A central role for gamma-glutamyl hydrolases in plant folate homeostasis

A central role for gamma-glutamyl hydrolases in plant folatehomeostasis

Tariq A. Akhtar1,†,*, Giuseppe Orsomando2, Payam Mehrshahi3, Aurora Lara-Nunez4, Malcolm J. Bennett3, Jesse F. Gregory III4

and Andrew D. Hanson1

1Horticultural Sciences Department, University of Florida, Gainesville, FL 32611, USA,2Dipartimento Patologia Molecolare e Terapie Innovative, Universita Politecnica delle Marche, Ancona, 60131, Italy,3Plant Sciences Division, School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough

LE12 5RD, UK, and4Food Science and Human Nutrition, University of Florida, Gainesville, FL 32611, USA

Received 23 May 2010; accepted 28 July 2010; published online 16 September 2010.*For correspondence (fax +734 647 0884; e-mail [email protected]).†Present address: University of Michigan, Department of Molecular, Cellular and Developmental Biology, Kraus Natural Sciences Building, 830 North University,

Ann Arbor, MI 48109, USA.

SUMMARY

Most cellular folates carry a short poly-c-glutamate tail, and this tail is believed to affect their efficacy and

stability. The tail can be removed by c-glutamyl hydrolase (GGH; EC 3.4.19.9), a vacuolar enzyme whose role in

folate homeostasis remains unclear. In order to probe the function of GGH, we modulated its level of

expression and subcellular location in Arabidopsis plants and tomato fruit. Three-fold overexpression of GGH

in vacuoles caused extensive deglutamylation of folate polyglutamates and lowered the total folate content by

approximately 40% in Arabidopsis and tomato. No such effects were seen when GGH was overexpressed to a

similar extent in the cytosol. Ablation of either of the major Arabidopsis GGH genes (AtGGH1 and AtGGH2)

alone did not significantly affect folate status. However, a combination of ablation of one gene plus RNA

interference (RNAi)-mediated suppression of the other (which lowered total GGH activity by 99%) increased

total folate content by 34%. The excess folate accumulated as polyglutamate derivatives in the vacuole. Taken

together, these results suggest a model in which: (i) folates continuously enter the vacuole as polyglutamates,

accumulate there, are hydrolyzed by GGH, and exit as monoglutamates; and (ii) GGH consequently has an

important influence on polyglutamyl tail length and hence on folate stability and cellular folate content.

Keywords: folate, polyglutamate, gamma-glutamyl hydrolase, vacuole, tomato.

INTRODUCTION

Tetrahydrofolate (THF) and its derivatives, collectively

termed folates, are indispensable cofactors in one-carbon

(C1) metabolism. Folates provide C1 units at various oxida-

tion levels (methyl, methylene, or formyl) to acceptor mol-

ecules during the synthesis of purines, thymidylate,

pantothenate, formyl-methionyl tRNA, and methionine

(Scott et al., 2000). Moreover, as methionine is required to

drive the activated methyl cycle, the synthesis of methylated

products such as choline, lignin, and chlorophyll depends

indirectly upon folates (Hanson and Roje, 2001; Rebeille

et al., 2006).

Plants and microbes synthesize folates de novo, whereas

animals have a strict dietary requirement, most of which

comes from plant sources. Plant folates are assembled in the

mitochondrion from pterin, p-aminobenzoate (pABA), and

glutamate precursors (Figure 1). In vivo, the majority of

folates are conjugated to a c-linked polyglutamyl tail of up to

eight residues. The tail is added, one glutamate at a time, by

folylpolyglutamate synthetase (FPGS; EC 6.3.2.17). In ani-

mals and plants, multiple FPGS isoforms exist, each tar-

geted to a distinct subcellular location (Chen et al., 1996;

Ravanel et al., 2001). The polyglutamyl tail is shortened or

removed by c-glutamyl hydrolase (GGH; EC 3.4.19.9), a

vacuolar endo- and/or exo-peptidase found in animals and

plants (Wang et al., 1993; Orsomando et al., 2005). As part of

the folate salvage pathway, GGH also hydrolyzes polygluta-

mates of the folate breakdown product, pABA polygluta-

mate (Orsomando et al., 2006). The relative activities of

FPGS and GGH are considered to determine the extent of

intracellular folate polyglutamylation.

256 ª 2010 The AuthorsJournal compilation ª 2010 Blackwell Publishing Ltd

The Plant Journal (2010) 64, 256–266 doi: 10.1111/j.1365-313X.2010.04330.x

Polyglutamylation can directly affect C1 metabolism,

because folate-dependent enzymes prefer polyglutamates

to monoglutamyl forms. For example, methionine synthases

from various organisms cannot use monoglutamyl

5-methyl-THF (5-CH3-THF) (Whitfield et al., 1970; Cherest

et al., 2000; Ravanel et al., 2004). The polyglutamyl tail may

also help to ‘channel’ folates between the active sites of

folate-dependent enzymes, many of which exist as com-

plexes (Schirch and Strong, 1989). Furthermore, because

polyglutamylation favors binding to enzymes, and enzyme-

bound polyglutamates are protected from oxidative break-

down, folates tend to be stabilized by polyglutamylation

(Suh et al., 2001). The ability of folate transporters to shuttle

folates across membranes decreases as polyglutamyl tail

length increases (Shane and Stokstad, 1975). Polyglutamy-

lation also increases the anionic nature of folates and

impedes the passive diffusion of folates across biomem-

branes (Appling, 1991). Hence, polyglutamylation generally

favors the retention of cellular and organellar polyglutamates in

the compartments in which they reside (Shane, 1989).

In plants, however, almost nothing specific is known

about the in vivo significance of polyglutamylation. Nor is

the functional role of GGH clear in relation to folate

homeostasis. In bacteria, overexpression of GGH causes

extensive deconjugation of polyglutamates and decreased

folate retention, without a major effect on growth rate

(Sybesma et al., 2003; Akhtar et al., 2008). In animal sys-

tems, enhanced GGH expression decreases polyglutamate

abundance and intracellular folate levels (Li et al., 1993;

Rhee et al., 1993; Cole et al., 2001), whereas increased FPGS

activity is associated with higher intracellular folate levels

(Lowe et al., 1993; Sakamoto et al., 2008). Taken together,

these observations imply that intracellular folate levels are

positively correlated with polyglutamate tail length.

