, engineering, salvage
Abstract
Folates are essential cofactors for one-carbon transfer reactions and are
needed in the diets of humans and animals. Because plants are major
sources of dietary folate, plant folate biochemistry has long been of in-
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 inrelation 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.
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THF:tetrahydrofolate
C1: one-carbon
Biofortification: thebreeding of crops toincrease theirnutritional value byusing conventionalcrossing and selectionor genetic engineering
Pterin: a heterocycliccompound containinga pyrazine ring fused toa pyrimidine ring thathas a carbonyl oxygenand an amino group
pABA:p-aminobenzoate
DHF: dihydrofolate
Contents
INTRODUCTION . . . . . . . . . . . . . . . . . . 4.2
FOLATE BIOSYNTHESIS . . . . . . . . . . 4.3Pterin 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
Cloning Plant Folate Transporters
by Homology . . . . . . . . . . . . . . . . . . . 4.12
ENGINEERING FOLATE
L E V E L S . . . . . . . . . . . . . . . . . . . . . . . . . . 4 . 1 2
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,glycine, and serine), purines, thymidylate, pan-
tothenate, and formylmethionyltransfer RNA
(14, 38). THF and its C1-substituted deriva-
tives are collectively termed folates (vitamin
B9). Humans and animals are unable to make
folates de novo and hence depend on dietary
sources, especially plants (11, 83). The health
impacts of folate deficiency in humans can be
severe; they include anemia, spina bifida and
other birth defects, and a higher risk of car-
diovascular disease and certain cancers (11, 87).
Because most plant foods are rather low in fo-
lates and folates are lost during processing andcooking, dietary folatedeficiencycan occur eas-
ilyand is widespread in poorer countries as well
as in some populations within richer ones (11,
83). Folate deficiency is consequently a signifi-
cant worldwide public health problem.
The prevalence of folate deficiency has led
theUnited States (since1998) andsubsequently
many other Western countries to mandate fo-
late fortificationthe addition to cereal grainproducts 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 implementin poorercountries andmay
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 driveto 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
forthe much lessexploredareas of homeostasis,
catabolism, transport, and storage. Through-
outthis 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, whichas
noted aboveis 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-
callyattachedtotheN5and/orN10positionsofTHF (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
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Polyglutamyl tail: ashort chain of-linked
glutamate residuesadded enzymatically tothe glutamate moietyof folates
GCHI: GTPcyclohydrolase I
oxidative or photooxidative cleavage of the C9
N10 bond that links the pterin and pABA moi-
eties. This inherent lability underlies the need
of humans and animals for a continual supplyof folate; what is degraded must be replaced, or
deficiency ensues.
A short, -linked chain of additional
glutamate residues (up to approximately six)
is typically attached to the first glutamate
(Figure 1). This polyglutamyl tail is important
to folate function because folate-dependent en-
zymesgenerally prefer polyglutamates, whereas
folate transporters prefer monoglutamyl forms
(89). The tail thus affects both cofactor activity
and membrane transport. Moreover, because
the tail promotes enzyme binding and folates
are far more stable to oxidative cleavage whenbound than when free, polyglutamylation can
indirectly enhance folate stability (89).
FOLATE BIOSYNTHESIS
Folates are present throughout plant cellsin
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 AC) 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 homodecamerof26-kDasubunits,
whereas in plantsGCHI is a homodimerof 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 howplant 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
N
HNNH
5
NH
H2N
O
CH2
H
H
HC
10NH
CH2CH
COOHO
CH2 COOHNH
Tetrahydropterin p-Aminobenzoate Glutamate
C1 unit
N5CH3
OHCN10
N5CHN10
N5CH2N10
N5CHO
Name
5-methyl-THF
10-formyl-THF
5,10-methenyl-THF
5,10-methylene-THF
5-formyl-THF
p-Aminobenzoylglutamate
N
HNN
NH
H2N
O
CH2
H
H
Dihydropterin moietyof dihydrofolate
THF
a
b c
9
Figure 1
Structure of folates. (a) Chemical structure of tetrahydrofolate (THF),monoglutamyl form. The red arrowhead marks the oxidatively labile C9N10bond. A polyglutamyl tail can be attached via the -carboxyl group of theglutamate 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 stepin 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-
neopterinmonophosphateto dihydroneopterin
maybe carriedout by a nonspecific phosphatase
in plants, as in Escherichia coli (91), but there is
as yet no biochemical or genetic evidence forthis hypothesis for plants, so a specific enzyme
remains a possibility.
