J. Biol. Macromol., 9(2), 49-62 (2009)

ArticleIdentification and characterization of γ-glutamyltransferase knockout mutants in
Arabidopsis
Rafael Prieto1*, Yoshihiro Nakano2 and Jiro Sekiya1
1

Department of Bioscience and Biotechnology, Faculty of Bioenvironmental Science,

Kyoto Gakuen University, Sogabe-cho, Kameoka, Kyoto 621-8555, Japan.
2

Shikoku Research Center, National Agriculture Research Center for Western Region, NARO,

Zentsuji, Kagawa 765-8508, Japan.

Received February 6, 2009; Accepted March 6, 2009
Arabidopsis mutants harboring T-DNA or transposon insertions in γ-glutamyltransferase(GGT) genes AtGGT1, AtGGT2 and AtGGT3 were characterized.

Gene expression analysis

indicated that the identified atggt1-1, atggt1-2, atggt2-1, atggt2-2 and atggt3-1 plants were
GGT-null mutants.

GGT activity analysis indicated that the atggt1 mutant alleles were defi-

cient in cell wall bound GGT, whereas the two atggt2 mutant alleles and the atggt3 mutant
allele were deficient in soluble GGT.

GSH contents and GSH catabolic rates in wild typeand atggt1 plants suggest that AtGGT1 is involved in GSH catabolism.
Keywords: Arabidopsis, γ-glutamyltransferase, glutathione, glutathione catabolism, mutant
analysisand Escherichia coli, GGTs are well

Introduction
γ-Glutamyltransferase (GGT, EC.

characterized and have been shown to

2.3.2.2.) is found in plants, mammals andpossess several physiologically important

microorganisms.

functions including glutathione (GSH)

In mammals, yeast

catabolism (Taniguchi and Ikeda, 1998;*

Correspo ndi ng aut ho r:
Tel: +81 -7 71- 29-3493
Fax: +8 1-771-29-3429
E- ma il: r apr iet o @kyot ogakuen. ac. jp

Suzuki et al., 1993; Mehdi et al., 2001).
The function of plant GGTs has not yet
been clearly defined, although they areArabidopsis γ-Glutamyltransferase Knockout Mutants

50

thought to be involved in GSH catabol-terization of Arabidopsis GGT knockoutism (Bergmann and Rennenberg 1993;

mutants suggested that GGT is involved

Martin and Slovin, 2000; Shaw et al.,in GSH degradation in the apoplast and2005; Nakano et al., 2006a, Ohka-thema-Ohtsu et al., 2007a,b).

2007a,b; Martin et al., 2007; Grzam et al.,Plant GGTs are classified into so-

vacuole(Ohkama-Ohtsu

et

al.,

2007).

luble GGTs and bound GGTs, the latter

Plant cDNAs encoding heterodi-of which are solubilized by high ionicmeric GGTs that have molecular struc-strength buffer containing 0.5-1 M NaCltures very similar to those of animals and(Nakano et al., 2004).

microorganisms have

Several plants,

been characte-

including radish, Arabidopsis and rice,

rized in Arabidopsis (AtGGT; Storoz-

have both soluble and bound isoforms,

henko et al., 2002; GGT1-3; Ohka-

whereas tobacco and onion have only the

ma-Ohtsu et al., 2007a,b; Martin et al.,bound isoforms (Nakano et al., 2004).

2007; Grzam et

In radish, bound GGT is mainly asso-(AcGGT; Shaw et al., 2005); and radish

ciated with the cell wall (Nakano et al.,(RsGGT1-3; Nakano et al., 2006c).

2004), whereas soluble GGT is mainly

Heterodimeric plant GGTs are composed

localized to the vacuole (Nakano et al.,of one large and one small subunit that2006b).

Taking into account that: a)are thought to be generated by the pro-oxidized glutathione (GSSG) and GSH

teolytic cleavage of a single polypeptide

S-conjugate (GSX) are transported to thechain precursor.

vacuole by ABC-type transporters (Rea,

revealed that, besides AtGGT, which is1999), b) both GSX and GSH are thought

also named At4g39640, in the Arabidop-

to be transported to the extracellular

sis genome there are at least two more

space, and c) a small amount of mainly

genes, which are named At4g39650 and

GSSG are localized to the apoplast (Va-

At4g29210,

nacker et al., 1999; Ohkama-Ohtsu et al.,

GGTs.

2007a, Okawa and Nakano unpublished),tains another potential heterodimeric

the subcellular localization of radish

GGT gene that is named At1g69820.

