the plant journal a perturbation in glutathione ...a perturbation in glutathione biosynthesis...

A perturbation in glutathione biosynthesis disruptsendoplasmic reticulum morphology and secretory membranetraffic in Arabidopsis thaliana

Kenneth K. C. Au1,†, Jose Perez-Gomez1,†, Helia Neto1,‡, Christopher Muller2, Andreas J. Meyer2,§, Mark D. Fricker1 and Ian

Moore1,*

1Department of Plant Sciences, University of Oxford, South Parks Rd, Oxford OX1 3RB, UK, and2Heidelberg Institute of Plant Sciences, University of Heidelberg, Im Neuenheimer Feld 360, D-69120 Heidelberg, Germany

Received 11 January 2012; revised 20 March 2012; accepted 22 March 2012; published online 12 July 2012.

*For correspondence (e-mail [email protected]).†These authors contributed equally to this work.‡Present address: The Henry Wellcome Laboratory for Cell Biology, College of Medical Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK.§Present address: INRES – Chemical Signalling, University of Bonn, Friedrich-Ebert-Allee 144, D-53113 Bonn, Germany.

SUMMARY

To identify potentially novel and essential components of plant membrane trafficking mechanisms we

performed a GFP-based forward genetic screen for seedling-lethal biosynthetic membrane trafficking mutants

in Arabidopsis thaliana. Amongst these mutants, four recessive alleles of GSH2, which encodes glutathione

synthase (GSH2), were recovered. Each allele was characterized by loss of the typical polygonal endoplasmic

reticulum (ER) network and the accumulation of swollen ER-derived bodies which accumulated a soluble

secretory marker. Since GSH2 is responsible for converting c-glutamylcysteine (c-EC) to glutathione (GSH) in

the glutathione biosynthesis pathway, gsh2 mutants exhibited c-EC hyperaccumulation and GSH deficiency.

Redox-sensitive GFP revealed that gsh2 seedlings maintained redox poise in the cytoplasm but were more

sensitive to oxidative challenge. Genetic and pharmacological evidence indicated that c-EC accumulation

rather than GSH deficiency was responsible for the perturbation of ER morphology. Use of soluble and

membrane-bound ER markers suggested that the swollen ER bodies were derived from ER fusiform bodies.

Despite the gross perturbation of ER morphology, gsh2 seedlings did not suffer from constitutive oxidative ER

stress or lack of an unfolded protein response, and homozygotes for the weakest allele could be propagated.

The link between glutathione biosynthesis and ER morphology and function is discussed.

Keywords: secGFP, UPR, reticulon, roGFP, rml 1, GSH1, BIP, calreticulin, Arabidopsis thaliana

INTRODUCTION

Membrane trafficking is a tightly regulated and complex

process that transports macromolecules such as proteins,

polysaccharides, and lipids between endomembrane

organelles and plasma membrane. The biosynthetic and

trafficking functions of the secretory pathway organelles are

responsible for some of the most biologically important

functions of plant cells, such as the biogenesis of the plasma

membrane, vacuoles, cell walls, and cell plate. Membrane

trafficking involves the formation of a variety of specific

transport vesicles that package transported macromolecules

at one membrane compartment, followed by fusion of these

vesicles to a target membrane. This process can result in

either exchange between stable and biochemically distinct

compartments or in maturation of membrane compart-

ments over time (Huotari and Helenius, 2011). Much of our

fundamental knowledge comes from forward genetic stud-

ies in yeast, which have played a key role in uncovering

conserved regulators of the secretory pathway (Novick and

Schekman, 1979).

Although the basic molecular framework of the secretory

pathway is similar across eukaryotes, molecular and cyto-

logical studies suggest that plant cells have acquired

distinctive features in various aspects of membrane traffick-

ing, for example in relation to cell polarity and cytokinesis

(Bednarek and Falbel, 2002; Chow et al., 2008; Dettmer and

Friml, 2011; Muller et al., 2003). The existence of plant-

specific trafficking activities is also supported by phylo-

genetic studies of plant genomes which have shown extensive

diversification of important membrane trafficking protein

families such as Rab GTPases, Arf-guanine-nucleotide

ª 2012 The Authors 881The Plant Journal ª 2012 Blackwell Publishing Ltd

The Plant Journal (2012) 71, 881–894 doi: 10.1111/j.1365-313X.2012.05022.x

exchange factors (Arf-GEFs), and soluble N-ethylmaleimide-

sensitive factor attachment protein receptors (SNAREs)

(Kienle et al., 2009; Richter et al., 2007; Sanderfoot, 2007;

Woollard and Moore, 2008). In some cases, for example the

Arabidopsis syntaxin KNOLLE or the Arf-GEFs GNOM and

GNOMLIKE1 (GNL1), this reflects plant-specific neofunction-

alization (Muller et al., 2003; Richter et al., 2007; Teh and

Moore, 2007). Mutational studies have revealed significant

functional redundancy in many of these gene families,

however, which complicates reverse genetic analysis (Ebine

et al., 2011; Pinheiro et al., 2009; Sanderfoot, 2007). Conse-

quently, to identify genes with important functions in plant

membrane trafficking, several laboratories have used for-

ward genetic approaches employing fluorescent proteins as

trafficking markers in Arabidopsis thaliana.

Screens for mutants in which organelle markers showed

atypical localization have identified components that regu-

late organelle morphology (Faso et al., 2009; Matsushima

et al., 2003; Nakano et al., 2009; Rojo et al., 2001) or protein

trafficking in the endomembrane system (Agee et al., 2010;

Fuji et al., 2007; Tanaka et al., 2009; Teh and Moore, 2007).

We previously described the use of a secreted green

fluorescent protein (secGFP) as a marker to identify mem-

brane trafficking mutants in Arabidopsis (Teh and Moore,

2007; Zheng et al., 2004). Secreted GFP does not accumulate

in fluorescent form when secreted to the apoplast in wild-

type plants, but in mutants that perturb the secretory

pathway fluorescent secGFP accumulates in the cytoplasmic

organelles along the pathway and enhanced seedling fluo-

rescence is observed (Teh and Moore, 2007; Zheng et al.,

2004). Previous forward screens for membrane trafficking

mutants have identified viable plants, but it is to be expected

that perturbation of essential secretory functions will be

lethal in plants as in yeast (Novick and Schekman, 1979). It

has been argued that seedling-lethal mutants are more likely

than embryo-lethals to identify plant-specific functions that

underpin development rather than functions required sim-

ply for cell maintenance (Berna et al., 1999; Jurgens et al.,

1991). We reasoned that the same may be true of plant-

specific aspects of membrane trafficking where conserved,

core, eukaryotic trafficking functions are more likely to

mutate to embryo or gamete lethality. In support of this,

insertion mutants in conserved SNARE or Rab GTPases have

been shown to mutate to embryo- or gamete-lethal pheno-

types once redundancy is eliminated (e.g. Surpin et al., 2003;

Pinhiero et al., 2009) while the plant-specific trafficking loci

KNOLLE and GNOM were initially identified as seedling-

lethal mutants (Jurgens et al., 1991).