The side chain of dihydroneopterin is then
cleaved to 6-hydroxymethyldihydropterin
(HMDHP) and glycolaldehyde by
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ADC
pABA
Chorismate
pABA
pABA-Glc
pABA-Glc
THF
DHF
GTP
DHN-P3
DHN-P
DHNDHM
HMDHP
Pi
PPi
HMDHP
HMDHP-P2
DHPGlu
Glu
Folate-Glun
THFTHFGlu
Folate-Glun
Glu
Folate-Glun
pABA
Folate-Glun
Folates
Folates
[Glycosides]
Folates
A
B
C
D
E
F
G
H
I
J1J2J3
L
1
23
4
5 6
7
8
K
*
*
*
Pterins
Pterins
9
Mitochondrion
Plastid
Vacuole
Figure 2
The folate biosynthesis pathway and its compartmentation. Red letters denote enzymes that mediatebiosynthetic 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);J1J3, isoforms of folylpolyglutamate synthase (EC 6.3.2.17); K, UDP-glucosep-aminobenzoate (pABA)glucosyltransferase; L, -glutamyl hydrolase (EC 3.4.19.9). Circled numbers designate known or inferredtransporters; 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-D-glucopyranosyl ester; THF,tetrahydrofolate.
ADCS: aminodeoxy-chorismatesynthase
dihydroneopterin aldolase; this enzyme
also mediates the epimerization of dihy-
droneopterin to dihydromonapterin, which
it likewise cleaves to yield HMDHP (31).Arabidopsis and other plants have small, di-
verged dihydroneopterin aldolase families;
their members lack obvious targeting signals
and therefore are presumably cytosolic (31).
Dihydroneopterin and dihydromonapterin can
be metabolized to -D-glycosides, at least intomato fruit engineered to overproduce pterins
(20). Neither the sugar moiety nor the glyco-
syltransferase(s) involved have been identified,
nor is it known whether the glycosides serve as
a mobilizable reserve of pterins.
p-Aminobenzoate Synthesis
pABA is synthesized from chorismate, theprod-
uct of the shikimate pathway, in two steps
(Figure 2, steps D, E). Both steps are localized
in plastids, as is the shikimate pathway (6, 7).
First, chorismate is converted to aminodeoxy-
chorismate by aminodeoxychorismate synthase
(ADCS), a bipartite protein with tandem do-
mains that are homologous to the PabA and
PabB subunits ofE. coliADCS (6, 57). Second,
aminodeoxychorismate lyase converts amin-
odeoxychorismate to pABA (7). pABA can be
esterified to glucosein a reversiblereaction me-
diated by a cytosolic UDP-glucosyltransferase
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FPGS:
folylpolyglutamatesynthase
GGH: -glutamylhydrolase
(ArabidopsisAt1g05560, 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
The HMDHP and p ABA precursors are as-
sembled into THF in the mitochondrion
(Figure 2, steps FI). 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 twodomains of a single bifunc-
tional protein in plants (57, 77). Aside from the
canonical, mitochondrially targeted HMDHP
pyrophosphokinasedihydropteroate 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 glutamateto 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 polyglutamatetail 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 FPGSisoforms,and otherhigherplants appearto 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
cytosol, mitochondria, and plastids (Figure 2,
steps J1J3) (74). However, data from single
and double FPGS knockouts strongly suggest
that one or more FPGS isoforms are targetedto multiple organelles (58). Multiple targeting
could accountfor how, in plants such as tomato,
two FPGS genes suffice (94). In any case, the
presence of FPGS in cytosol, mitochondria, and
plastids is consistentwith thepresenceof 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 notFPGS or theATP required forthe
