A perturbation in glutathione biosynthesis disrupts ... - WordPress.com

The Plant Journal (2012)

doi: 10.1111/j.1365-313X.2012.05022.x

A perturbation in glutathione biosynthesis disrupts endoplasmic reticulum morphology and secretory membrane traffic in Arabidopsis thaliana Kenneth K. C. Au1,†, Jose´ Pe´rez-Go´mez1,†, He´lia Neto1,‡, Christopher Mu¨ller2, Andreas J. Meyer2,§, Mark D. Fricker1 and Ian Moore1,* 1 Department of Plant Sciences, University of Oxford, South Parks Rd, Oxford OX1 3RB, UK, and 2 Heidelberg 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. *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 compartments over time (Huotari and Helenius, 2011). Much of our ª 2012 The Authors The Plant Journal ª 2012 Blackwell Publishing Ltd

fundamental knowledge comes from forward genetic studies 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 cytological studies suggest that plant cells have acquired distinctive features in various aspects of membrane trafficking, 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 plantspecific trafficking activities is also supported by phylogenetic studies of plant genomes which have shown extensive diversification of important membrane trafficking protein families such as Rab GTPases, Arf-guanine-nucleotide 1

2 Kenneth K. C. Au et al. exchange factors (Arf-GEFs), and soluble N-ethylmaleimidesensitive 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 neofunctionalization (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). Consequently, to identify genes with important functions in plant membrane trafficking, several laboratories have used forward 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 regulate 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 membrane 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 wildtype plants, but in mutants that perturb the secretory pathway fluorescent secGFP accumulates in the cytoplasmic organelles along the pathway and enhanced seedling fluorescence 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 simply for cell maintenance (Berna´ et al., 1999; Ju¨rgens et al., 1991). We reasoned that the same may be true of plantspecific 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 phenotypes 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 seedlinglethal mutants (Ju¨rgens et al., 1991). Here we report the use of secGFP and improved ratiometric 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, including cell division (Vernoux et al., 2000), heavy metal detoxification (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; Shanmugam 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 seedlings 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 fluorescence intensities, which can be affected by variation in secGFP expression or imaging efficiency, putative mutants could be identified by comparing the intensity ratio of secGFP and a stoichiometrically expressed nuclear- (nlsRm) or Golgi-targeted (STN-Rm) red fluorescent protein (RFP) marker. 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 siblings (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

ª 2012 The Authors The Plant Journal ª 2012 Blackwell Publishing Ltd, The Plant Journal, (2012), doi: 10.1111/j.1365-313X.2012.05022.x

Arabidopsis glutathione synthase mutant and ER morphology 3

(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).

subsequent sequencing showed that all mutations were allelic (see below and Figure 4). Although the mutants showed similar secGFP accumulation phenotypes, they exhibited widely different developmental 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 membrane markers Various fluorescent-protein-tagged markers of the endomembrane 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 transGolgi-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


4 Kenneth K. C. Au et al.

(a)

(b)

(c)

(d)

(k)

(l)

(m) (e)

(f)

(g)

(h)

(n)

(o) (i)

(j)

(p)

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 thiolconjugated 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).


Arabidopsis glutathione synthase mutant and ER morphology 5 suggests that the endocytic and biosynthetic tonoplast markers FM4-64 and BobTIP-GFP labeled different compartments in the mutant. To test this further we labeled the vacuolar lumen using monochlorobimane (MCB), which becomes fluorescent only after it conjugates reduced cytosolic thiols, principally GSH in WT cells. The conjugated product, glutathione-S-bimane (GSB), is then actively transported to the vacuole (Fricker et al., 2000). In N37, fluorescent 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 fulllength form, typical of the ER, rather than the C-terminally truncated form normally observed in the vacuole or apoplast (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 lossof-function at this locus and this is accompanied by a reduced efficiency of biosynthetic membrane trafficking without gross perturbation of other endomembrane organelles.

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)

(j)

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 (Pasternak 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

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 (gsh26) (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 (gsh26) 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).

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


6 Kenneth K. C. Au et al.

(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.

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 predominant 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 deficiency. 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 (Figure 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 abundance 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


Arabidopsis glutathione synthase mutant and ER morphology 7

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.

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 considerable quantities of c-EC. Although GSH is associated with a variety of cellular functions, it is primarily known for its role in redox homeostasis 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.