Here we report the use of secGFP and improved ratio-

metric fluorescent secretory markers (Samalova et al., 2006)

in a screen for seedling-lethal secretory mutants. We

describe the identification of four allelic mutations that

reveal an unexpected link between endoplasmic reticulum

(ER) organization and glutathione (GSH) biosynthesis. Glu-

tathione is a tripeptide composed of glutamate, cysteine,

and glycine and is the most abundant small thiol molecule

in cells with cytosolic concentration of 2–3 mM (Meyer et al.,

2001). It serves a diverse range of cellular functions, includ-

ing cell division (Vernoux et al., 2000), heavy metal detox-

ification (Cobbett et al., 1998), and regulation of disulfide

bond formation in the ER (Chakravarthi et al., 2006). Most

importantly, GSH plays a major role in maintenance of the

redox state and oxidative stress signaling (Meyer, 2008;

Noctor et al., 2000). Glutathione biosynthesis is a two-step

process that first combines glutamate and cysteine to form

gamma-glutamylcysteine (c-EC), followed by the addition

of glycine to form GSH. These two steps are catalyzed by

glutamate-cysteine ligase (GSH1) and glutathione synthase

(GSH2), respectively. Mutant alleles of GSH1 and GSH2 have

been described previously (Ball et al., 2004; Cairns et al.,

2006; Cobbett et al., 1998; Dubreuil-Maurizi et al., 2011; Jobe

et al., 2012; Parisy et al., 2007; Pasternak et al., 2008; Shan-

mugam et al., 2012; Vernoux et al., 2000) but their effects on

endomembrane function were not documented. Here we

show that ER morphology, but not protein folding, is

unexpectedly sensitive to a perturbation in the final step of

the GSH biosynthesis pathway.

RESULTS

To identify secretory membrane trafficking mutants we

screened for enhanced intracellular fluorescence in seed-

lings expressing the secGFP reporter (Teh and Moore, 2007;

Zheng et al., 2004) or the ratiometric secretory trafficking

markers nlsRm-2A-secGFPf and STN-Rm-2A-secGFPf

(Samalova et al., 2006). The latter two markers had the

advantage that instead of comparing absolute secGFP fluo-

rescence intensities, which can be affected by variation in

secGFP expression or imaging efficiency, putative mutants

could be identified by comparing the intensity ratio of sec-

GFP and a stoichiometrically expressed nuclear- (nlsRm) or

Golgi-targeted (STN-Rm) red fluorescent protein (RFP) mar-

ker. In previous screens for secGFP accumulators (Teh and

Moore, 2007), the brightest mutants were seedling lethal and

attempts to isolate temperature-sensitive alleles yielded few

mutants, so to recover the seedling-lethal mutations we

screened individual M2 families for secGFP accumulators

and propagated recessive mutations via heterozygous sib-

lings (see Experimental Procedures).

Four mutants accumulated secGFP in swollen bodies

We selected four independent mutants – G4, N37, S6, and

S10 (Figure 1a)-for detailed study because they all mapped

to the same arm of chromosome V and confocal microscopy

revealed that they each accumulated secGFP in swollen

bodies 5–50 lm in diameter (Figure 1b). G4 was isolated

from a line expressing STN-Rm-2A-secGFPf, N37 from a line

expressing nlsRm-2A-secGFPf, and S6 and S10 from secGFP

line S76 (Zheng et al., 2004). Complementation tests and

882 Kenneth K. C. Au et al.

ª 2012 The AuthorsThe Plant Journal ª 2012 Blackwell Publishing Ltd, The Plant Journal, (2012), 71, 881–894

subsequent sequencing showed that all mutations were

allelic (see below and Figure 4).

Although the mutants showed similar secGFP accumula-

tion phenotypes, they exhibited widely different develop-

mental abnormalities after 7 days of growth (Figure 1a). G4

and S10 terminated growth soon after germination, while

N37 had severely reduced root growth and died within

3 weeks. Despite the gross disruption of ER morphology, S6

was surprisingly similar to the wild type (WT) in morphology

and was viable, though it was always smaller and produced

far fewer seeds.

Swollen bodies accumulate ER markers and some mem-

brane markers

Various fluorescent-protein-tagged markers of the endo-

membrane system were expressed in the N37 mutant

background after the original secGFP marker had been

removed by segregation following a backcross. The ER

lumen marker (GFP-HDEL) (Boevink et al., 1998; Crofts et al.,

1999) was observed exclusively in the swollen bodies rather

than the typical tubular fusiform bodies and polygonal ER

network of WT siblings (Figure 2a,b).

To further test the hypothesis that these structures were

derived from the ER, an ER membrane marker, DER2-YFP (L.

Frigerio, University of Warwick, UK, pers. comm.), was

co-expressed with the ER lumen marker GFP-HDEL. In WT,

the ER network was labeled by both DER2-YFP and GFP-

HDEL and there was a high degree of colocalization at the ER

network (Figures 2k and S1a,b in Supporting Information).

In N37, both markers labeled the swollen bodies, with

GFP-HDEL in the lumen and DER2-YFP at the perimeter

(Figures 2l and S1c). Occasionally, DER2-YFP was seen to

label a tubular ER network (Figure S1d), suggesting the

presence of a residual ER network in N37 despite its failure to

label with GFP-HDEL.