To further examine this relationship, we turned to the

recently described GGH systems of Arabidopsis and tomato

(Solanum lycopersicum) (Orsomando et al., 2005; Akhtar

et al., 2008). The Arabidopsis GGH family comprises three

genes, AtGGH1, AtGGH2, and AtGGH3, arranged in tandem

on chromosome 1. Tomato also contains three GGH genes

(LeGGH1, LeGGH2, and LeGGH3) of which the latter encodes

a catalytically inactive isoform. Modulation of GGH activity

in Arabidopsis and tomato fruit revealed that total cellular

folate levels are influenced by the extent of GGH-mediated

hydrolysis of vacuolar polyglutamates. These results thus

demonstrate a direct impact of polyglutamylation on intra-

cellular folate content.

RESULTS

Overexpression of GGH in Arabidopsis and tomato fruit

The metabolic significance of GGH was first explored in

Arabidopsis and tomato fruit by overexpressing AtGGH2

and LeGGH2, respectively. These proteins exhibit both

endo- and exo-peptidase activity towards polyglutamates.

Two versions of these GGHs were introduced: the native full-

length (FL) protein containing the predicted signal peptide or

a truncated version (TR) missing the predicted signal pep-

tide. In Arabidopsis, a hexahistidine (His6) epitope was fused

to the C-terminus of AtGGH2-FL and to the N-terminus of

AtGGH2-TR, respectively. The constitutive 35S promoter

from figwort mosaic virus was used to drive expression in

Arabidopsis (Richins et al., 1987), while in tomato the E8

promoter was utilized to restrict expression to late stages of

fruit development (Kneissl and Deikman, 1996). From

among the independent transgenic lines obtained for each

species and each construct we selected three for further

analysis; these lines showed the largest increase in total

GGH activity, which was about threefold (Figure 2a). The

lines expressing the truncated GGHs were indistinguishable

from the wild type; tomato fruit from the LeGGH2-FL lines

and siliques from the AtGGH2-FL lines were consistently

smaller, with few seeds.

Targeting of the overexpressed GGHs was checked in

Arabidopsis by assaying GGH activity in vacuoles isolated

from wild-type leaves and those expressing AtGGH2-FL or

AtGGH2-TR. The truncated protein was predicted to be

cytosolic, as is usual for vacuolar proteins without their

targeting peptide (Frigerio et al., 1998). Vacuolar GGH

activity was about fourfold higher for AtGGH2-FL transfor-

mants than for the wild type, whereas that for AtGGH2-TR

transformants was the same as the wild type (Figure 2b).

-Glu tail

COOH

CH

COOH

CH2

CH2

NH

CH

COOH

CH2

CH2

COOH

n

C

O

O

C

C

N

NHNR

5

NH

NH2

O

CH2

9

HH

HNR′

10CHN

HCH2

CH2

NH

O

pABAPterin Glu

Folate R R′

THF

5-Methyl-THF

H H

CH3 H

5-Formimino-THF CH = NH

5-Formyl-THF

10-Formyl-THF

CHO H

H CHO

5, 10-Methylene-THF

5, 10-Methenyl-THF

-CH2-

-CH-

H

Figure 1. Structures of tetrahydrofolate (THF) polyglutamates and their

C1-substituted derivatives.

Folates are composed of pterin, p-aminobenzoate (pABA) and glutamate

moieties. A c-linked polyglutamate tail of up to eight residues is attached to

the first glutamate. One-carbon (C1) units at various oxidation levels are

attached to the N5 and/or N10 position of the pterin moiety. Folates readily

undergo oxidative cleavage of the C9–N10 bond yielding pterin and pABA-

glutamate moieties. Arrows indicate the bonds cleaved by c-glutamyl

hydrolase (GGH).

Gamma-glutamyl hydrolases and folate homeostasis 257

ª 2010 The AuthorsJournal compilation ª 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 64, 256–266

Moreover, when vacuolar protein extracts from both trans-

genic lines were probed for the His6 epitope, only those from

AtGGH2-FL gave positive signals (Figure 2b). The full-length

native protein was thus sent to the vacuole and the truncated

one was not. Since the fourfold increase in activity in the

AtGGH2-FL vacuoles is similar to that in whole-leaf extracts

it may be further concluded that native AtGGH2 was sent

solely to vacuoles. We observed no difference in GGH

activity in protoplasts isolated from the lines overexpressing

the native or truncated enzymes, suggesting that the

enhanced activity was intracellular in both cases.

Folate content and polyglutamylation status in plants

overexpressing GGH

To examine the relationship between GGH and folate

homeostasis, we measured total folate levels and the degree

of polyglutamylation in both Arabidopsis and tomato lines

with enhanced GGH activity. In Arabidopsis leaves and

tomato fruit expressing vacuole-targeted, native GGH, folate

levels dropped by 39 and 46%, respectively (Figure 3a). In

contrast, overexpression of the truncated AtGGH2 or

LeGGH2 did not significantly alter folate content. The major

folate species in wild-type Arabidopsis leaf tissue was

5,10-methenyl-THF (5,10 = CH-THF), whereas 5-CH3-THF

dominated in tomato fruit; these species were 70 and 90%

polyglutamylated, respectively (Figure 3c). The degree of

polyglutamylation of these folate species was severely

impaired only in lines expressing the native versions of GGH

(Figure 3b,c). In these plants, the trend toward an increas-

ingly monoglutamylated folate pool was at the expense of

the longer chain folates (six or more glutamates).

T-DNA insertion mutants of GGH and expression of the

Arabidopsis GGH family

The consequences of GGH inactivation were investigated in

Arabidopsis (Orsomando et al., 2005). We first used real-

time PCR to probe the expression of each family member in

various tissues (Figure 4a). AtGGH1 and AtGGH2 were

expressed in all tissues examined, with AtGGH1 predomi-

nating in roots and AtGGH2 in leaves. Conversely, AtGGH3

was expressed at very low levels in all tissues, suggesting

that the corresponding isoform makes little contribution to

total cellular GGH activity and is consequently not physio-

logically relevant. Moreover, when AtGGH3 was expressed

in Escherichia coli, we failed to detect any GGH activity (data

not shown).