GGT isoforms is in agreement with a

However, analysis of the At1g69820 de-possible involvement of GGT in GSH

duced amino acid sequence (accession no.

catabolism. According with this, charac-

NP177140) revealed a putative proteinal.,2007); onion

Gene database search

encoding

heterodimeric

The Arabidopsis genome con-

R. Prieto, Y. Nakano and J. Sekiya

51

that apparently lacks approximately 70%

was provided by the Cold Spring Harborof the GGT large subunit.

Laboratory (http://genetrap.cshl.edu). The

The functional characterization of

atggt3-1 transposon insertion mutant

GGT genes is important to understand the(N161036, ecotype Larnsberg erecta, Ler)

physiological role of the GGT isoforms.

was identified from the Exotic database

We have named the At4g39640 cDNA

(http://www.jic.bbsrc.ac.uk/science/cdb/esequence as AtGGT1 (accession no.

xotic) and was provided by the Notting-

Z49240), the At4g39650 cDNA sequence

ham Arabidopsis Stock Centre (Univer-as AtGGT2 (accession no. AK220806)

sity of Nottingham,

and the At4g29210 cDNA sequence as

Bulk seeds were grown on gardening soil

AtGGT3 (accession no. NM119065). In

supplemented

the present study, we describe the identi-(Hyponex Japan) once a month.

fication and characterization of Arabi-

natively, previously sterilized seeds were

dopsis knockout mutants having affected

planted on 1/2 Murashige and Skoog agar

GGT isoforms that may differ in theirmedium supplemented with 1.5% sucrose

functions and cellular localizations.

(MSS).

with

Loughborough).

1/1000

Hyponex
Alter-

After 3 days at 4oC, plants weregrown at 25oC under continuous light.
Materials and Methods
Plant Materials

Genotype Analysis

The atggt1-1, atggt2-1 and atggt2-2
T-DNA

insertion

mutants

Arabidopsis genomic DNA was(SALK_

prepared from 12 to 15 T4 segregants ac-

080363, SALK_069311 and SALK_

cording to Liu et al. (1995). The geno-

147881, ecotype Columbia, Col, Alonsotypes of the T-DNA insertion lines were

et al., 2003) were identified by a homol-

verified by genomic PCR, using theogy search of the Salk Institute Genomic

T-DNA

Analysis Laboratory database (http://(5'-GCGTGGACCGCTTGCTGCAACT-

signal.salk.edu/cgi-bin/tdnaexpress), and3') and AtGGT specific primers.

were provided by the Arabidopsis Bio-

genotype of the atggt1-1 mutant was con-logical Resource Center (Ohio State

firmed using LBb1, At1-5.1 (5’-GCAG-

University, Columbus).

AGAGTCTGAACAATCGCT-3’)

The atggt1-2

leftborderprimer

LBb1

The

and

transposon insertion mutant (CSHL_

At1.2-3.1 (5’-TTCCCCGGGAACGCCT-GT1235, ecotype Larnsberg erecta, Ler)

ACTGA-3’) primers, and the two atggt2

Arabidopsis γ-Glutamyltransferase Knockout Mutants

52

alleleswereverified

using

LBb1,

3’ and 5’-CAAGACTTCTGGGCATCT-

At2-5g.1 (5’-ACATTGACCAACAATA-3’) was used to amplify a 425 bp frag-GATGGA-3’) and At1.2-3.1 primers.

ment of Actin2 cDNA as the internal

On the other hand, the genotype of thestandard of gene expression.

transposon insertion line atggt1-2 was

AtGGT1 cDNA fragment, a 1725 bp

confirmed by genomic PCR, using the

AtGGT2 cDNA fragment, and a 456 bp

transposon primer DS3-2 (5’-CGATTA-AtGGT3 cDNA fragment were amplified

CGCTATTTATCCCGTTC-3’), At1-5.1

using primer sets AtGGT1 (At1-5.3:

and At1.2-3.2 (5’-TCAATATCCTGAA-

5’-GGGATTCCATCAGGTGTTTC-3’

GGGAACC-3’) primers. The genotype ofandthe atggt3 mutant was verified using

5’-ATGTCGCTCGTTCGTACTGC-3’

DS3.2 and the AtGGT3 specific primers

and At1.2-3.2) and AtGGT3 (At3-5.2:

At3-5.1 (5’-CTAGCTCAGAGTCTTGA-

5’-CCTCAACCGTGAATTACCGT-3’

GACT-3’) and At4g29210 3’-2 (5’-CTC-

and At4g29210 3’-2), respectively. The

CACCTGATAGCTCCT-3’).