Interestingly, the trafficking of Golgi (ST-RFP) and trans-

Golgi-network (TGN) markers (VHA-a1:GFP) (Dettmer et al.,

2006) and the morphology of these compartments in

confocal laser scanning microscopy appeared unaffected

in the N37 mutant (Figure 2g–j). In contrast, the tonoplast

(BobTIP-GFP) (Reisen et al., 2003) and plasma membrane

proteins (PIP-GFP) (Cutler et al., 2000) were trapped in the

membrane of swollen bodies in N37 (Figure 2c–f). The fact

that these two markers exhibited significant accumulation

in the ER, even in WT, suggests they were exported

relatively inefficiently from the ER and accumulated in

swollen ER bodies. The lipophilic styryl dye FM4-64 was

used to label the endosomes and tonoplast (Bolte et al.,

2004; Dettmer et al., 2006; Ueda et al., 2001). After 60 min of

incubation, FM4-64 labeled the tonoplast of N37 and WT

seedlings, suggesting that the endocytic pathway was not

perturbed by the mutation (Figure 2m). Notably, FM4-64 did

not completely colocalize with BobTIP-GFP, which was

clearly trapped in the membrane of swollen bodies. This

(a)

(b)

Figure 1. Four putative membrane trafficking mutants of similar phenotype identified in a seedling lethal screen.

(a) Bright field images of 7-day-old seedlings of the wild-type secGFP line (S76), and the four mutants S6, N37, G4 and S10.

(b) Confocal images of root epidermal cells of the same genotypes as in (a). Green represents secGFP; red in G4 represents a Golgi marker (ST-RFP).

Bars = 1 cm in (a) and 50 lm in (b).

Arabidopsis glutathione synthase mutant and ER morphology 883


(a) (b) (k)

(l)

(m)

(n)

(o)

(c) (d)

(e) (f)

(g) (h)

(i)

(p)

(j)

Figure 2. Fluorescent organellar markers in wild-type (WT) and mutant seedlings.

Confocal images (a–o) of fluorescent organellar markers.

(a–h) Epidermal root cells of wild type (a, c, e, and g) and N37 (gsh2-5) (b, d, f, and h) expressing either an endoplasmic reticulum marker (GFP-HDEL) (a and b), a

tonoplast marker (BobTIP-GFP) (c and d), a plasma membrane marker (PIP-GFP) (e and f) or a trans-Golgi network marker (VHA-a1:GFP) (g and h). (i, j) WT (i) and G4

(gsh2-4) (j) expressing ST-RFP-2A-secGFP. ST-RFP-2A (Golgi and vacuole) and secGFP are represented in red and green, respectively. (k, l) GFP-HDEL (green) and

DER2-YFP (red) in WT (k) and N37 (gsh2-5) (l). Refer to Figure S1 for higher-resolution images. (m) N37 (gsh2-5) expressing BobTIP-GFP (green) and stained for

60 min with FM4-64 (red). Arrows indicate points at which the FM4-64 labeled tonoplast is not appressed to the swollen bodies that are labeled by BobTIP-GFP. (n),

(o) Wild-type (n) and N37 (gsh2-5) (o) expressing mCHERRY-HDEL (red) and incubated in 100 lM monochlorobimane for 15 min. The fluorescence of thiol-

conjugated bimane is shown in green. (p) Immunoblot analysis with anti-GFP antibodies of proteins extracted from untransformed control plants (lane 1), WT and

mutant siblings of N37 (lanes 2 and 3, respectively), S6 mutants (lane 4), and the mutagenized parental line secGFP(S76) (lane 5). Black and white arrowheads

indicate the position of bands corresponding to truncated and non-truncated secGFP, respectively. Molecular weight markers are indicated on the left.

Bars = 20 lm in (b, d h, l and o); 10 lm in (f); 5 lm in (j and m).



suggests that the endocytic and biosynthetic tonoplast

markers FM4-64 and BobTIP-GFP labeled different compart-

ments in the mutant. To test this further we labeled the

vacuolar lumen using monochlorobimane (MCB), which

becomes fluorescent only after it conjugates reduced cyto-

solic thiols, principally GSH in WT cells. The conjugated

product, glutathione-S-bimane (GSB), is then actively trans-

ported to the vacuole (Fricker et al., 2000). In N37, fluores-

cent bimane-labeled vacuoles did not colocalize with the ER

luminal marker mCHERRY-HDEL (Nelson et al., 2007) in the

swollen bodies (Figure 2n,o). Hence, we concluded that the

swollen bodies were distinct from vacuoles. This conclusion

is supported by immunoblot analysis (Figure 2p), which

showed that in N37 and S6, secGFP accumulated in its full-

length form, typical of the ER, rather than the C-terminally

truncated form normally observed in the vacuole or apop-

last (Zheng et al., 2004).

Electron microscopy revealed that swollen bodies in the

epidermal cells at the root tips of the strong allele (S10) and

weaker allele (S6) were surrounded by electron-dense

structures that resembled ribosomes (Figure 3). In younger

cells of S6, we also found that the ER tubules were dilated

compared with the WT (Figure 3g). Similar structures were

also observed with confocal microscopy (Figure 3h–j).

Together, these findings using three different alleles

indicate that ER morphology is grossly perturbed by loss-

of-function at this locus and this is accompanied by a reduced

efficiency of biosynthetic membrane trafficking without

gross perturbation of other endomembrane organelles.

All four mutations reside in the GSH2 locus

Fine mapping of N37 and DNA sequencing of candidate

genes identified mutations in the GSH2 locus (At5g27380) of

all four mutants. This locus encodes GSH2 which catalyses

the final step in the biosynthesis of GSH. Two T-DNA alleles,

gsh2-1 and gsh2-2, have been described at this locus (Pas-

ternak et al., 2008), so S10, G4, N37, and S6 were renamed

gsh2-3 to gsh2-6, respectively, in decreasing order of

severity of the developmental phenotype (Figure 1a). Each

mutant carried a single base pair substitution that resulted

either in a non-sense mutation W130Stop in S10 (gsh2-3), or

missense mutation G434E in G4 (gsh2-4), P364S in N37

(gsh2-5), and G94E in S6 (gsh2-6) (Figures 4a and S2). The

stop codon in the gsh2-3 allele is predicted to truncate GSH2

downstream of the dimerization unit but before the active

site residues and thus is expected to be a null allele (Herrera

et al., 2007). Mutations in gsh2-4, gsh2-5, and gsh2-6

resulted in non-conservative changes in conserved amino

acid residues (Figure 4b).

The gsh2 mutants exhibit c-EC hyperaccumulation and GSH

depletion, leading to reduced redox buffer capacity

Glutathione biosynthesis is a two-step process that first

combines glutamate and cysteine to form c-EC followed by

the addition of glycine to c-EC to form GSH. These two steps

are catalyzed by GSH1 and GSH2, respectively (Figure 5a).

To examine whether GSH biosynthesis was affected by the

(a) (b)

(c) (d)

(e) (f)

(g) (h)

(i) (j)

Figure 3. Electron microscopy of wild-type (WT) and mutant seedlings.