Based on the premise that AtGGH1 and AtGGH2 are

essentially the sole contributors to Arabidopsis GGH activity,

two independent T-DNA mutant lines were obtained for each

gene (Figure 4b). Wild type segregants and plants homozy-

gous for the T-DNA insertion were identified by PCR using

primers flanking the insertion site along with a third primer

specific for the T-DNA left border (Figure 4c). The position of

the T-DNAs within the exon regions of each corresponding

Vac

uola

r G

GH

act

ivity

(nm

ol m

in–1

mg–

1 pr

otei

n)

0%

4%

10%

50 ─

37 ─

Anti-His6

TR FL

0

10

20

30

40

50

0

2

4

6

WT 7-8

WT

AtGGH2-

TR

AtGGH2-

FL

7-10 7-5 2-4 2-12 2-9

Arabidopsis

WT0

0.1

0.2

0.3

0.4

T-4 T-6 T-11

Wild type TR-GGH2

GG

H a

ctiv

ity(n

mol

min

–1 m

g–1

prot

ein)

GG

H a

ctiv

ity(n

mol

min

–1 m

g–1

prot

ein)

Tomato

FL-GGH2

F-2 F-4 F-14

(a)

(b)

Figure 2. Overexpression of c-glutamyl hydrolase (GGH) in tomato and

Arabidopsis.

(a) Overexpression of tomato and Arabidopsis cDNAs encoding full-length

GGH2 protein (FL-GGH2) or a truncated version missing the signal peptide

(TR-GGH2). Expression in tomato was driven by the fruit-specific E8

promoter and in Arabidopsis by the figwort mosaic virus 35S promoter.

Data for three independent lines exhibiting significantly (P < 0.05) higher

GGH activity in tomato fruit (T2 generation) and Arabidopsis leaf tissue (T3

generation) are presented. The GGH activity was assayed in desalted

tissue extracts from red tomato fruit or 4-week-old Arabidopsis rosette

leaf tissue. Specific activity values are the means � SE of three indepen-

dent experiments from three to five different plants from each indepen-

dent line.

(b) Arabidopsis vacuolar GGH activity in wild-type (WT) and AtGGH2

overexpressing lines. Vacuoles were purified on a Ficoll gradient and

harvested from the 0 to 4% Ficoll interface as shown. The neutral red dye

staining indicates the presence of intact vacuoles. Note the enhanced GGH

activity only in vacuoles from AtGGH2-FL lines. Vacuolar protein extracts

(8–10 lg) from AtGGH2-TR (TR) and AtGGH2-FL (FL) lines were also

subject to immunoblot analysis using antibodies against the His6 epitope

(inset). Equal protein loading was verified by staining the immunoblot with

Ponceau red. Specific activity values are the means � SE of three

independent vacuole preparations.

258 Tariq A. Akhtar et al.


AtGGH gene was confirmed by sequencing the left border

junction. The absence of additional T-DNA insertions within

each line was confirmed by PCR designed to amplify the nptII

marker gene carried by each T-DNA; amplicons were not

seen in any wild-type segregant but were present in all the

mutants (Figure 4c). The individual GGH mutant alleles were

named ggh1-1, ggh1-2, ggh2-1, and ggh2-2.

GGH activity and bond cleavage specificity in the GGH

mutants

To probe the effects of T-DNA insertion in AtGGH1 and

AtGGH2, total GGH activity was measured in crude protein

extracts from roots and leaves from the mutant lines

(Figure 5a). In both ggh1-1 and ggh1-2, total activity was

unaltered in leaves and reduced by only 10% in roots. Con-

versely, both ggh2-1 and ggh2-2 showed large reductions in

Roots Siliques Stems Youngleaves

Matureleaves

0

1

2

3

4 At1g78660 (GGH1)

At1g78680 (GGH2)

At1g78670 (GGH3)

0.5 kb

SAIL 258C07

SALK 018537

At1g78660

SALK 145009

GABI-Kat 109A04

At1g78680

(ggh1-2)

(ggh1-1)

(ggh2-2)

(ggh2-1)

800

400

600

ggh1-1

41 2 3

WT KO WT KO

ggh2-2

1 2 3 4

WT KO WT KO

ggh2-1

1 2 3 4

WT KO WT KO

1 2 3 4

WT KO WT KO

ggh1-2

Cop

ies

× 1

06/

250

ng to

tal R

NA

(a)

(b)

(c)

Figure 4. Expression of the Arabidopsis c-glutamyl hydrolase (GGH) family

and identification of T-DNA insertion mutants.

(a) Expression levels of the three Arabidopsis GGH mRNAs. Young leaves

were collected from 2 to 3-week-old plants; stems, siliques, and mature leaves

were from 4 to 6-week-old plants. Roots were from 2-month-old liquid

cultures. Absolute quantification was performed by real time PCR.

(b) Structures of the Arabidopsis GGH1 (At1g78660) and GGH2 (At1g78680)

genes and orientation of the T-DNA insertions. Introns, exons, and untrans-

lated regions of the mature mRNAs are represented by lines, white, and black

boxes, respectively. T-DNA insertion lines were selected from the SALK, SAIL,

and GABI-Kat collections. Arrows represent each T-DNA (right–left border

orientation), verified by sequencing of the left border junction, but are not

drawn to scale.

(c) Analysis of GGH mutant genotypes by PCR. Pooled genomic DNA samples

(‡20 individuals) from wild-type segregants (WT) and homozygous mutants

(KO) were used as PCR templates; primers either flanked the T-DNA insertion

plus a left-border T-DNA-specific primer (lanes 1 and 2) or targeted the

selectable marker gene (lanes 3 and 4). Sizes of the amplicons (in bp) are

indicated on the left.

Det

ecto

r re

spon

se (

µA)

0

0.3

0.2

0.1

WT GGH2-TR GGH2-FL

1

2

3 45

67

8

20 24 28 32Retention time (min)

5-CH3-THFTHF 5,10 = CH-THF

5-CHO-THF

Fol

ate

(nm

ol g

–1 fr

esh

wt)

0

0.4

0.8

1.2

1.6Arabidopsis

*

Fol

ate

(nm

ol g

–1 fr

esh

wt)

0

0.2

0.4

0.6

0.8Tomato

*

# glutamates1 2 + 3 4 + 5 >6

LeGGH2-FL

Wild typeLeGGH2-TR

0

20

40

60

80

% o

f 5-C

H3-

TH

F

Tomato

**

% o

f 5, 1

0 =

CH

-TH

F

AtGGH2-TRAtGGH2-FL

Wild type

0

20

40

60

80Arabidopsis

*

* **

(a) (b)

(c)

Figure 3. Folate content and polyglutamylation status in plants overexpress-

ing c-glutamyl hydrolase (GGH).

(a) Total folate content in 4-week-old Arabidopsis rosette leaves (upper panel)

and red tomato fruit (lower panel) overexpressing the truncated (TR) or full-

length (FL) GGH2, relative to wild-type (WT) controls. The major folate species

detected were tetrahydrofolate (THF), 5-methyl-THF (5-CH3-THF), 5,10-methe-

nyl-THF (5,10 = CH-THF), and 5-formyl-THF (5-CHO-THF). For analysis, 1–2 g

of Arabidopsis or tomato fruit tissue was pooled from three independent

transformants; wild-type samples were pooled from three individual plants.