8 Kenneth K. C. Au et al. 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 swollen 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 deficiency in gsh2. To distinguish the effects of hyperaccumulation 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

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(j)

(k)

(l)

(m)

(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 microscopy 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.

Figure 6. The swollen endoplasmic reticulum (ER) phenotype of gsh2 is caused by accumulation of c-glutamylcysteine rather than glutathione (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-resolution 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).

(i)


Arabidopsis glutathione synthase mutant and ER morphology 9 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 promoting 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 response (UPR), so we analyzed the accumulation of the ERresident 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 tunicamycin (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 maintaining 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 thiolmodifying 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 reticulon (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, mCHERRYHDEL 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.


10 Kenneth K. C. Au et al. 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 trafficking mutants. We show that a perturbation in GSH biosynthesis 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 meristematic 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 developmental 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 phenotype 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 characterized. The regulated formation of disulfide bridges is an essential 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 (Banhegyi 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 protein 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 (STRFP) 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, however, 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 inefficiently exported and thus more sensitive to general perturbation 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


Arabidopsis glutathione synthase mutant and ER morphology 11 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 proteins 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 surrounded 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, GFPHDEL 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 (Ba´nhegyi 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 highlight 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) into gsh2-5 or rml1 were performed by crossing previously described transgenic Arabidopsis homozygous for the fluorescent protein markers with plants heterozygous for gsh2-5 or rml1. Constructs of RTNLB1-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 into Agrobacterium tumefaciens and subsequently to Arabidopsis by the floral dipping method (Clough and Bent, 1998). Seeds were sterilized with 70% ethanol, stratified at 4C for 2 days on agar plates containing MS medium (pH 5.7) with 1% sucrose and cultivated at 20C under a 16-h photoperiod.

Genetic screen Seeds from transgenic Arabidopsis lines expressing secGFP (line S76; Zheng et al., 2004), nlsRm-2A-secGf, or STN-Rm-2A-secGf (Samalova et al., 2006) were mutagenized with ethane-methyl-sulfonate (EMS) as described (Teh and Moore, 2007) and seed was collected separately from over 7000 individual M1 plants. Threeday-old seedlings from 7000 M2 families, germinated and grown on MS agar, were screened for enhanced GFP fluorescence using a Leica MZFLIII Stereo Microscope (Leica Microsystems (UK) Ltd, Milton Keynes, Bucks, UK). Initially, seedlings were screened in pools comprising approximately 30 seeds from each of 10 M2 families. Individual M2 families from pools segregating fluorescent seedlings were subsequently rescreened to identify the segregating M2 family. Intracellular accumulation of secGFP in cytoplasmic organelles of the mutant seedlings was confirmed by confocal microscopy. The screen identified 97 independent putative mutants, and 73 of these could be propagated to the next generation via their phenotypically WT heterozygous siblings. Initially eight phenotypically 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 and crossed to Ler for approximate mapping by bulk segregant analysis (BSA) (Lukowitz et al., 2000). To do this, four phenotypically WT plants from each mutant family were selected for crossing and their heterozygosity was subsequently assessed by scoring their selfed progeny for mutant phenotypes; another four plants were used if none of the initial set were heterozygotes. Following BSA, four of the mutants that mapped to the top of ChrV and each exhibited similar secGFP accumulation phenotypes were selected for detailed characterization.

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. Samples were washed with 50 mM K2HPO4 solution before transferring to 2% osmium tetroxide for 2 h. After washing with distilled water, the samples were dehydrated in an ethanol series and embedded in


12 Kenneth K. C. Au et al. Spurr’s resin. Sections were examined with a Hitachi H7650 transmission electron microscope (Hitachi High-Technologies Europe GmbH, Maidenhead, Berks, UK).