Transmission electron microscopy (a–g) and confocal (h–j) images of root tip

epidermal cells of 7-day-old seedlings.

(a, b) Wild-type cells showing a tubular endoplasmic reticulum (ER) and the

absence of swollen bodies. (c–f) Swollen bodies present in S10 seedlings

carrying a strong allele (gsh2-3) (c, d) and S6 carrying the weakest allele (gsh2-

6) (e, f). The swollen bodies were surrounded by ribosomes as indicated by

the black dots around the swollen bodies. (g) Some younger cells of S6 (gsh2-

6) had dilated ER tubules. (h–j) Confocal images of epidermal cells in the

meristematic regions of S6 (gsh2-6) (h, i) and S10 (gsh2-3) (j) expressing

secGFP. The apparently dilated tubules caused by the weaker allele in S6 are

indicated by arrows in (h).

T, tubular ER; S, swollen bodies; V, vacuole. Bars = 500 nm in (a, c, e, and g);

100 nm in (b, d, and f); 5 lm in (j).



GSH2 mutations, we measured the content of cytosolic c-EC

and GSH in 10-day-old seedlings of gsh2 alleles. In this

analysis, we also included the null T-DNA allele gsh2-1

(Pasternak et al., 2008) and rml1 which is a GSH1 mutant

(Vernoux et al., 2000). While the level of GSH in WT was, as

expected, much higher than that of c-EC, c-EC was the pre-

dominant small thiol in all gsh2 mutants (Figure 5b). Among

the gsh2 mutants, gsh2-1, gsh2-3, and gsh2-4 showed the

greatest degree of c-EC hyperaccumulation and GSH defi-

ciency. The c-EC content in these mutants was 10–13 lmol

g)1 which is about 400-fold greater than in WT whilst GSH

was undetectable, suggesting that there was little or no

GSH2 activity. Hyperaccumulation of c-EC was also

observed in gsh2-5 and gsh2-6, although to a lesser extent

(170-fold greater than in WT), and their GSH content was

higher than that of rml1. Germination of gsh2-5 on medium

supplemented with 1 mM GSH rescued root growth (Fig-

ure 5c), indicating that the developmental abnormality was

attributable to the perturbation in GSH biosynthesis. Thus

each of the mutations in gsh2-3 to gsh2-6 apparently caused

a reduction in GSH2 activity, thereby increasing the abun-

dance of its substrate, c-EC, and decreasing the abundance

of its product, GSH.

To investigate the small thiol content of the swollen ER

bodies, roots were stained with monobromobimane (MBB)

and MCB. Unlike MCB that preferentially labels reduced GSH

through a GST-catalysed reaction (Meyer et al., 2001), MBB

is more reactive and labels small thiols non-specifically

(Fahey et al., 1981). As the analysis of low molecular weight

thiols of gsh2 revealed that c-EC replaced GSH as the most

abundant small thiol molecule, we expected that MBB would

mostly label c-EC in gsh2 seedlings. Furthermore, because

(a)

(b)

Figure 4. Mutations in the GSH2 locus of mutants S10, G4, N37 and S6.

(a) Location in the GSH2 locus (At5g27380) of the mutation in each allele (numbering refers to codons). S10, G4, N37, and S6 were renamed gsh2-3, gsh2-4, gsh2-5,

and gsh2-6, respectively.

(b) Alignment of glutathione synthase (GSH2) protein sequence of Arabidopsis, human (Homo sapiens) and yeast (Saccharomyces cerevisiae) using CLUSTALW.

Residues affected by mutation in G4, N37, S6 and S10 are boxed. Symbols below the sequences denote the degree of conservation. Asterisks (*) indicate identical

residues; semi-colon (:) and full-stop (.) indicate conserved and semi-conserved substitutions, respectively.



bimane becomes fluorescent and membrane-impermeant

after it is conjugated to a thiol molecule, a c-EC-bimane

adduct would be trapped in the membrane-bound swollen

bodies if it formed there. Indeed, an increased bimane signal

was detected in swollen bodies of gsh2-5 root cells with

MBB labeling compared with MCB labeling (Figure S3),

suggesting that swollen ER-derived bodies contained con-

siderable quantities of c-EC.

Although GSH is associated with a variety of cellular

functions, it is primarily known for its role in redox homeo-

stasis by reversible formation of disulfide bridges (Jessop

and Bulleid, 2004; Meyer, 2008). Cytosolic redox-sensitive

GFP (c-roGFP) has been shown to report GSH redox

potential in planta (Marty et al., 2009; Schwarzlander et al.,

2008). To assess the ability of gsh2 plants to maintain a

reduced intracellular environment, we expressed c-roGFP in

WT and gsh2-5. In time-lapse experiments (Figure 5d,e)

seedlings were perfused in half-strength MS medium, and

upon challenge with 10 mM H2O2, both WT and the mutant

showed full oxidation of roGFP. Washout of H2O2 resulted in

full recovery of the highly reduced redox state in WT after

15–20 min, while the mutant remained significantly more

oxidized (P < 0.01). Compared with WT, the redox state of

gsh2-5 was highly variable under steady-state conditions.

Assuming that c-roGFP1 responds to c-EC as it does to GSH,

it thus appeared that the gsh2-5 mutant had a weaker redox

buffer capacity than WT even at steady state (Figure 5e). It

also appears that despite the abundance of c-EC in gsh2

plants, this low-molecular-weight thiol cannot replace GSH

in regulation of the redox state.

Figure 5. Low-molecular-weight thiol content and response to oxidative stress in gsh2-5.

(a) Schematic diagram of the glutathione (GSH) biosynthesis pathway. Buthionine sulfoximine (BSO) is a reversible inhibitor of glutamate-cysteine ligase (GSH1).

rml1 and gsh2 are mutant alleles of GSH1 and GSH2, respectively.

(b) Low-molecular-weight thiol analysis of wild type (WT), gsh2 and rml1. Glutathione and c-glutamylcysteine are represented by black and grey bars, respectively.

(c) Primary root length of WT and gsh2-5 grown with or without GSH. Asterisks indicate values that were significantly different from WT under the same conditions

(P < 0.01). Error bars represent SD. n = number of seedlings.

(d) Representative time course traces showing the dynamic response of the GSH redox potential in WT (black) and gsh2-5 (grey) to transient oxidation. Samples

were first perfused in half-strength MS (MS1) for 10 min, then in medium containing 10 mM H2O2 for 15 min, followed by half-strength MS (MS2) for 20 min.