Data are the mean values � SE from six independent extractions; asterisks

indicate significant difference (P < 0.05, t-test) from wild type.

(b) Representative HPLC chromatograms obtained from folate polyglutamate

analysis of either Arabidopsis leaf or tomato fruit tissue. The detector channel

that specifically responds to 5-CH3-THF at 0 mV is illustrated. Numbers above

each peak represent the length of the polyglutamate tail associated with this

folate species.

(c) Folate polyglutamylation in Arabidopsis and tomato fruit. The number of

glutamates in the polyglutamyl tail of the major folate species in Arabidopsis

(5,10 = CH-THF) and tomato (5-CH3-THF) are shown as percentages of the

total. Wild-type and transgenic tissue was prepared as above. Asterisks

indicate significant difference (P < 0.05, t-test) when compared with the wild

type within each category of tail length.



GGH activity in both leaves and roots, averaging approxi-

mately 90 and 82%, respectively. Additionally, with primers

designed to measure expression of AtGGH1 or AtGGH2, no

mRNA signal in either corresponding GGH mutant plant

could be detected by semi-quantitative RT-PCR (Figure 5a).

These data show clearly that AtGGH2 is the main contributor

to total Arabidopsis GGH activity.

Because AtGGH1 and AtGGH2 exhibit distinct c-glutamate

bond cleavage patterns (Orsomando et al., 2005), their

individual contributions to total GGH activity can be mon-

itored in total protein extracts. Specifically, GGH1 cleaves

folate pentaglutamate (PteGlu5) to PteGlu2 and PteGlu3,

while GGH2 yields mainly PteGlu1 and a modest amount of

PteGlu2. On this basis, comparison of wild-type tissues

revealed that leaves contain almost exclusively GGH2 and

roots both GGH1 and GGH2 isozymes (Figure 5b). Likewise,

inspection of the cleavage patterns in mutant lines showed

that AtGGH1 and AtGGH2 mutants exhibited bond cleavage

profiles identical to recombinant AtGGH2 and AtGGH1,

respectively (Figure 5b). The reciprocal consistency of these

data further reinforces the probability that the third gene,

AtGGH3, makes little if any contribution to total GGH

activity.

RNA interference (RNAi)-mediated knockdown of residual

GGH in individual GGH knockouts

As the single GGH mutants retained substantial GGH activity

and the tandem organization of the Arabidopsis GGH genes

precludes generation of double mutants, we pursued RNAi-

mediated downregulation of AtGGH1 and AtGGH2 in the

ggh2-1 and ggh1-2 mutant backgrounds, respectively.

In ggh2-1 mutants targeted for AtGGH1 knockdown (At-

GGH1RNAi ggh2-1), three independent lines were obtained

that exhibited an average knockdown in AtGGH1 mRNA of

79%, relative to wild-type levels. Among three independent

lines targeted for AtGGH2 knockdown (AtGGH2RNAi ggh1-2),

AtGGH2 mRNA levels were reduced on average by 88%

(Figure 6a). In these lines with lowered GGH gene expres-

sion, total GGH activity in rosette leaf tissue was measured

and compared with the wild type (Figure 6b). The average

residual GGH activities in the three independent RNAi lines

targeting AtGGH1 and AtGGH2 were 0.008 and 0.02 nmol

min)1 mg)1 protein, respectively, representing 99 and 98%

knockdown in activity.

Total leaf folate content in the RNAi lines was measured

and compared with wild type and with the individual mutant

lines into which each RNAi construct was introduced.

Relative to wild-type plants, there were no significant

changes in total folate content in either ggh1-2 or ggh2-1

single mutants (Figure 6c) even though the latter had only

approximately 10% of wild-type GGH activity. However, both

AtGGH1RNAi ggh2-1 and AtGGH2RNAi ggh1-2 lines exhibited

significantly higher folate content (approximately 30%),

the increase being due mainly to 5,10 = CH-THF, the

PteGlu1 PteGlu2 PteGlu3

Extent of reaction

Rel

ativ

e pr

oduc

t con

cent

ratio

n 0 0.2 0.40

0.1

0.2

0.3Wild type root

0.10

0 0.05

0.025

0.05

0.075Wild type leaf

0 0.05 0.10

0.025

0.05

0.075GGH2 KO

0 0.20

0.1

0.2

0.3GGH1 KO

0.4

0 0.2 0.40

0.1

0.2

0.3

RecombinantGGH1

0 0.2 0.40

0.1

0.2

0.3

RecombinantGGH2

GG

H a

ctiv

ity(n

mol

min

–1 m

g–1

prot

ein)

ggh1-2 ggh2-1

KOWT

ggh2-2

WT KO

ggh1-1

KOWT KO WT

WT ggh1-1 ggh1-2 ggh2-1 ggh2-2

RootLeaf

0

1

2

10

8——

600 —500 —

* *

*

* *

*

(a)

(b)

Figure 5. c-Glutamyl hydrolase (GGH) activity and bond cleavage specificity

in the GGH mutants.

(a) Total GGH activity in wild-type (WT) and individual GGH mutant lines.

Roots were collected from 6 to 8-week-old plants grown in liquid medium and

leaf tissue was from rosette leaves from 4 to 6-week-old plants grown in

potting soil. Semiquantitative RT-PCR indicates the presence and absence of

functional mRNAs coding for GGH1 and GGH2 in WT and individual mutant

(KO) lines, respectively (inset). Data are the means and SE from three

independent experiments and asterisks indicate significant difference

(P < 0.05, t-test) when compared with the wild type.

(b) Specificity of GGH bond cleavage in wild-type and individual mutant lines.

The GGH activity was assayed in desalted tissue extracts. Progress curves for

the initial stages of hydrolysis are presented as plots of relative concentration

of each reaction product formed (folic acid monoglutamate, PteGlu1, folic acid

diglutamate, PteGlu2, or folic acid triglutamate, PteGlu3) versus the extent of

reaction (see Experimental Procedures). Progress curves from wild-type leaf

and root tissue (upper panel) are compared with those from the individual

GGH mutants (middle panel) and those from purified recombinant GGH1 and

GGH2 (lower panel). Data for the individual GGH mutants were derived from

pooled leaf and root tissue from the two GGH1 mutant lines (ggh1-1 and

ggh1-2) or from the two GGH2 mutant lines (ggh2-1 and ggh2-2). Data are

representative of results obtained in three independent experiments.



predominant folate species in rosette leaf tissue (Figure 3a).