The PCR

expression of AtGGT genes in atggt mu-

products were analyzed as described in atant genotypes was analyzed by RT-PCR,

previous paper (Nakano et al., 2006c).

using the following primers: At1-5.1 andAt1.2-3.2),

AtGGT2

An 855 bp(At2-5.1:

At1.2-3.1 primers to amplify a 451 bp
AtGGT1

RNA isolation and RT-PCR analysis
Total RNA was prepared from 4

cDNA

fragment,

At1-5.2:

5’-ATGTCGCTGGTTCGAACAGT-3’

week-old soil grown plants using RNeasy

and At1.2-3.2 primers to amplify a 1719

Plant mini kit (QIAGEN, USA) follow-

bp AtGGT1 cDNA fragment, At2-5.1 anding manufacturer’s instructions.

The

At1.2-3.2 primers to amplify a 1725 bp

total RNA was treated with RNase free

AtGGT2 cDNA fragment, and At4g29210

DNase (Takara, Japan) and reverse tran-

5’-UTR

scription was performed using oligo dTG-3’) and At4g29210 3’-2 primers toprimer and ReverTraAce (TOYOBO, Ja-

amplify a 1772 bp AtGGT3cDNA frag-pan) as described by the manufacturer.

ment.

(5’-GAGATAGAGATTGAA-

The synthesized first strand cDNA wastreated with RNase H (Takara).

Enzyme Assay

For RT-PCR, the primer set

Soluble and bound GGTs were ex-

Act2 (5’-GTTGGTGATGAAGCACAA-

tracted from the samples as previously

R. Prieto, Y. Nakano and J. Sekiya

described by Nakano et al. (2004).

53

al., 2004).

Briefly, the samples were homogenizedwith 10 vol. of L buffer (20 mM Tris-HCl,

Measurement of thiol compounds

pH 8.0, 5 mM 2-mercaptoethanol, 1 mM

Thiol compounds were extracted

EDTA and 1 mM PMSF) supplemented

and analyzed from rosette leaves ofwith 5% (w/w) insoluble polyvinylpirro-

3-week-old Arabidopsis plants grown on

lidone (PVP) and centrifuged at 10,000 x

MSS, and from 15+2 mm siliques andg for 10 min.

The supernatant (S-1 frac-dry seeds from soil grown plants as de-tion), which represents soluble plus mi-

scribed elsewhere (Nakano et al., 2006b).

crosomal fractions, was recovered andfurther centrifuged at 100,000 x g for 1 h.

Results and Discussion

After that, the soluble protein fraction

Identification of atggt mutants(S-2) was recovered from the supernatant.

In order to undertake the functional

The pellet (P fraction), which representscharacterization of Arabidopsis GGTthe microsomal fraction, was resuspended

isoforms, we obtained one T-DNA inser-

in L buffer.

The cell debris pellet de-tion mutant and one transposon insertion

rived from the initial 10,000 x g centri-

mutant alleles of AtGGT1, two T-DNA

fugation was washed twice with L buffer

insertion mutant alleles of AtGGT2, andand resuspended in either H buffer (20one transposon insertion mutant allele ofmM Tris-HCl, pH 8.0, 1 M NaCl) or T

AtGGT3 (Fig. 1A).

buffer (20 mM Tris-HCl, pH 8.0, 1%

putative GGT insertion mutants was con-

Triton X-100).

After centrifugation atfirmed by genomic PCR, and homozyg-

10,000 x g for 10 min, the protein frac-ous lines were recovered at the expectedtions extracted with H buffer (CW frac-

1/4 ratio for single gene insertion.

tion) and T buffer (PS fraction) were re-sequencing

covered from their corresponding super-

T-DNA-GGT and transposon-GGT chi-natants.

meric PCR products indicated that 1)of

The genotype of the

the

DNA

corresponding

atggt1-1 had T-DNA inserted in the third
Preparation of Arabidopsis protoplasts

AtGGT1 exon, 2) atggt1-2 had the trans-

Arabidopsis protoplasts were pre-poson inserted in the fifth AtGGT1 exon,

pared and the enzyme activity was meas-3) both atggt2-1 and atggt2-2 mutant al-ured as described previously (Nakano etleles had T-DNA inserted in the second