Confocal laser scanning microscopy Except for simultaneous imaging of GFP and YFP, samples were examined using Zeiss LSM 510 META laser scanning microscope (Carl Zeiss Ltd, Welwyn Garden City, Herts, UK). The configuration of the instrument was described previously (Samalova et al., 2006; Teh and Moore, 2007; Zheng et al., 2004) except for imaging mCHERRY, bimane and c-roGFP1. The mCHERRY was excited with a 543 nm HeNe laser and a HFT 458/543 primary dichroic mirror, and was detected with a 565–615 nm band pass filter. Bimane was excited with a 405 nm blue diode laser and a HFT 458/543 primary dichroic mirror, and was detected with a 475–525 nm band pass filter. For simultaneous imaging of mCHERRY and bimane, the multi-track line-sequential imaging mode with the configuration mentioned above was used. Images were processed using Zeiss LSM Image Browser. In the time course experiment with c-roGFP1, the perfusion chamber was connected to a Gilson Minipuls 2 peristaltic pump (Gilson Inc., Middleton, WI, USA) with HPLC tubing and was mounted on the confocal microscope. Imaging and ratiometric analysis of roGFPs was performed as described (Schwarzlander et al., 2008). Samples coexpressing GFP and YFP were examined using Leica TCS SP5 II. The GFP was excited with the 458 nm argon laser line and was detected with a PMT channel set to collect signal between 475 and 510 nm. The YFP was subsequently excited with the 514 nm argon laser line and was detected with a second PMT channel set to collect signal between 572 and 613 nm. Images were processed 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 label the 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 sapiens) and yeast (Saccharomyces cerevisiae) were obtained from GenBank (Arabidopsis, CAB51027.1; human, NP_000169.1; yeast, CAA 74136.1) and were aligned using CLUSTALW (http://www.ebi.ac.uk/Tools/clustalw2/) to assess the conservation of eukaryotic GSH2. Putative conserved domains of Arabidopsis GSH2 were identified 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 seedlings and analyzed by HPLC as described previously (Cairns et al., 2006).

Immunoblot analysis For the secGFP immunoblot, proteins from 6-day-old seedlings were 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 medium with either DMSO or 5 lg ml)1 tunicamycin (Sigma-Aldrich, Poole, UK). Proteins were extracted first by homogenization in two volumes of extraction buffer: 4% SDS, 100 mM 2-amino-2-(hydroxymethyl)-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 then boiled 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 membrane as described (Batoko et al., 2000). Before incubating with primary antibody, the blot was stained with Ponceau S solution (0.5% Ponceau S and 1% acetic acid) for 5 min and subsequently washed with 1% acetic acid. An image of the blot was taken with a Nikon 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 (a gift from J. Denecke, University of Leeds, UK) were diluted to 1:5000 and 1:10 000, respectively. Alkaline phosphatase-conjugated secondary antibody (anti-rabbit IgG) (Sigma-Aldrich) was used at 1:10 000 dilution.

ACKNOWLEDGEMENTS We thank Markus Schwarzlander, University of Oxford, for assistance on roGFP analysis and comments on the manuscript, Lorenzo Frigerio, University of Warwick, for the anti-BiP antibody, RTNLB1YFP and DER2-YFP constructs, Ju¨rgen Denecke, University of Leeds, for the anti-CRT antibody, Barry Martin and Chris Hawes, Oxford Brookes University for assistance with electron microscopy and for PIP-GFP seeds, and Ru¨diger Hell, Heidelberg University, Germany for access to HPLC. KKCA was funded by the Clarendon Fund and Linacre Canadian Alumni Scholarship. This work was supported by grant BBS/B/09562 from the UK Biotechnology and Biological Sciences Research Council.

SUPPORTING INFORMATION Additional supporting information may be found in the online version 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 labeling of 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 journal provides supporting information supplied by the authors. Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.

REFERENCES Agee, A.E., Surpin, M., Sohn, E.J. et al. (2010) MODIFIED VACUOLE PHENOTYPE1 is an arabidopsis myrosinase-associated protein involved in endomembrane protein trafficking. Plant Physiol. 152(1), 120–132. Ball, L., Accotto, G.P., Bechtold, U. et al. (2004) Evidence for a direct link between glutathione biosynthesis and stress defense gene expression in Arabidopsis. Plant Cell, 16(9), 2448–2462. Banhegyi, G., Benedetti, A., Csala, M. and Mandl, J. (2007) Stress on redox. FEBS Lett. 581(19), 3634–3640. Ba´nhegyi, G., Lusini, L., Puska´s, F., Rossi, R., Fulceri, R., Braun, L., Mile, V., di Simplicio, P., Mandl, J. and Benedetti, A. (1999) Preferential transport of glutathione versus glutathione disulfide in rat liver microsomal vesicles. J. Biol. Chem. 274(18), 12213–12216. Batoko, H., Zheng, H., Hawes, C. and Moore, I. (2000) A Rab1 GTPase is required for transport between the endoplasmic reticulum and golgi apparatus and for normal golgi movement in plants. Plant Cell, 12(11), 2201–2218. Bednarek, S.Y. and Falbel, T.G. (2002) Membrane trafficking during plant cytokinesis. Traffic, 3(9), 621–629.