The degree of polyglutamylation of this folate was strikingly

enhanced in both RNAi lines, where >96% of it was

polyglutamylated (Figure 6c).

Folate content and polyglutamylation status in Arabidopsis

leaf vacuoles

The increase in total folate content observed upon GGH

downregulation coupled with the decrease in total folate

resulting from GGH overexpression in its native vacuolar

location implies a significant role for the vacuole in folate

homeostasis. We therefore measured the vacuolar folate

content in Arabidopsis plants with up- or downregulated

GGH activity.

Intact vacuoles were isolated from leaf protoplasts

derived from plants overexpressing GGH (AtGGH2-FL), from

plants with 1% of normal GGH activity (AtGGH1RNAi ggh2-1),

and from wild-type controls. The total vacuolar folate

contents essentially mirrored those of whole-leaf extracts

from the same plants, i.e. vacuolar folate contents decreased

by 48% in AtGGH2-FL plants and increased by 42% in

AtGGH1RNAi ggh2-1 plants, relative to vacuoles from wild-

type plants (Figure 7a). In lines expressing cytosolic GGH

(AtGGH2-TR), no changes in vacuolar folate content or

degree of polyglutamylation were observed (not shown).

The two main folate species in all vacuoles isolated were

10-formyl-dihydrofolate (10-CHO-DHF), an oxidation

product routinely formed from 10-formyl-tetrahydrofolate

(10-CHO-THF) during organelle isolation (Goyer et al., 2005)

and 5-CH3-THF, which is the predominant folate in pea leaf

vacuoles (Orsomando et al., 2005). The extent of 5-CH3-THF

polyglutamylation was less in vacuoles from AtGGH2-FL

plants than from wild-type plants (33% versus 52%) but was

greater in vacuoles from AtGGH1RNAi ggh2-1 plants (>97%)

(Figure 7b).

DISCUSSION

The widely held notion that polyglutamylation has a critical

impact on folate homeostasis is only indirectly supported in

animal systems (Suh et al., 2001) and has not been explored

in plants at all. Additionally, the few studies in animal sys-

tems and bacteria that examined relationships between

GGH activity and folate content have yielded conflicting

results (Cole et al., 2001; Sybesma et al., 2003; Sakamoto

et al., 2008). This study accordingly aimed to assess whether

GGH contributes to the control of folate polyglutamylation

in vivo, and to clarify the relationship between the degree

of polyglutamylation and plant folate content.

Our results are consistent with a model (Figure 8) in which

folate polyglutamates are continuously imported into the

vacuole. The resulting vacuolar polyglutamate pool is

hydrolyzed by GGH, and the monoglutamates produced

are exported to the cytosol. This model parallels one

proposed for folate polyglutamate turnover in mammalian

AtGGH2RNAi ggh1-2

0

20

40

60

80

100

120

WT

AtGGH1 AtGGH2

AtGGH1RNAi ggh2-1R

elat

ive

expr

essi

on (

% W

T)

WT1–38 1–6 1–29 2–37 2–29 2–22

GG

H a

ctiv

ity (

nmol

min

–1 m

g–1 )

AtGGH1RNAi ggh2-1

AtGGH2RNAi ggh1-2

——

0.08

0

0.04

0.12

2.0

2.2

1–38 1–6 1–29 2–37 2–29 2–22

5, 10 = CH-THF

5-CHO-THF

Fol

ate

(nm

ol g

–1 fr

esh

wt)

THF

5-CH3-THF

0

0.5

1.0

1.5

2.0

2.5

**

% o

f 5, 1

0 =

CH

-TH

F

0

10

20

30

40

50

60

70

80

# glutamates

AtGGH1RNAi ggh2-1

Wild type

AtGGH2RNAi ggh1-2

1 2 + 3 4 + 5 > 6

*

*

*

**

** *

(a)

(b)

(c)

Figure 6. RNA interference (RNAi)-mediated knockdown of residual c-glut-

amyl hydrolase (GGH) in the individual GGH knockouts and its effect on folate

status.

(a) Transcript abundance of GGH1 (left) and GGH2 (right) in wild type (WT)

and three independent RNAi lines targeting GGH1 or GGH2 in the ggh2-1 or

ggh1-2 mutant backgrounds, respectively. Relative quantification of GGH

gene expression was by real-time PCR.

(b) Total GGH activity in the three RNAi lines described above. For compar-

ison, GGH activity in the wild type and in the individual GGH mutants is

presented (right panel).

(c) Folate content and polyglutamyl tail length distribution. Total folate and its

major forms [tetrahydrofolate (THF), 5-methyl-THF (5-CH3-THF), 5,10-methe-

nyl-THF (5,10 = CH-THF), 5-formyl-THF (5-CHO-THF)] in the wild type and

individual GGH mutants are compared with those in the RNAi lines (left

panel). Asterisks indicate significant difference (P < 0.05, t-test) when com-

pared with the wild type. Polyglutamyl tail length (right panel) as a percentage

of the major folate species (5,10 = CH-THF) in the same plants. Data are the

means � SE from six independent extractions from rosette leaf tissue

randomly sampled from three different plants for each genetic background.

For folate analysis of each RNAi line, leaf tissue from the three independent

lines described above was pooled. Asterisks indicate significant difference

(P < 0.05, t-test) when compared with the wild type within each category of

tail length.



lysosomes (Sirotnak and Tolner, 1999; Schneider and Ryan,

2006). Because monoglutamates are generally poor enzyme

substrates, they tend to remain unbound and are conse-

quently more susceptible to oxidative breakdown (Suh

et al., 2001; Gregory and Quinlivan, 2002). It might therefore

be expected that the release of monoglutamates from the

vacuole – and, by extension, GGH activity – is subject to strict

control. In support of this idea, Arabidopsis plants and

tomato fruit with just a threefold elevation of vacuolar GGH

activity contained approximately 40% less total folate, while

knockdown of Arabidopsis GGH activity caused a approxi-

mately 30% increase in folate content.

Our model raises several important issues. First, it invokes

a tonoplast transport system for polyglutamates. This would

be unlike all folate carriers so far described in plants, which

accept only monoglutamates (Bedhomme et al., 2005; Klaus

et al., 2005; Raichaudhuri et al., 2009). An analogous polyg-

lutamate transport system has, however, been demon-

strated in mammalian lysosomes, although its molecular

basis has not been elucidated (Barrueco and Sirotnak, 1991).