(e) Based on the time course in (d), the average GSH redox potential in WT (white) and gsh2-5 (grey) was measured at the end of each perfusion step. Asterisks

indicate values that were significantly different from WT at each point (n = 10, P < 0.01).

All error bars represent SD.



The ER phenotype of gsh2 is caused by hyperaccumulation

of c-EC rather than depletion of GSH

To investigate the origin of the morphological defect of the

ER in gsh2 alleles, we first asked whether a supplement of

GSH could alleviate this aspect of the phenotype. The swol-

len ER body phenotype was rescued by germinating gsh2

with exogenous GSH (Figure 6a,b,d,e). Since synthesis of

c-EC can be inhibited by GSH through feedback inhibition on

GSH1 (Hell and Bergmann, 1990), exogenous GSH could

abolish further accumulation of c-EC as well as GSH defi-

ciency in gsh2. To distinguish the effects of hyperaccumula-

tion of c-EC and GSH deficiency we first used GFP-HDEL to

examine the ER morphology of rml1 seedlings which are

deficient in c-EC synthesis. Although rml1 resembled gsh2-5

in stature and showed a more severe GSH deficiency

(Figures 5b and 6g), its ER morphology appeared as WT

(Figure 6h,i). Furthermore, when production of c-EC and GSH

was inhibited in WT by germination on 1 mM buthionine

sulfoximine (BSO), which is an inhibitor of GSH1 (Figure 5a)

(Griffith and Meister, 1979; Cobbett et al., 1998), root growth

was significantly reduced (Figure 6j,k) but confocal micros-

copy revealed no change in ER morphology (Figure 6a,c). In

contrast, when gsh2-5 seedlings were germinated on BSO,

their developmental abnormalities were unchanged

(Figure 6l,m) but normal ER morphology was restored in

cotyledons and hypocotyls (Figure 6d,f). The recovery in the

root was partial: an ER network was observed but typical

fusiform bodies were absent in many cells. Because this

resembled WT treated with 10 mM H2O2 (Figure S4), we

suspect that the roots of gsh2-5 suffered oxidative stress

when grown on BSO. From these observations, we attributed

the swollen ER phenotype of gsh2 seedlings to accumulation

of c-EC rather than depletion of GSH.

(a) (b) (c)

(d) (e) (f)

(g)

(j)

(l) (m)

(k)

(h) (i)

Figure 6. The swollen endoplasmic reticulum

(ER) phenotype of gsh2 is caused by accumula-

tion of c-glutamylcysteine rather than glutathi-

one (GSH) deficiency.

(a–f) Confocal images of hypocotyls of wild type

(WT) (a–c) and gsh2-5 (d–f) expressing an ER

lumenal marker (GFP-HDEL). Seedlings were

grown either in normal growth medium (a, d),

or in growth medium supplemented with 1 mM

GSH (b, e) or 1 mM buthionine sulfoximine (BSO)

(c, f). (g–i) Bright field (g) and confocal images of

hypocotyls (h, i) of 7-day-old rml1 expressing the

ER lumen marker (N-YFP-HDEL). (i) Higher-reso-

lution image of cortical ER. (j–m) Bright field

images of 7-day-old WT (j, k) and gsh2-5 (l, m)

seedlings grown in media without (j, l) and with

1 mM BSO (k, m).

Bars = 5 mm in (g and m), 20 lm in (f and h), and

10 lm in (i).



gsh2-5 seedlings exhibit a normal unfolded protein

response

Correct formation of disulfide bridges is crucial for proteins

to attain their native conformation. In the ER a collection of

oxidoreductases and protein disulfide isomerases (PDI)

work with chaperones to catalyse protein folding by pro-

moting disulfide bond formation and reduction (Liu and

Howell, 2010; Onda et al., 2009). It has been shown that in

mammalian cells, oxidoreductases in the ER are reduced

by GSH (Jessop and Bulleid, 2004); thus GSH plays a key

role in the electron transfer process that is required for

disulfide bond formation in this organelle. Therefore we

asked whether gsh2 seedlings are compromised in protein

folding which may result in the accumulation of secreted

and endomembrane proteins in the ER. This situation

would be expected to trigger the unfolded-protein re-

sponse (UPR), so we analyzed the accumulation of the ER-

resident chaperones Binding Protein (BiP) and calreticulin

(CRT), whose expression is upregulated during the UPR

(Christensen et al., 2008; Costa et al., 2008; Koizumi et al.,

2001). As shown in Figure 7, the accumulation of BiP and

CRT in WT and gsh2-5 did not appear to differ, implying

that the mutant was not under constitutive ER stress. Next,

to determine whether gsh2 seedlings were capable of

eliciting the UPR we analysed the accumulation of CRT and

BiP in seedlings treated with the UPR-inducing drug tu-

nicamycin (Bertolotti et al., 2000; Iwata and Koizumi, 2005;

Koizumi et al., 2001). After a 7-h treatment, the abundance

of BiP and CRT was increased in both WT and gsh2-5 with

no apparent difference between the genotypes. Hence, the

ability to mount a UPR did not appear to be compromised

in gsh2-5.

Reticulon over-expression failed to suppress swelling

of the ER

A major contributor to the tubular morphology of the ER

membranes is the reticulon family. Reticulons localize in

tubular domains of the ER and are responsible for main-

taining the morphology of the ER by generating membrane

curvature (Sparkes et al., 2010; Tolley et al., 2008; Voeltz

et al., 2006; West et al., 2011). It has been shown in vitro that

a Xenopus reticulon, RTN4a, can be inhibited by thiol-

modifying reagents, resulting in perturbation of ER tubule

morphology (Voeltz et al., 2006). In Arabidopsis there are 21

genes encoding reticulon-like proteins, namely AtRTNLB1–

21 (Nziengui et al., 2007; Oertle et al., 2003). It has been

shown that AtRTNLB1–4 and -13 can remodel ER lumen

without affecting secretory transport (Sparkes et al., 2010;

Tolley et al., 2008). To test whether normal ER morphology

can be restored in gsh2 by over-expression of reticulons,

yellow fluorescent protein (YFP)-tagged Arabidopsis reticu-

lon (RTNLB1-YFP) (Sparkes et al., 2010) and the ER lumen

marker mCHERRY-HDEL (Nelson et al., 2007) were

co-expressed in WT and gsh2-5. While RTNLB1-YFP clearly

labeled the ER network in both WT and gsh2-5, mCHERRY-

HDEL labeled tubular fusiform bodies in WT but swollen ER

bodies in gsh2-5 (Figure S5). There was little colocalization

between RTNLB1-YFP and mCHERRY-HDEL. Although both

DER2-YFP and RTNLB1-YFP are ER membrane markers, the

localization patterns of RTNLB1-YFP and DER2-YFP were

noticeably different, such that the former localized exclu-

Figure 7. Unfolded protein response in gsh2 and wild-type (WT) seedlings.