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-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
thepresence of a potent GGHinhibitor, folate-
binding proteins that protect polyglutamatesfrom 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 andhence 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,
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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-
ent overall picture has yet to emerge. Present
knowledge is summarized in Figure 3. As in
other biosynthetic pathways, regulatory loopsappear to operate at both enzyme and gene lev-
els. Thus, in vitro studies have demonstrated
strong feedback inhibition of dihydropteroate
synthase by dihydropteroate, DHF, and THF
(59). Dihydropteroate and DHF also inhibit
the pABA synthesis enzyme ADCS (81), but it
seems unlikely that this effect enables feedback
control in vivo because dihydropteroate and
DHF pools are, presumably, mainly mitochon-
drial, whereas ADCS is plastidial. No other
feedback-regulatory properties of plant pABA
or pterin synthesis enzymes have been demon-
strated (5,6, 7, 31). However, forhumanFPGS,high folate substrate concentrations curtail the
formation of long-chain polyglutamates (93),
and the low average polyglutamyl tail lengths
in plants engineered to overproduce folates (21,
85) are consistent with a similar negative effect
of high folate levels on plant FPGS enzymes.
Transcriptomic analyses have provided
evidence for both feedback and feedforward
regulation of the expression of folate pathway
genes. Specifically, blocking folate synthesis in
Arabidopsis cells with the folate analog
methotrexate (a DHF reductase inhibitor)
causes folate depletion and an increase intranscript level of a single folate synthesis gene,
the cytosolic isoform of FPGS (51), which
suggests that folate polyglutamates exercise
feedback control over expression of this gene.
Conversely, overexpression of the GCHI and
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 thealdolase, andaccumulation 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
Mitochondrion
Plastid
GTP
DHN-P3
DHN
HMDHP
DHN-P
HMDHP-P2
DHP
DHF
THF
Glu
Folate-Glun
ADC
Chorismate
pABA
Glu +
+
+
THF
Folate-Glun
J2Glu
J1
I
H
G
F
C
A
B
D
E
Figure 3
Potential regulatory loops operating at the enzymeand gene levels in folate biosynthesis. Broken purplelines denote potential enzyme-level feedbackinhibition mechanisms identified by in vitro studies.Broken blue lines denote gene-level feedbackrepression (minus signs) or feedforward activation(plus signs) mechanisms inferred from transcriptomedata. Folate biosynthesis enzymes are shown byletters as in Figure 2. Dihydromonapterin has beenomitted for simplicity. Abbreviations: ADC,aminodeoxychorismate; DHF, dihydrofolate;DHN, dihydroneopterin; DHP, dihydropteroate;Glu, glutamate; Glun, polyglutamate; HMDHP,6-hydroxymethyldihydropterin; P, phosphate;P2, diphosphate; P3, triphosphate; pABA,p-aminobenzoate; THF, tetrahydrofolate.
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Turnover: the
dynamic balancebetween synthesis anddegradation of abiomolecule
pABA-Glu: p-amino-benzoylglutamate
transgenes, expression of the endogenous
GCHI and ADCS genes is unaffected (94).
Thus, neither gene-level nor enzyme-level
feedback appears to control the committingreactions of pterin and p ABA 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 p ABA (85). Such paradoxical
observations nicely illustrate how little the
regulation of folate biosynthesis is understood.