The model also envisions monoglutamate export from the

vacuole via a second transporter or perhaps via a single

transporter that exchanges mono- for polyglutamates. Nei-

ther the import nor export carrier appears to be identical

with the ATMRP1 vacuole transporter, since the latter is a

monoglutamate importer (Raichaudhuri et al., 2009).

A second issue is that overexpressing GGH in the cytosol

altered neither the degree of polyglutamylation nor the level

of folate. However, the cytosolic folate pool is relatively small

(Orsomando et al., 2005) and is probably protein bound, as in

animals. In animal cells, the concentration of folate-utilizing

enzymes is roughly equivalent to that of folates, implying a

very low proportion of free, unbound folate (Schirch and

Strong, 1989). Therefore, cytosolic GGH may simply have

been denied access to cytosolic folates, so that the rate of

deglutamylation was negligible. That little if any cytosolic

deglutamylation occurred as a result of cytosolic GGH

expression is supported by the observation that cytosolic

expression did not affect vacuolar folate polyglutamylation

or folate content. Assuming that vacuoles acquire polyglu-

tamates from the cytosol (Figure 8), a cytosolic polygluta-

mate deficit would have been mirrored in vacuoles.

Vacuole Cytosol

GGH

Folate-Glun

Folate-Glu1

Folate-Glun

2

3

Folate-Glun

Folate-Glu1

1

4

6

7

5

FPGS

Figure 8. A model for the role of c-glutamyl hydrolase (GGH) in folate

polyglutamate homeostasis.

Polyglutamates from the cytosol are continuously imported (1) into the

vacuole by a transport system. Vacuolar polyglutamates are either hydrolyzed

by GGH to the corresponding monoglutamates (2) or sequestered from GGH

attack and serve as potential storage forms of the cofactor (3). A second

transport system (4) exports monoglutamates from the vacuole to the cytosol

where they are routed to organelles (5) or polyglutamylated by folylpolyg-

lutamate synthetase (FPGS) (6). Polyglutamates represent the preferred

substrates for folate-dependent enzymes (7).

Monoglutamates

AtGGH2-FL

Wild type

AtGGH1RNAi

ggh2-1

Glu1 Glu2 Glu3 Glu4 Glu5+

Polyglutamates

Percent of 5-CH3-THF020406080100 20 40 60 80 100

0

2

4

6

8

10F

olat

e (n

mol

mg–

1 pr

otei

n)

THF 5-CH3-THF

5, 10 = CH + 10-CHO-THF

10-CHO-DHF

5-CHO-THF

*

*

(a)

(b)

Figure 7. Folate content and polyglutamylation status in Arabidopsis leaf

vacuoles.

(a) Total vacuolar folate content [tetrahydrofolate (THF), 5-methyl-THF (5-CH3-

THF), 10-formyl-dihydrofolate (10-CHO-DHF), 5-formyl-THF (5-CHO-THF),

10-formyl-THF (10-CHO-THF), 5,10-methenyl-THF (5,10 = CH-THF)] in the wild

type, c-glutamyl hydrolase (GGH2) overexpressing lines (FL-GGH2), and RNAi

lines (AtGGH1RNAi ggh2-1) with <1% of normal GGH activity.

(b) Vacuolar polyglutamyl tail length distribution. Data show the percentages

of vacuolar 5-CH3-THF that were polyglutamylated (2–6 glutamate residues) or

in the monoglutamyl form in the plants described above. Data are the

means � SE from three to six independent preparations from 4-week-old

rosette leaf tissue; asterisks indicate significant difference (P < 0.05, t-test)

compared with wild type.



A third and particularly enigmatic issue is why any

polyglutamates at all survive in the vacuole alongside

GGH, since vacuolar GGH activity in our experiments with

Arabidopsis (approximately 7 nmol min)1 mg)1 protein) is

sufficient to hydrolyze all vacuolar polyglutamates within

seconds, as was found in other studies (Orsomando et al.,

2005). Moreover, although tripling vacuolar GGH activity in

Arabidopsis lowered vacuolar polyglutamate levels, it did so

by only 19%. These observations imply that vacuolar

polyglutamates are in some way substantially protected

from attack by resident GGH. There is no evidence for plant

folate-binding proteins analogous to those in animals, for

vacuolar GGH inhibitors, or for rapid intravacuolar resyn-

thesis of polyglutamyl tails (Henderson, 1990; Scott et al.,

2000; Orsomando et al., 2005). An attractive alternative

possibility is that vacuolar folates are partially sequestered

in intravacuolar inclusions, such as those described for

anthocyanins (Pecket and Small, 1980).

Finally, the relationship established between polygluta-

mate tail length and folate content in this study is relevant to

metabolic engineering (‘biofortification’) of folate content in

plants (Bekaert et al., 2008). The strategies used to date have

all targeted the initial steps of the biosynthesis pathway, and

have not deliberately sought to manipulate polyglutamyla-

tion. In fact, however, engineering efforts in tomato and rice

resulted in enhanced folate content predominantly in mono-

glutamyl forms (Dıaz de la Garza et al., 2007; Storozhenko

et al., 2007). In theory, suppressing GGH activity could be

combined with engineering the synthesis pathway to pro-

mote vacuolar storage of polyglutamates.

EXPERIMENTAL PROCEDURES

Plant material and growth conditions

Wild-type Arabidopsis thaliana (Col-0 ecotype) seeds were fromLehle Seeds (http://www.arabidopsis.com/) and tomato (Solanumlycopersicum cv. Micro-Tom) seed were from the Ball seed com-pany (http://www.ballhort.com/). Arabidopsis and tomato plantswere grown in potting soil supplemented with Osmocote� in agrowth chamber at 22–25�C under a 12-h photoperiod (approxi-mately 80 and 200 lmol m)2 sec)1 fluence rate, respectively). Ara-bidopsis roots were obtained from liquid cultured plants grown inhalf-strength Murashige and Skoog medium containing 1% sucrose(Prabhu et al., 1996). Red and red-ripe tomato fruit were defined asbreaker plus 3 and 7 days, respectively.

Identification of Arabidopsis T-DNA mutants

Two GGH1 mutant lines, SALK 018537 and SAIL 258C07, and oneGGH2 mutant line, SALK 145009, were from the ArabidopsisBiological Resource Center (ABRC; http://abrc.osu.edu/). The GGH2mutant T-DNA insertion line GABI-Kat 109A04 was providedby Bernd Weisshaar (Max-Planck-Institute for Plant BreedingResearch, Cologne, Germany). Wild-type or homozygous mutantsegregants were identified by PCR analysis using gene-specificprimers flanking the T-DNA insertion and a T-DNA-specific primer.Genomic DNA was extracted according to Edwards et al. (1991).The insertion site was confirmed by sequencing the PCR ampliconobtained from screened mutant homozygotes. In each line, PCR

targeting the selectable marker gene (nptII for SALK lines, bar forSAIL lines, or dhps for GABI-Kat lines) on the T-DNA failed toamplify any products in wild-type segregants indicating theabsence of secondary T-DNA insertions. Homozygous mutantsand wild-type segregants were selfed and their progeny was usedfor experiments. All primers are listed in Table S1 in SupportingInformation.