Eight-day-old WT and gsh2-5 seedlings were incubated in liquid MS medium with either DMSO (control) or 5 lg ml)1 tunicamycin for 7 h before protein extraction.

(a, c) Immunoblot analysis of Binding Protein (BiP) and calreticulin (CRT) expression using anti-BiP and anti-CRT antibodies. Arrows indicate bands that are

detectable at a lower intensity before tunicamycin treatment while arrowheads indicate bands that are only visible after treatment. Molecular weight markers are

indicated on the left.

(b, d) Ponceau S stain of the blots in (a and c) to demonstrate equal loading.



sively in the tubular ER network and the latter localized in the

membrane of swollen bodies (compare Figures 2l and S5b).

Thus RTNLB1-YFP over-expression was apparently unable

to restore ER membrane morphology.

DISCUSSION

In this study we have characterized four alleles of GSH2 that

were identified in a screen for secretory membrane traffick-

ing mutants. We show that a perturbation in GSH biosyn-

thesis can affect both the operation of the secretory pathway

and the morphology of the ER. Although the four gsh2

alleles all showed a similar degree of perturbation in ER

morphology and secretory trafficking, they exhibited a wide

range of developmental abnormalities. Surprisingly, the

weakest allele, gsh2-6, was viable which indicates that gross

perturbation of ER morphology is compatible with viability

of the cell and organism. Consistent with this, the surface of

the swollen ER bodies was decorated with ribosomes and

was thus apparently competent for translocation.

The small thiol profile of the two strong alleles was

comparable to that of a previously described gsh2 null

mutant gsh2-1 (Pasternak et al., 2008). The intermediate

allele, gsh2-5, which undergoes post-germinative meriste-

matic development, had decreased redox buffer capacity

and increased sensitivity to oxidative stress. In addition,

roGFP1 expressed in this mutant exhibited a variable

cytosolic redox state that was substantially more oxidized

than WT (on average 21 and 47% oxidized in WT and gsh2-5,

respectively). We attempted to measure the GSH redox state

in the ER using modified roGFPs with a more oxidizing

midpoint potential (Lohman and Remington, 2008) but these

probes were still almost fully oxidized even in WT ER,

precluding reliable measurement of any increase in the

oxidation state in gsh2.

It has been reported previously that the complete absence

of GSH in gsh1 null mutants confers embryo lethality (Cairns

et al., 2006), whereas gsh2 null mutants are seedling lethal

owing perhaps to the accumulation of c-EC, which may

substitute for GSH in some instances (Pasternak et al., 2008).

Here, using genetic and pharmacological evidence, we show

that the deficiency in GSH determines the degree of devel-

opmental abnormality in gsh2 and rml1, whereas it is the

accumulation of c-EC, but not GSH deficiency, that affects

the morphology and secretory pathway of the ER. Thus tight

regulation of c-EC synthesis and conversion to GSH may be

required to maintain this aspect of cellular function. Indeed

GSH1 activity shows feedback inhibition by GSH (Hell and

Bergmann, 1990) and this fails when GSH2 is defective

(Pasternak et al., 2008). This also means that although

exogenous GSH can suppress the ER morphology pheno-

type of gsh2, it is likely to act indirectly by alleviating

accumulation of c-EC through feedback inhibition of GSH1

(Hell and Bergmann, 1990). This may explain why, in initial

tests, we failed to rescue the swollen ER phenotype of gsh2 if

we supplied GSH after germination when c-EC had already

accumulated to a significant level. Recently, Jobe et al.

(2012) reported a viable loss-of-function mutation at the

gsh2 locus. This allele, nrc2, was identified in a screen for

mutants with a reduced response to exogenous cadmium.

Based on growth and c-EC accumulation, ncr2 appears to be

weaker than any of the alleles described here but its

secretory activity and ER morphology have not been char-

acterized.

The regulated formation of disulfide bridges is an essen-

tial process for the proper folding and maturation of newly

synthesized proteins. Changes in the redox state of specific

thiol groups on some proteins can result in their aggregation

in the ER, inhibiting anterograde traffic (Kawagoe et al.,

2005; Orsi et al., 2001; Pompa and Vitale, 2006). Glutathione

is required directly or indirectly to reduce ER proteins via

enzymatic reactions (Jessop and Bulleid, 2004), and it has

been postulated that ER stress may lead to enlargement of

the lumen to accommodate more misfolded proteins (Banh-

egyi et al., 2007). Glutathione has been detected in the

Arabidopsis ER (Zechmann et al., 2008), but despite the

gross perturbation of small thiol accumulation, gsh2-5

apparently does not significantly accumulate unfolded pro-

tein or protein aggregates at steady state. Nevertheless, the

UPR can still be triggered in gsh2-5, showing that the

monitoring of protein misfolding is not prevented. It is

surprising that despite the expected need for GSH in

disulfide bond formation, it is ER morphology and protein

export rather than general protein folding that appear to be

most sensitive to perturbation of GSH metabolism.

Since the integral membrane markers of the Golgi (ST-

RFP) and TGN (VHA-a1:GFP) are able to reach their targeted

compartment but secGFP, the plasma membrane (PM)

marker (PIP-GFP) and the tonoplast marker (BobTIP-GFP)

are not, ER export appears to be partially blocked in gsh2.

One possible explanation is that the change in c-EC levels

causes a cargo-specific folding or selection process to be

disrupted. An alternative explanation is suggested, how-

ever, by the observation that even in WT cells PIP-GFP and

BobTIP-GFP were often detectable in the ER as well as their

resident membrane. This suggests that they may be ineffi-

ciently exported and thus more sensitive to general pertur-

bation of membrane trafficking by accumulation of c-EC.

Indeed it has been shown that early steps of secretory

membrane traffic in tobacco protoplasts are sensitive to

exogenously applied thiol-containing reagents (Pompa and

Vitale, 2006).