Lastly, mammalian GCHI is regulated by a
feedback mechanism that involves a regulatory
protein (99) and also by phosphorylation
(49). Such mechanisms could not have beendetected 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 C9N10 bond, which yields a pterinplus p-aminobenzoylglutamate (pABA-Glu) or
its polyglutamyl forms (Figure 4) (34). Such
nonenzymatic cleavage is thought to be the
mainrouteof folate breakdownin all organisms
(89). However, evidence on this point is lacking
for plants, so enzyme-mediated cleavage can-
not be excluded; an active cleavage process is
suggested by the unusually high folate break-
down rates in plants. Folate-cleaving enzymesfrom microorganisms have been reported (84),
and mammalian ferritin facilitates folate cleav-
age in vitro and in vivo (90). Moreover, the
action of the folate-dependent COG0354 pro-
tein, which participates in synthesis or repair of
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
fullyoxidized,aromatic formpterin-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 ofpABA-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, p ABA-Glu is
hydrolyzed to yieldpABA 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, tobe 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
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showed it to be unstable and probably a
metalloenzyme (12). The corresponding gene
has not been cloned, although seven potential
candidates were tested and ruled out (12). Thepartially purified Arabidopsis root activity also
releases glutamate from folic acid, raising the
possibility of enzymatic folate degradation
a
N
HNN
NH
H2N
O
CHO
HH
Dihydropterin-6-aldehyde
N
HNN
NH2N
O
CH2OH
6-Hydroxymethylpterin
N
HNN
NH2N
O
CHO
Pterin-6-aldehyde
N
HNN
NH2N
OCOOH
Pterin-6-carboxylate
C
-Glu tail
N
HNNH
NH
H2N
O
CH29
H
H
H
NH
10NH
O
pABA-Glu
THF
Pterin
N
HNN
NH
H2N
O
CH2OH
HH
6-HMDHP
O
C
CH
COOH
(CH2)2
NH CH
COOH
NH
CH
COOH
COOH
n
C
O
(CH2)2
(CH2)2
bGTP
Chorismate
HMDHPHMDHP-P2
DHP
DHF
THF
Folate-Glun
Glu
pABA
Glu
pABA-Glun DHPAld
pABA-Glu
GGH
PGH
PTAR
PTAR
PTAR
PTAD
in vivo (12). Another possible way to recycle
p ABA-Gludirect reincorporation into DHF
viathe actionof dihydropteroatesynthasewas
excluded by a kinetic study of this enzyme (66).Salvage of the pterin cleavage product is
less well understood, but certain points are
fairly clear. First, dihydropterin-6-aldehyde
can be salvaged in vivo by reduction of its
side chain to give the folate synthesis inter-
mediate HMDHP (Figure 4b) (66). In pea
leaves, this reaction appears (a) to take place
predominantly in the cytosol and (b) to be
catalyzed by multiple NADPH-dependent
pterin aldehyde reductase (PTAR) isoforms
that can reduce both dihydropterin-6-aldehyde
and pterin-6-aldehyde; similar multiple PTAR
isoforms also occur in Arabidopsis seeds (62).One Arabidopsisisoform (At1g10310) has been
cloned; it belongs to the short-chain dehydro-
genase/reductase family and attacks diverse
aromatic and aliphatic aldehydes (62). Like-
wise, all thepea isoforms attack other aldehydes
(62). Dihydropterin-6-aldehyde thus seems not
to be salvaged by a single dedicated enzyme but
rather by a battery of broad-specificity alde-
hyde reductases. In support of this hypothesis,
Figure 4
Folate breakdown and salvage reactions.(a) Structures and relationships of folate breakdownproducts. Arrowheads mark the oxidatively labileC9N10 bond (red), the bond cleaved byp-aminobenzoylglutamate hydrolase (black), andbonds cleaved by-glutamyl hydrolase (GGH;yellow). Dotted arrows show (photo)chemicaloxidations. Solid arrows show enzymatic reactions;red crosses mark those that appear not to occur inplants (although they occur in other organisms).
The folate synthesis intermediate6-hydroxymethyldihydropterin (HMDHP) iscolored blue. (b) Folate salvage reactions (purplearrows) in relation to folate biosynthesis.
Abbreviations: DHF, dihydrofolate;DHP, dihydropteroate; DHPAld, dihydropterin-6-aldehyde; GGH, -glutamyl hydrolase; Glu,glutamate; Glun, polyglutamate; P2, diphosphate;pABA-Glu, p-aminobenzoylglutamate; PGH,p-aminobenzoylglutamate hydrolase; PTAD, pterinaldehyde dehydrogenase; PTAR, pterin aldehydereductase; THF, tetrahydrofolate.