54

Arabidopsis γ-Glutamyltransferase Knockout Mutants

Fig. 1
A, Genomic structure of Arabidopsis T-DNA and transposon insertion
alleles. Rectangles and lines represent the exons and the introns of putative
GGT genes, respectively. Triangles indicate T-DNA or transposon. AtGGT
RT-PCR expression analysis. B, AtGGT mRNA accumulation in siliques of
atggt mutants and wild type plants. C, Differential expression patterns of the
AtGGT gene family in Arabidopsis organs. The number of PCR cycles is indicated. The figures illustrate representative results from three independent experiments.
AtGGT2 exon, and 4) the transposon in-

On the other hand, RT-PCR gene

sertion in the atggt1-3 mutant allele wasexpression analysis of atggt mutants in-localized to the third AtGGT3 exon (Fig.

dicated that atggt1-1, atggt1-2, atggt2-1,

1A).

atggt2-2, and atggt3-1 showed no de-R. Prieto, Y. Nakano and J. Sekiya

55

tectable AtGGT1, AtGGT2, and AtGGT3

ma-Ohtsu et al., (2007a,b). Besides, themRNA accumulation, respectively, and

AtGGT expression profiles indicate simi-

therefore, they appeared to be null mutant

larities to those of different radish

alleles (Fig. 1B). With the exception ofRsGGT genes (Nakano et al., 2006c), andatggt1, which, as described by Ohka-

suggest that GGT isoforms may play

ma-Ohtsu et al. (2007a) showed increased

various roles at the germination, vegeta-

chlorosis compared with the wild typetive growth and reproductive stages.

(data not shown), the rest of atggt mu-

the other hand, GGT::GUS expression

tants grew normally on soil and MSS

analysis indicated that GGT1 expression

medium.

is limited to the vascular tissue, whereas

On

GGT2 expression is limited to embryo,endosperm, outer integument, and a small

AtGGT gene expression analysis
AtGGT gene expression in different
Arabidopsis organs was analyzed by the

portion of the funiculus in developing
siliques (Ohkama-Ohtsu et al., 2007a).

semi-quantitative RT-PCR method. BothAtGGT1 and AtGGT3 were expressed inDistribution of GGT activity in Arabi-

all the tested organs (Fig. 1C).

dopsisIn con-trast, the AtGGT2 gene was mainly ex-By

low-speed

centrifugationpressed in siliques, at a significant level(10,000 x g for 10 min) of rosette leaf

in flowers, but not in roots, stems or ro-

homogenates, we found that Arabidopsis

sette leaves (Fig. 1C).

GGT activity was localized to both the

These results are

similar to the ones described by Ohka-

supernatant(S-1

fraction),

which

56

Arabidopsis γ-Glutamyltransferase Knockout Mutants

contained soluble and microsomal protein

al bound GGT, in rosette leaves.

As afractions, and the corresponding cell de-consequence, the GGT activity that loca-

bris pellet, which contained the crude cell

lized to the Arabidopsis S-1 fraction will

wall fraction and other cellular compo-

be subsequently referred to as soluble

nents (Nakano et al., 2004; Table 1).

GGT activity.

Here, we proceeded further with the

As in the case of radish bound GGTanalysis of Arabidopsis GGT distribution

activity, which is mainly associated withby conducting high-speed centrifugation

the cell wall (Nakano et al., 2004), the(100,000 x g for 1 h) of the S-1 protein

Arabidopsis GGT activity that localized

fraction in order to obtain soluble protein

to the cell debris pellet could be solubi-

fraction (S-2) and microsomal fraction

lized with high ionic strength buffer con-(P). The results indicate that the GGTtaining 0.5 M NaCl (CW fraction), but

activity of the S-1 fraction was essentially

not with 1% Triton X-100 (PS fraction)

recovered in the S-2 fraction, but not in(Table 1).

the P fraction (Table 1).

These results

dish protoplast (Nakano et al., 2004), cell

are in agreement with the distribution ofwall degradation of Arabidopsis cells also

GGT activity in radish cotyledons (Na-

led to the disappearance of NaCl extract-

kano et al., 2004) and indicate the exis-

able GGT activity but not that of the so-tence of soluble GGT, but not microsom-

luble one (Table 2). These results indi-Moreover, as reported in ra-

R. Prieto, Y. Nakano and J. Sekiya

57

cate that this type of Arabidopsis GGTmutants was not significant compared

activity is associated with the cell wall.

with those of wild type plants (Figs. 2B).

The GGT activity that localized to the

Similar results were found when com-

CW fraction will be subsequently re-paring soluble and bound GGT activities

ferred to as bound GGT activity.

in leaves of wild type and atggt1 plants(data not shown). These results indicate

Fractionation analysis of GGT activity

that AtGGT1 encodes the main Arabi-

in atggt mutants

dopsis bound GGT.