It is still unclear how c-EC accumulation affects ER

morphology. Animal reticulon proteins are required for the

formation of tubular ER and they are known to affect ER

morphology in plants (Sparkes et al., 2010; Tolley et al.,

2008; Voeltz et al., 2006). The function of animal reticulon

can be perturbed by treatment with thiol-modifying reagents

such as N-ethyl-maleimide (NEM), so we hypothesized that



the accumulated c-EC in gsh2 seedlings may affect ER

morphology by interacting with one or more of the 21 plant

reticulons in Arabidopsis (Nziengui et al., 2007; Oertle et al.,

2003). However, we were unable to eliminate the swollen

bodies in gsh2 by overexpressing reticulons, nor could we

phenocopy the swollen bodies of gsh2 plants by growing

WT with various concentrations of NEM (Figure S5).

The ER is a morphologically dynamic structure that also

changes during development (Ridge et al., 1999). In many

Brassicaceae plants including Arabidopsis, parts of the ER

also exist as spindle-shaped structures known as ER bodies

(Behnke and Eschlbeck, 1978; Iversen, 1970; Matsushima

et al., 2003). These structures accumulate fluorescent pro-

teins with a C-terminal H/KDEL retrieval motif (Hawes et al.,

2001; Toyooka et al., 2000; Yamada et al., 2008). Indeed,

such markers predominantly label ER bodies in WT but

swollen ER in gsh2. Similar to fusiform ER bodies (Hawes

et al., 2001), the swollen bodies in gsh2 were also sur-

rounded by ribosomes, suggesting that they were functional

for protein translocation. Because we do not see any

spindle-shaped ER bodies in gsh2, the swollen bodies in

gsh2 may be derived from these ER bodies.

Interestingly, of all the ER markers that were tested,

RTNLB1-YFP was the only one that exclusively labeled the ER

network in gsh2-5, while all other markers (DER2-YFP, GFP-

HDEL and mCHERRY-HDEL) were localized predominantly in

swollen bodies. This may be attributed to the fact that

reticulons preferentially localize in membranes of high

curvature such as the tubular network so are excluded from

less curved surfaces like the spherical swollen bodies.

RTNLB1-YFP may have preferentially labeled the residual

ER network in gsh2. One possibility is that accumulation of

c-EC in the ER may directly cause swelling of the ER bodies.

In fact, results from MBB and MCB labeling suggested that

c-EC was abundant in the swollen bodies of gsh2-5. Even in

the weak alleles of gsh2 the total quantity of c-EC in the tissue

is approximately 20-fold greater than the total WT GSH

content. The GSH content of WT plants corresponds to a

cytosolic concentration of 2–3 mM, so c-EC may exceed

50 mM in gsh2. We speculate that the accumulation of such

quantities of c-EC in the relatively small volume of the ER

may perhaps be sufficient to induce swelling. The apparent

absence of reticulons from the surface of the spindle-shaped

ER bodies may make these regions more susceptible to

swelling if the ER volume increases, giving rise to the

swollen bodies in gsh2. It is unclear how c-EC may enter the

ER. In WT cells c-EC is undetectable, being rapidly converted

to GSH by cytosolic GSH2, so a specific transporter is

unlikely. Glutathione has been immunologically detected,

although not quantified, in the ER of Arabidopsis (Zechmann

et al., 2008) but is present in the ER lumen of animal cells at a

similar concentration to that of the cytosol (Banhegyi et al.,

1999; Hwang et al., 1992; Le Gall et al., 2004), so perhaps

c-EC could utilize the same transport pathway to enter the ER.

The phenotypes of the gsh2 alleles reported here high-

light the need for significant further investigation to fully

account for the unexpected influence of GSH metabolism on

form and function of the ER.

EXPERIMENTAL PROCEDURES

Plant material and growth conditions

Introgressions of c-roGFP1 (Schwarzlander et al., 2008), GFP-HDEL(Zheng et al., 2004), ST-RFP (Teh and Moore, 2007), BobTIP-GFP(Reisen et al., 2003), PIP-GFP (a gift of C. Hawes), VHAa1-GFP(Dettmer et al., 2006), and N-YFP-HDEL (Teh and Moore, 2007) intogsh2-5 or rml1 were performed by crossing previously describedtransgenic Arabidopsis homozygous for the fluorescent proteinmarkers with plants heterozygous for gsh2-5 or rml1. Constructs ofRTNLB1-YFP and DER2-YFP were made available by L. Frigerio,University of Warwick, UK; mCHERRY-HDEL (Nelson et al., 2007)was obtained from the Arabidopsis Biological Resource Centre(ABRC), Columbus, OH, USA. The constructs were transformed intoAgrobacterium tumefaciens and subsequently to Arabidopsis bythe floral dipping method (Clough and Bent, 1998). Seeds weresterilized with 70% ethanol, stratified at 4�C for 2 days on agar platescontaining MS medium (pH 5.7) with 1% sucrose and cultivated at20�C under a 16-h photoperiod.

Genetic screen

Seeds from transgenic Arabidopsis lines expressing secGFP (lineS76; Zheng et al., 2004), nlsRm-2A-secGf, or STN-Rm-2A-secGf

(Samalova et al., 2006) were mutagenized with ethane-methyl-sul-fonate (EMS) as described (Teh and Moore, 2007) and seed wascollected separately from over 7000 individual M1 plants. Three-day-old seedlings from 7000 M2 families, germinated and grown onMS agar, were screened for enhanced GFP fluorescence using aLeica MZFLIII Stereo Microscope (Leica Microsystems (UK) Ltd,Milton Keynes, Bucks, UK). Initially, seedlings were screened inpools comprising approximately 30 seeds from each of 10 M2

families. Individual M2 families from pools segregating fluorescentseedlings were subsequently rescreened to identify the segregatingM2 family. Intracellular accumulation of secGFP in cytoplasmicorganelles of the mutant seedlings was confirmed by confocalmicroscopy. The screen identified 97 independent putative mu-tants, and 73 of these could be propagated to the next generationvia their phenotypically WT heterozygous siblings. Initially eightphenotypically WT plants were propagated for each mutant family;if no heterozygote was found, an additional eight were tested.Heterozygous M3 or M4 plants were backcrossed to Col-0 andcrossed to Ler for approximate mapping by bulk segregant analysis(BSA) (Lukowitz et al., 2000). To do this, four phenotypically WTplants from each mutant family were selected for crossing and theirheterozygosity was subsequently assessed by scoring their selfedprogeny for mutant phenotypes; another four plants were used ifnone of the initial set were heterozygotes. Following BSA, four ofthe mutants that mapped to the top of ChrV and each exhibitedsimilar secGFP accumulation phenotypes were selected for detailedcharacterization.