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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 aliphaticaldehyde 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).
Second, if PTAR activity fails to intercept
dihydropterin-6-aldehyde before it is oxidized
to pterin-6-aldehyde, then the pterin moietycan 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 capacityis surprising inasmuch
as enzymes with this activity occur in other or-
ganisms; pteridine reductase 1 ofLeishmania 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 bea 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 THFand 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
compound can undergo rapid cleavage at
acidic pH to p ABA-Glu and a methylated
pterin (55). Presumably the levels of ascorbate,
glutathione, and other reductants are sufficientin 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 C1substituent 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 seemsanomalous, given that 3050% 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 19). Thesesteps, 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,
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mitochondria probably export pterins
resulting from folate degradation (see
above). Pterin transport has not been
studied in plant mitochondria, but itseems likely to be carrier mediated given
that the only well-studied pterin uptake
process, in the protistan parasiteLeishma-
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 whenfolate 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-
latetransportisoneofthemostimportant
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
beencloned and extensivelycharacterized
(68); it is a member of the mitochondrialcarrier family. However, the closest ho-
mologofMFTinArabidopsis(At5g66380,
AtFOLT1), although it demonstrates fo-
late transport activity in two heterologous
systems, is targeted to the chloroplast
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 folateuptakewhen expressedinE.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). Thislack 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-
bidopsisFBT 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 theeightArabidopsisFBTpro-
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-affinityS-adenosylmethionine
transporter (22).
5. Vacuolar p ABA glucose ester import.
It is necessary to invoke a tonoplast
transporter for p ABA glucose ester
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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 p ABA 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.
The argument for a tonoplast transport
system for folate polyglutamates is
outlined above (see the section entitledFolate 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
resistanceassociated 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). Polyglutamylfolates 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
accumulation of, methotrexate (73),
indicating that this protein functions in
planta, at least in folate analog transport.
Whether it plays a significant role infolate 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, likeother MRPs, AtMRP1trans-
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 atleast 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 theeffects of folatesyn-
thesis inhibitors (16, 51). There is like-
wise direct evidence for uptake of theclose folate analog methotrexate (16, 51,
70), including a 1993 study ofDatura 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-
ateresistanceresultedinanincreaseinthe
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 andmetabolize 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).
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Cloning Plant Folate Transportersby Homology
The cloning-by-homology approach that
yielded AtFOLT1, At2g32040, and AtMRP1may 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
protoncoupled 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. Thesixth class is the FBT family, one of whose
plant homologs (At2g32040) also transports
folates. The seventh class, members of the
bacterial energycoupling 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 exceptionis 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.
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 pterinsynthesis (Figure 2, step A), alone or together
withADCS, the first enzyme ofpABA 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, butit 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 p ABA levels (i.e., p ABA plus
its glucose ester) were analyzed in GCHI-
overexpressing tomato fruit (20), severe pABAdepletion was observed, indicating that the
pABA supplylimits further folateaccumulation.
Consistent with this view, adding anArabidopsis
ADCS transgene greatly increases the total
p ABA 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 inthe 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
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Ta
ble1
Pro
jec
tsa
iming
tome
tab
oli
ca
llyeng
ineer
folate
con
ten
t
Year
Sp
ec
ies
Organ
(s)teste
d
Gene
(s)ad
de
dora
blate
d
Ch
ange
infolate
Commen
ts
Re
ference
2004
Arabidopsis
Leaves
+
Escherichia
coliGCHI
+2-to+4-fold
1,250-foldincreaseinpterins
40
2004
Tomato
Fruit
+
MouseGC
HIa
+2-fold
Up-to-140-foldincreaseinpterins
20
2005
Arabidopsis
Leaves
5-F
CLablated
+2-fold
Growthslowed20%;floweringdelayed
30
2007
Tomato
Fruit
+
ArabidopsisADCS
None
Up-to-40-foldincreaseinpABA
21
+
MouseGC
HIa/
Upto+25-fold
>20-foldincreasesinpterinsandpABA
+
ArabidopsisADCS
2007
Rice
Grain
+
ArabidopsisGCHI
None
Up-to-29-foldincreaseinpterins
85
+
ArabidopsisADCS
Upto6-fold
Up-to-89-foldincreaseinpABA
+
ArabidopsisGCHI/ADCS
Upto+100-fold
Average4-foldincreaseinpterinsand
25
-foldincreaseinpABA
2008
Rice
Leaves
+
WheatHP
PK/DHPS
+75%
Pre
cursorsnotanalyzed
29
Grain
+40%
2009
Maize
Grain
+
E.