Arabidopsis glutathione synthase mutant and ER morphology 13 Behnke, H.-. and Eschlbeck, G. (1978) Dilated cisternae in capparales: an attempt towards the characterization of a specific endoplasmic reticulum. Protoplasma, 97(4), 351–363. Berna´, G., Robles, P. and Micol, J.L. (1999) A mutational analysis of leaf morphogenesis in Arabidopsis thaliana. Genetics, 152(2), 729–742. Bertolotti, A., Zhang, Y., Hendershot, L.M., Harding, H.P. and Ron, D. (2000) Dynamic interaction of BiP and ER stress transducers in the unfolded protein response. Nat. Cell Biol. 2(6), 326–332. Boevink, P., Oparka, K., Cruz, S.S., Martin, B., Betteridge, A. and Hawes, C. (1998) Stacks on tracks: the plant golgi apparatus traffics on an actin/ER network? Plant J. 15(3), 441–447. Bolte, S., Talbot, C., Boutte, Y., Catrice, O., Read, N.D. and Satiat-Jeunemaitre, B. (2004) FM-dyes as experimental probes for dissecting vesicle trafficking in living plant cells. J. Microsc. 214(2), 159–173. Cairns, N.G., Pasternak, M., Wachter, A., Cobbett, C.S. and Meyer, A.J. (2006) Maturation of Arabidopsis seeds is dependent on glutathione biosynthesis within the embryo. Plant Physiol. 141(2), 446–455. Chakravarthi, S., Jessop, C.E. and Bulleid, N.J. (2006) The role of glutathione in disulphide bond formation and endoplasmic-reticulum-generated oxidative stress. EMBO Rep. 7(3), 271–275. Chow, C., Neto, H., Foucart, C. and Moore, I. (2008) Rab-A2 and Rab-A3 GTPases define a trans-golgi endosomal membrane domain in Arabidopsis that contributes substantially to the cell plate. Plant Cell, 20(1), 101–123. Christensen, A., Svensson, K., Persson, S., Jung, J., Michalak, M., Widell, S. and Sommarin, M. . (2008) Functional characterization of Arabidopsis Calreticulin1a: a key alleviator of endoplasmic reticulum stress. Plant Cell Physiol. 49(6), 912–924. Clough, S.J. and Bent, A.F. (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16(6), 735–743. Cobbett, C.S., May, M.J., Howden, R. and Rolls, B. (1998) The glutathionedeficient, cadmium-sensitive mutant, cad2-1, of Arabidopsis thaliana is deficient in gamma-glutamylcysteine synthetase. Plant J. 16(1), 73–78. Costa, M.D.L., Reis, P.A.B., Valente, M.A.S., Irsigler, A.S.T., Carvalho, C.M., Loureiro, M.E., Aragao, F.J.L., Boston, R.S., Fietto, L.G. and Fontes, E.P.B.. (2008) A new branch of endoplasmic reticulum stress signaling and the osmotic signal converge on plant-specific asparagine-rich proteins to promote cell death. J. Biol. Chem. 283(29), 20209–20219. Crofts, A.J., Leborgne-Castel, N., Hillmer, S., Robinson, D.G., Phillipson, B., Carlsson, L.E., Ashford, D.A. and Denecke, J. (1999) Saturation of the endoplasmic reticulum retention machinery reveals anterograde bulk flow. Plant Cell, 11(11), 2233–2248. Cutler, S.R., Ehrhardt, D.W., Griffitts, J.S. and Somerville, C.R. (2000) Random GFP-cDNA fusions enable visualization of subcellular structures in cells of Arabidopsis at a high frequency. Proc. Natl Acad. Sci. 97(7), 3718–3723. Dettmer, J. and Friml, J. (2011) Cell polarity in plants: when two do the same, it is not the same. Curr. Opin. Cell Biol. 23(6), 686–696. Dettmer, J., Hong-Hermesdorf, A., Stierhof, Y. and Schumacher, K. (2006) Vacuolar H+-ATPase activity is required for endocytic and secretory trafficking in Arabidopsis. Plant Cell, 18(3), 715–730. Dubreuil-Maurizi, C., Vitecek, J., Marty, L., Branciard, L., Frettinger, P., Wendehenne, D., Meyer, A.J., Mauch, F. and Poinssot, B. (2011) Glutathione deficiency of the Arabidopsis mutant pad2-1 affects oxidative stressrelated events, defense gene expression, and the hypersensitive response. Plant Physiol. 157(4), 2000–2012. Ebine, K., Fujimoto, M., Okatani, Y. et al. (2011) A membrane trafficking pathway regulated by the plant-specific RAB GTPase ARA6. Nat. Cell Biol. 13(7), 853–859. Fahey, R.C., Newton, G.L., Dorian, R. and Kosower, E.M. (1981) Analysis of biological thiols: quantitative determination of thiols at the picomole level based upon derivatization with monobromobimanes and separation by cation-exchange chromatography. Anal. Biochem. 111(2), 357–365. Faso, C., Chen, Y.N., Tamura, K. et al. (2009) A missense mutation in the Arabidopsis COPII coat protein Sec24A induces the formation of clusters of the endoplasmic reticulum and Golgi apparatus. Plant Cell, 21(11), 3655– 3671. Fricker, M.D., May, M., Meyer, A.J., Sheard, N. and White, N.S. (2000) Measurement of glutathione levels in intact roots of Arabidopsis. J. Microsc. 198, (Pt 3) 162–173. Fuji, K., Shimada, T., Takahashi, H., Tamura, K., Koumoto, Y., Utsumi, S., Nishizawa, K., Maruyama, N. and Hara-Nishimura, I. (2007) Arabidopsis