Electron microscopy

Ten-day old seedlings were fixed in 2% paraformaldehyde and 1%glutaraldehyde in 20 mM K2HPO4 buffer (pH 7.0) for 3–5 h. Sampleswere washed with 50 mM K2HPO4 solution before transferring to 2%osmium tetroxide for 2 h. After washing with distilled water, thesamples were dehydrated in an ethanol series and embedded in



Spurr’s resin. Sections were examined with a Hitachi H7650 trans-mission electron microscope (Hitachi High-Technologies EuropeGmbH, Maidenhead, Berks, UK).

Confocal laser scanning microscopy

Except for simultaneous imaging of GFP and YFP, samples wereexamined using Zeiss LSM 510 META laser scanning microscope(Carl Zeiss Ltd, Welwyn Garden City, Herts, UK). The configurationof the instrument was described previously (Samalova et al., 2006;Teh and Moore, 2007; Zheng et al., 2004) except for imagingmCHERRY, bimane and c-roGFP1. The mCHERRY was excited with a543 nm HeNe laser and a HFT 458/543 primary dichroic mirror, andwas detected with a 565–615 nm band pass filter. Bimane wasexcited with a 405 nm blue diode laser and a HFT 458/543 primarydichroic mirror, and was detected with a 475–525 nm band passfilter. For simultaneous imaging of mCHERRY and bimane, themulti-track line-sequential imaging mode with the configurationmentioned above was used. Images were processed using ZeissLSM Image Browser.

In the time course experiment with c-roGFP1, the perfusionchamber was connected to a Gilson Minipuls 2 peristaltic pump(Gilson Inc., Middleton, WI, USA) with HPLC tubing and wasmounted on the confocal microscope. Imaging and ratiometricanalysis of roGFPs was performed as described (Schwarzlanderet al., 2008). Samples coexpressing GFP and YFP were examinedusing Leica TCS SP5 II. The GFP was excited with the 458 nm argonlaser line and was detected with a PMT channel set to collect signalbetween 475 and 510 nm. The YFP was subsequently excited withthe 514 nm argon laser line and was detected with a second PMTchannel set to collect signal between 572 and 613 nm. Images wereprocessed using LAS AF (Leica Microsystems (UK) Ltd).

Fluorescent dye treatment

Seedlings were immersed in 5 lM FM4-64 (Invitrogen Ltd, Paisley,UK; from 5 mM stock in water) aqueous solution for 60 min to labelthe tonoplast. Seedlings were immersed in 100 lM MCB or MBB(Invitrogen; from 100 mM stock in ethanol) aqueous solution.

Protein domain analysis

Protein sequences of GSH2 from Arabidopsis, human (Homo sapi-ens) and yeast (Saccharomyces cerevisiae) were obtained fromGenBank (Arabidopsis, CAB51027.1; human, NP_000169.1; yeast,CAA 74136.1) and were aligned using CLUSTALW (http://www.e-bi.ac.uk/Tools/clustalw2/) to assess the conservation of eukaryoticGSH2. Putative conserved domains of Arabidopsis GSH2 wereidentified using the Basic Local Alignment Sequence Tool program(BLAST; http://blast.ncbi.nlm.nih.gov/).

Low-molecular-weight thiol molecule analysis

Low-molecular-weight thiols were extracted from 10-day-old seed-lings and analyzed by HPLC as described previously (Cairns et al.,2006).

Immunoblot analysis

For the secGFP immunoblot, proteins from 6-day-old seedlingswere extracted and analyzed by immunoblot as described(Samalova et al., 2006). For the unfolded protein response analysis,10-day-old seedlings were incubated in half-strength MS mediumwith either DMSO or 5 lg ml)1 tunicamycin (Sigma-Aldrich, Poole,UK). Proteins were extracted first by homogenization in twovolumes of extraction buffer: 4% SDS, 100 mM 2-amino-2-(hydrox-ymethyl)-1,3-propanediol (TRIS)-HCl (pH 6.8), 400 lg ml)1 brom-

ophenol blue, 200 mM DTT, 20% glycerol, 0.2% b-mercaptoethanol,and protease inhibitor cocktail (Sigma-Aldrich). Samples were thenboiled for 10 min followed by centrifugation at 10 000 g for 5 min.Then SDS-PAGE was run as described (Samalova et al., 2006).Proteins were then blotted onto a polyvinylidene fluoride mem-brane as described (Batoko et al., 2000). Before incubating withprimary antibody, the blot was stained with Ponceau S solution(0.5% Ponceau S and 1% acetic acid) for 5 min and subsequentlywashed with 1% acetic acid. An image of the blot was taken with aNikon D700 camera (Nikon UK Ltd, Kingston upon Thames, Surrey,UK) before Ponceau S was de-stained with 1% acetic acid. Anti-BiP(a gift from L. Frigerio, University of Warwick, UK) and anti-CRT (agift from J. Denecke, University of Leeds, UK) were diluted to 1:5000and 1:10 000, respectively. Alkaline phosphatase-conjugated sec-ondary antibody (anti-rabbit IgG) (Sigma-Aldrich) was used at1:10 000 dilution.

ACKNOWLEDGEMENTS

We thank Markus Schwarzlander, University of Oxford, for assis-tance on roGFP analysis and comments on the manuscript, LorenzoFrigerio, University of Warwick, for the anti-BiP antibody, RTNLB1-YFP and DER2-YFP constructs, Jurgen Denecke, University of Leeds,for the anti-CRT antibody, Barry Martin and Chris Hawes, OxfordBrookes University for assistance with electron microscopy and forPIP-GFP seeds, and Rudiger Hell, Heidelberg University, Germanyfor access to HPLC. KKCA was funded by the Clarendon Fund andLinacre Canadian Alumni Scholarship. This work was supported bygrant BBS/B/09562 from the UK Biotechnology and Biological Sci-ences Research Council.

SUPPORTING INFORMATION

Additional supporting information may be found in the online ver-sion of this article:Figure S1. GFP-HDEL and DER2-YFP labeling in N37 (gsh2-5).Figure S2. Domain structure of Arabidopsis glutathione synthase.Figure S3. Monobromobimane and M monochlorobimane labelingof small thiols in root epidermal cells.Figure S4. Buthionine sulfoximine-treated gsh2-5 root cells.Figure S5. RTNLB1-YFP and NEM treatment in N37 (gsh2-5).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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