coliGCHI
+2-fold
Pre
cursorsnotanalyzed;-caroteneand
ascorbatepathwaysalsoengineered
60
2009
Lettu
ce
Leaves
+
ChickenG
CHIa
+2-to+9-fold
Pre
cursorsnotanalyzed
64
2010
Arabidopsis
Leaves
+
AtGGH2
39%
Polyglutamatetaillengthdecreased
3
2010
Tomato
Fruit
+
LeGGH2
46%
Polyglutamatetaillengthdecreased
3
2010
Arabidopsis
Leaves
+
GGHRNAi
+30%
Polyglutamatetaillengthincreased
3
aCodonusagemodifiedtoimproveplantexpression.
bAbbreviations:5-FCL,5-formyltetrahydrofolatecycloligase;ADCS,am
inodeoxychorismatesynthase;GCHI,GTPcyclohydrolaseI;HHPK/DHPS,6-hydroxymethyldihydropterin
pyrophosphokinasedihydropteroatesynthase;pABA,p-aminobenzoate;RNA
i,RNAinterference.
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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 compoundsthat 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 insertHMDHP 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 GGHwas over- or underexpressed in Arabidopsisand
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
relied on simple one- or two-gene strategies,
and almost all have overexpressed enzymes at
thehead of thepathway, which drivesflux 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-ityand 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 predictablenor understood and may not
occur in other systems, meaning that theGCHI-
plus-ADCS strategy may not necessarily workas 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
typeof GCHI (23), whereas others havea 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 principleenable 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).
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Finally, conventional breeding basedon nat-
ural variation may be a good alternative to
metabolic engineering and such breeding could
be informed by the outcomes of engineeringexperiments (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,couldform thebasis forbreed-
ing programs. Given the engineering evidence
that both pterin and p ABA supplies limit fo-
late synthesis (Table 1), such programs might
profitably analyze precursors as well as folates
in parents and progenies.
FOLATE FRONTIERS
The frontiers include all the topics in this re-
view, includingdespite the effort invested in
itthe biosynthesis pathway itself. Specifically,
only one step in this pathway (DHF synthase)
hasbeenvalidatedbymutantstudies(42);allthe
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 somedepend 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 bemetabolic profiling of folates and
folate precursors in xylem and phloem sap.
Other frontiers are at least implicit in the
foregoing sections on biosynthesis regulation,
turnover, and transport but bear reempha-
sizing. Regarding biosynthesis regulation, thegeneral lack of biochemical studies of enzymes
isolated fromplants(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 notnecessarily 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-
plain by spontaneous chemical processes alone,
and other hintsparticularly from compara-tive biochemistrythat 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 uptakeinto the cell.
Although homology to known folate trans-
portersisnearlyifnotcompletelyminedoutasa
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.
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3. There is evidence that plantsexhibit relativelyhigh folatebreakdown rates andcan salvage
the breakdown products for reuse in folate synthesis. Salvageenzyme activities have been
identified, and one salvage enzyme has been cloned.4. Three plant folate transporterstwo chloroplastic and one vacuolarhave 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.
FUTURE ISSUES
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, Oceane Frelin,
and Kenneth Ellens for comments on the manuscript.
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