vacuolar sorting mutants (green fluorescent seed) can be identified efficiently by secretion of vacuole-targeted green fluorescent protein in their seeds. Plant Cell, 19(2), 597–609. Griffith, O.W. and Meister, A. (1979) Potent and specific inhibition of glutathione synthesis by buthionine sulfoximine (S-n-butyl homocysteine sulfoximine). J. Biol. Chem. 254, 7558–7560. Hawes, C., Saint-Jore, C., Martin, B. and Zheng, H.Q. (2001) ER confirmed as the location of mystery organelles in Arabidopsis plants expressing GFP!. Trends Plant Sci. 6(6), 245–246. Hell, R. and Bergmann, L. (1990) k-Glutamylcysteine synthetase in higher plants: catalytic properties and subcellular localization. Planta, 180(4), 603–612. Herrera, K., Cahoon, R.E., Kumaran, S. and Jez, J.M. (2007) Reaction mechanism of glutathione synthetase from arabidopsis thaliana. J. Biol. Chem. 282(23), 17157–17165. Huotari, J. and Helenius, A. (2011) Endosome maturation. EMBO J. 30(17), 3481–3500. Hwang, C., Sinskey, A.J. and Lodish, H.F. (1992) Oxidized redox state of glutathione in the endoplasmic reticulum. Science, 257(5076), 1496–1502. Iversen, T. (1970) The morphology, occurrence, and distribution of dilated cisternae of the endoplasmic reticulum in tissues of plants of the Cruciferae. Protoplasma, 71(4), 467–477. Iwata, Y. and Koizumi, N. (2005) An Arabidopsis transcription factor, AtbZIP60, regulates the endoplasmic reticulum stress response in a manner unique to plants. Proc. Natl Acad. Sci. USA, 102(14), 5280–5285. Jessop, C.E. and Bulleid, N.J. (2004) Glutathione directly reduces an oxidoreductase in the endoplasmic reticulum of mammalian cells. J. Biol. Chem. 279(53), 55341–55347. Jobe, T.O., Sung, D.-Y., Akmakjian, G., Pham, A., Konives, E.A., MendozaCo´zatl, D.G. and Schroeder, J.I. (2012) Feedback inhibition by thiols outranks glutathione depletion: a luciferase-based screen reveals glutathione-deficient c-ECS and glutathione synthetase mutants impaired in cadmium-induced sulfate assimilation. Plant J. 70, 783–795. Ju¨rgens, G., Mayer, U., Ramon, A.T.R., Berleth, T. and Mise´ra, S. (1991) Genetic analysis of pattern formation in the Arabidopsis embryo. Development, 113, (Supplement 1) 27–38. Kawagoe, Y., Suzuki, K., Tasaki, M., Yasuda, H., Akagi, K., Katoh, E., Nishizawa, N.K., Ogawa, M. and Takaiwa, F. (2005) The critical role of disulfide bond formation in protein sorting in the endosperm of rice. Plant Cell, 17(4), 1141–1153. Kienle, N., Kloepper, T. and Fasshauer, D. (2009) Phylogeny of the SNARE vesicle fusion machinery yields insights into the conservation of the secretory pathway in fungi. BMC Evol. Biol. 9(1), 19. Koizumi, N., Martinez, I.M., Kimata, Y., Kohno, K., Sano, H. and Chrispeels, M.J. (2001) Molecular characterization of two Arabidopsis Ire1 homologs, endoplasmic reticulum-located transmembrane protein kinases. Plant Physiol. 127(3), 949–962. Le Gall, S., Neuhof, A. and Rapoport, T. (2004) The endoplasmic reticulum membrane is permeable to small molecules. Mol. Biol. Cell 15(2), 447– 455. Liu, J. and Howell, S.H. (2010) Endoplasmic reticulum protein quality control and its relationship to environmental stress responses in plants. Plant Cell, 22(9), 2930–2942. Lohman, J.R. and Remington, S.J. (2008) Development of a family of redoxsensitive green fluorescent protein indicators for use in relatively oxidizing subcellular environments. Biochemistry, 47(33), 8678–8688. Lukowitz, W., Gillmor, C.S. and Scheible, W.R. (2000) Positional cloning in Arabidopsis. why it feels good to have a genome initiative working for you. Plant Physiol. 123(3), 795–805. Marty, L., Siala, W., Schwarzla¨nder, M., Fricker, M.D., Wirtz, M., Sweetlove, L.J., Meyer, Y., Meyer, A.J., Reichheld, J. and Hell, R. (2009) The NADPHdependent thioredoxin system constitutes a functional backup for cytosolic glutathione reductase in Arabidopsis. Proc. Natl Acad. Sci. 106(22), 9109– 9114. Matsushima, R., Hayashi, Y., Yamada, K., Shimada, T., Nishimura, M. and Hara-Nishimura, I. (2003) The ER body, a novel endoplasmic reticulumderived structure in Arabidopsis. Plant Cell Physiol. 44(7), 661–666. Meyer, A.J. (2008) The integration of glutathione homeostasis and redox signaling. J. Plant Physiol. 165(13), 1390–1403. Meyer, A.J., May, M.J. and Fricker, M. (2001) Quantitative in vivo measurement of glutathione in Arabidopsis cells. Plant J. 27(1), 67–78.