Plasmids and constructs

Standard molecular methods were used to generate overexpressionand RNAi constructs, as summarized below. The pFMV, pHK1001,and pMON10086 vectors were kindly provided by Dr H. J. Klee(University of Florida, Gainesville, FL, USA); Arabidopsis andtomato cDNAs were as previously described (Orsomando et al.,2005; Akhtar et al., 2008). RNAi constructs targeting AtGGH1 andAtGGH2 were prepared as follows. From GGH1 cDNA, two PCRproducts corresponding to bases 532–863 and 532–1068 of the openreading frame (ORF) were amplified by high-fidelity PCR withprimers 1AB-5¢/1AB-3¢ and 1AC-5¢/1AC-3¢, respectively. The PCRproducts were digested with KpnI and ligated in a sense/antisenseorientation to form the hairpin construct targeting AtGGH1. Like-wise, primers 2AB-5¢/2AB-3¢ and 2AC-5¢/2AC-3¢ were used to PCR-amplify two fragments from AtGGH2 cDNA (corresponding to bases232–518 and 232–721 of the ORF, respectively), digested with KpnIand ligated to form the hairpin construct targeting AtGGH2. Eachhairpin was digested with BamHI/XbaI and ligated into the corre-sponding sites of pFMV, which places each hairpin under the con-trol of the constitutively active figwort mosaic virus (FMV) promoterand nopaline synthase (nos) terminator. To overexpress AtGGH2,the full-length AtGGH2 cDNA (At1g78680) was amplified usingprimers AtG2FLF/AtG2FLR, digested with NcoI/XhoI and ligatedinto the corresponding sites in pET28b (Novagen, http://www.emdchemicals.com/), which adds a C-terminal His6 epitope to thecorresponding protein. Using this construct as a template, a C-ter-minally tagged His6 full-length AtGGH2 sequence was amplifiedusing HISFLG2F/HISFLG2R, digested with XbaI/KpnI, and ligated tothe corresponding sites of pFMV. To overexpress a truncated ver-sion of AtGGH2 without the signal peptide, the short version of thiscDNA as described by Orsomando et al. (2005) was used as a tem-plate to amplify a N-terminally His6-tagged cDNA with FMVATG2F/FMVATG2R primers. This amplicon was digested with XbaI/KpnIand ligated to the corresponding sites of pFMV. The pFMV con-structs, containing either RNAi or overexpression cassettes, weredigested with NotI and ligated to the NotI-digested binary vectorpHK1001 and subsequently introduced into Agrobacterium tum-efaciens (ABI strain) by electroporation. To overexpress LeGGH2 intomato, full-length and truncated versions of LeGGH2 (Akhtar et al.,2008) were amplified using primers FLLEGGH2F/FLLEGGH2R orTRCLEGGH2F2/FLLEGGH2R, digested with NotI/AscI, and ligatedinto the corresponding sites of pMON10086. Each construct wasintroduced into A. tumefaciens (ABI strain) by electroporation.

Arabidopsis and tomato transformation

Transformation of wild-type or individual Arabidopsis GGH mutantswas performed by the ‘floral-dip’ method (Clough and Bent, 1998;Zhang et al., 2006). Kanamycin-resistant seedlings were recoveredfrom surface-sterilized T1 seeds plated on solid Murashige andSkoog medium containing 1% sucrose and 0.6% agar supplementedwith 50 mg L)1 kanamycin. T2 progenies segregating 3:1 (kanamy-cin-resistant:kanamycin-sensitive) were identified and resistantplants were screened for GGH activity (see below). Several inde-pendent lines with enhanced GGH activity were selfed and homo-zygous T3 plants exhibiting 100% resistance on selection medium



were used for analysis. Agrobacterium-mediated tomato transfor-mation was performed according to the method of Clough and Bent,1998. Transformants were selected and regenerated on mediumsupplemented with 100 mg L)1 kanamycin. Plantlets containingthe ProE8::LeGGH2-FL or ProE8::LeGGH2-TR construct wereidentified by PCR performed on genomic DNA prepared usingprimers located in the E8 promoter and within the coding region ofLeGGH2 (see Table S1). Independent primary transformants weretransferred to potting soil and those with T0 fruit exhibiting signif-icantly higher GGH activity were identified and selfed. Transgenic T1

plants were confirmed by PCR as above, and T1 fruit was used foranalysis.

GGH activity assay

Plant tissues were pulverized in liquid nitrogen and extracted in100 mM potassium phosphate, pH 6.0, containing 10% (v/v) glyc-erol, 10 mM b-mercaptoethanol, and 3% (w/v) polyvinylpyrrolidone(PVP-40). After centrifugation (20 000 g, 4�C, 20 min), supernatantswere desalted on PD-10 columns (Amersham Biosciences, http://www.gelifesciences.com) equilibrated in extraction buffer, withoutPVP-40. Protein was estimated by the method of Bradford (1976).The GGH assays were performed using 0.1 mM folic acid pentag-lutamate (PteGlu5) substrate as previously described (Orsomandoet al., 2005). The GGH activity was calculated as the sum of allproducts (from PteGlu1 to PteGlu4), linearly accumulating as afunction of time. The extent of hydrolysis and the relative concen-tration of each PteGlu reaction product were calculated and plottedaccording to Orsomando et al. (2005). The slope of the line corre-sponding to each product measures the relative extent of its for-mation, thus indicating the c-glutamyl bond specificity of theenzyme.