The atggt1 mutant alleles presented

rologous

expression

Based on the heteof

Arabidopsis

soluble GGT activity similar to those of

AtGGT1 cDNA in tobacco plants, it was

wild type plants in siliques (Fig. 2A).

In

proposed that AtGGT1 encodes a hetero-contrast, bound GGT activity in atggt1

dimeric GGT that is localized to theFig. 2
GGT activity in wild type and atggt mutants.
GGT activity in soluble (A) and bound (B) protein fractions was extracted from siliques as described in the manuscript. Values are means+SD (n=3).

Arabidopsis γ-Glutamyltransferase Knockout Mutants

58

plasma membrane outside tobacco trans-2007).

In contrast, Ohkama-Ohtsu et al.

genic cells (Storozhenko et al., 2002).

(2007a) suggested that, in a similar way

However, our results clearly indicate thatto AtGGT1, AtGGT2 is an apoplastic

AtGGT1 is not localized to the Arabi-

enzyme that is only partially extracted

dopsis plasma membrane; rather, it isunder low-salt conditions.

likely localized to the cell wall, similar to

As illustrated in Fig. 2A, atggt3-1

radish bound GGT (Nakano et al., 2004).

and wild type plants had similar levels of

These results agree with those of Martinbound GGT activity in siliques.

etsuggested thattrast, GGT soluble activity in the atggt3-1

AtGGT1 is associated with a particulate

knockout plant was approximately 85%

fraction via an ionic interaction, and par-of that in wild type.

tially agree with those published by Oh-

al. (2007b) and Grzam et al. (2007) havekama-Ohtsu et al. (2007a) who proposed

proposed that AtGGT3 is a soluble pro-that AtGGT1 is a protein that may betein that catalyzes the first step in thebound to both the cell wall and the plas-

metabolism of GSH conjugates in thema membrane.

vacuole.

al.

(2007),

who

In con-Ohkama-Ohtsu et

Soluble GGT activity in siliques of
atggt2-1 and atggt2-2 was ca. 25% of
those of wild type plants (Fig. 2A).

GSH degradation analysis

In

Ohkama-Ohtsu et al. (2007a) havecontrast, bound GGT activity was iden-suggested that AtGGT1 is important intical in wild type and atggt2 mutants (Fig.

preventing oxidative stress by metaboliz-

2B).

Results indicate that AtGGT2 is a

ing extracellular GSSG in the apoplast.

soluble GGT that is mainly expressed in

However, they could not find significant

silique. Analysis of an atggt2 knockup

differences between wild type and atggt1

mutant suggested that AtGGT2 is a so-plants in terms of the GSH levels inluble protein which is extracellularly lo-

leaves, roots, stems, flowers, flower buds

calized when expressed in leaves (Martinand siliques.

et al., 2007). On the other hand, analysis

termination of GSH contents revealedof atggt2 knockout mutants suggested

that there were no significant differences

that, when expressed in siliques, AtGGT2in the steady-state levels of GSH in ro-

is membrane bound or soluble but asso-

sette leaves, siliques and mature seeds

ciated with storage bodies (Martin et al.,between atggt1 and wild type plants (dataIn a similar way, our de-R. Prieto, Y. Nakano and J. Sekiya

not shown).

However, these results may

presence

59ofL-buthionine-[S,be explained by the low GSH degradationR]-sulfoximine (BSO), a widely used

rate in atggt1 mutants that might be

GSH biosynthesis inhibitor that targets

compensated by a decrease in the GSH

γ-L-glutamyl-

biosynthetic rate.

In order to verify such

Rosette leaves of non-treated wild typea possibility, we monitored the timeand atggt1-1 plants presented similarcourse of GSH degradation in the

GSH contents (Fig. 3A).

L-cystein

Fig. 3
GSH (A) and cysteine (B) contents in Arabidopsis leaves. Rosette leaves of MSS grown wild
type (circles) and atggt1-1 mutant (squares) were excised and transferred onto either MSS (closed symbols) or MSS supplemented with 1 mM BSO (opensymbols) and incubated for 4-8 h at 25oC under continuous light. Values are means+SD (n=3).

synthetase.

In contrast,

Arabidopsis γ-Glutamyltransferase Knockout Mutants

60

after 8 h of BSO treatment, the GSH

the Promotion of Science to R.P.

contents in wild type and atggt1-1 leaves
gradually decreased to 44% and 67% of
the contents of the untreated counterparts.
We could not detect a significant amount

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Communicated by Ishijima Sumio