Quantitative real-time RT-PCR

Absolute quantification of individual GGH gene expression duringdevelopment was performed on the Applied Biosystems Gene-Amp 5700 PCR system (ABI, http://www.appliedbiosystems.com/).Total RNA from Arabidopsis tissues was extracted using theRNeasy Plant Mini Kit (Qiagen, http://www.qiagen.com/), andtreated with DNase (DNA-free� Kit, Ambion, http://www.ambi-on.com/) according to the manufacturer’s instructions. The RT-PCRreactions (25-ll final volumes) included the Taq-Man One-Step RT-PCR Master Mix reagents (ABI), plus 250 ng total RNA, 300 nM ofeach primer, and 150–250 nM of Taq-Man probe. Primers and Taq-Man probes were designed using the Applied Biosystems PRIMER

EXPRESS software. Standard curves for each GGH amplicon wereconstructed according to Goyer et al. (2004). Plasmid templateswere from RZPD (Deutsches Ressourcenzentrum fur Genomfors-chung GmbH, Berlin, Germany) for GGH1 (clone IDMPIZp2001J243Q, GenBank CF653065) and from ABRC for bothGGH2 (clone IDs 89D12, GenBank AY096428) and GGH3 (clone IDU12630, GenBank BT000554). In vitro transcription in the presenceof [5,6-3H]UTP was performed using the MAXIscript� kit (Ambion),according to the manufacturer’s instructions. Controls withoutreverse transcriptase were run in parallel to exclude genomic DNAcontamination. Relative quantification of GGH gene expression intransgenic Arabidopsis plants was conducted using an AppliedBiosystems 7300 Real-time PCR system (ABI). Total RNA wasextracted from 4-week-old rosette leaf tissue as above, except thatDNase treatment was performed using the RNase-Free DNase Set(Qiagen). The RNA samples were reverse transcribed using theHigh-Capacity cDNA Reverse Transcription Kit (ABI) with randomhexamers. The PCR employed ABI universal cycling conditionsusing SYBR GREEN PCR Master Mix (ABI) in a 25 ll reaction con-taining 6.25 ll of diluted cDNA (1:20) and a 300 nM concentration of

each primer. The UBI10 gene (At4g05320) served as an internalcontrol and expression values were calculated according to the2)DC¢T method (Livak and Schmittgen, 2001). SemiquantitativeRT-PCR was also performed on cDNA prepared as described aboveunder the following cycling conditions: 95�C for 10 min, 35 cyclesof 95�C for 1 min, 4�C or 58�C (depending on primer pairs for GGH1or GGH2, respectively) for 1 min, 72�C for 1 min, then 72�C for10 min. All primers are listed in Table S1.

Vacuole isolation and immunoblot analysis

Arabidopsis rosette leaf vacuoles were isolated as described byRobert et al. (2007), with the following modifications. Leaf tissuewas digested in 30-cm Petri plates containing protoplast enzymesolution without vacuum infiltration for a period of 3–4 h. Protop-lasts were recovered by passing digested tissue through a 70-lM

nylon mesh (Spectrum Laboratories, http://www.spectrumlabs.com/). The vacuolar marker enzyme, a-mannosidase, was assayedas described (Nok et al., 2000) to assess the relative enrichment andpurity of each vacuole preparation relative to the protoplasts fromwhich they were isolated. For immunoblot analysis, proteins from asingle vacuole preparation (approximately 8–10 lg) were precipi-tated using trichloroacetic acid and applied to pre-cast 2-amino-2-(hydroxymethyl)-1,3-propanediol (TRIS)-HCl gels formulated to15% acrylamide (Bio-Rad, http://www.bio-rad.com/). Followingelectrophoretic separation, proteins were transferred to polyviny-lidene difluoride membranes (Immobilon P, Millipore, http://www.millipore.com/) using a Trans-Blot SD Semi-Dry transfer system(Bio-Rad). Transfer efficiency was verified by staining the mem-branes with Ponceau S (Sigma, http://www.sigmaaldrich.com/).Blotted membranes were incubated in StartingBlock BlockingBuffer (Pierce Biotechnology, http://www.piercenet.com/) in TRIS-buffered saline (TBS; 20 mM TRIS–HCl, 150 mM NaCl, pH 7.5) con-taining 0.05% Tween 20 for 1 h, washed in TBS and incubatedovernight with anti-His5 antibody (1:1000) (Qiagen) in TBS at 4�C.Membranes were then washed in TBS containing 0.05% Tween 20(TBS-t) and incubated with horseradish peroxidase (HRP)-conju-gated anti-mouse IgG (1:2500) (Affinity Bioreagents, http://www.bioreagents.com/) for 1 h. Membranes were washed in TBS-t,rinsed in TBS, and HRP activity was detected using the ImmobilonWestern Chemiluminescent HRP Substrate (Millipore), according tothe manufacturer’s instructions. The chemiluminescent signal wascaptured with an Alpha Innotech Fluorchem SP imager (AlphaInnotech, http://www.alphainnotech.com/).

Folate analysis

Folates were extracted from leaf tissue (approximately 0.5 g) andred-ripe tomato fruit (approximately 2 g) by Polytron homogeniza-tion in 10–15 ml of 50 mM Na-HEPES, 50 mM N-cyclohexyl-2-aminoethanesulfonic acid (CHES), adjusted to pH 7.85 with HCl,containing 2% (w/v) sodium ascorbate, 1 mM CaCl2, and 10 mM

b-mercaptoethanol. Extracts were immediately incubated in a 100�Cwater bath for 10 min and then centrifuged (13,000 g, 15 min).Clarified crude extracts were split in two and treated plus or minus ratplasma conjugase to determine total folate or degree of polyglut-amylation. Folates were purified on folate-binding columns andanalyzed as previously described (Gregory and Toth, 1988). Forvacuolar folate analysis, the folate-binding column was scaled downto 1 ml and washing and elution buffers were reduced proportion-ately. Folates were quantified by HPLC with electrochemical detec-tion as previously described (Goyer et al., 2005; Orsomando et al.,2005). Detector response was calibrated with authentic THF, 5-for-myl-THF, 5-methyl-THF, 5,10-methenyl-THF, and 10-formyl-DHF,obtained from Schircks Laboratories (http://www.schircks.com/).



ACKNOWLEDGEMENTS

We thank Drs Denise Tieman and Mark Taylor for advice on tomatotransformation and construct design, Drs Valeria Naponelli andRocıo Dıaz de la Garza for help with folate analysis, Hazel Lees forArabidopsis vacuole analysis, and Drs Harry Klee and Kevin Folta forcritical reading of the manuscript. This project was supported byNational Science Foundation grant no. MCB-0443709 and by anendowment from the C. V. Griffin, Sr Foundation.

SUPPORTING INFORMATION

Additional Supporting Information may be found in the onlineversion of this article:Table S1. Synthetic oligonucleotides used in this study.Please note: As a service to our authors and readers, this journalprovides supporting information supplied by the authors. Suchmaterials are peer-reviewed and may be re-organized for onlinedelivery, but are not copy-edited or typeset. Technical supportissues arising from supporting information (other than missingfiles) should be addressed to the authors.

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A central role for gamma-glutamyl hydrolases in plant folate homeostasis

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