14 Kenneth K. C. Au et al. Muller, I., Wagner, W., Volker, A., Schellmann, S., Nacry, P., Kuttner, F., Schwarz-Sommer, Z., Mayer, U. and Jurgens, G. (2003) Syntaxin specificity of cytokinesis in Arabidopsis. Nat. Cell Biol. 5(6), 531–534. Nakano, R.T., Matsushima, R., Ueda, H., Tamura, K., Shimada, T., Li, L., Hayashi, Y., Kondo, M., Nishimura, M. and Hara-Nishimura, I. (2009) GNOM-LIKE1/ERMO1 and SEC24a/ERMO2 are required for maintenance of endoplasmic reticulum morphology in Arabidopsis thaliana. Plant Cell, 21(11), 3672–3685. Nelson, B.K., Cai, X. and Nebenfu¨hr, A. (2007) A multicolored set of in vivo organelle markers for co-localization studies in Arabidopsis and other plants. Plant J. 51(6), 1126–1136. Noctor, G., Veljovic-Jovanovic, S. and Foyer, C.H. (2000) Peroxide processing in photosynthesis: antioxidant coupling and redox signalling. Philos. Trans. R. Soc. Lond. B Biol. Sci. 355(1402), 1465–1475. Novick, P. and Schekman, R. (1979) Secretion and cell-surface growth are blocked in a temperature-sensitive mutant of saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA, 76(4), 1858–1862. Nziengui, H., Bouhidel, K., Pillon, D., Der, C., Marty, F. and Schoefs, B. (2007) Reticulon-like proteins in arabidopsis thaliana: structural organization and ER localization. FEBS Lett. 581(18), 3356–3362. Oertle, T., Klinger, M., Stuermer, C.A. and Schwab, M.E. (2003) A reticular rhapsody: phylogenic evolution and nomenclature of the RTN/Nogo gene family. FASEB J. 17(10), 1238–1247. Onda, Y., Kumamaru, T. and Kawagoe, Y. (2009) ER membrane-localized oxidoreductase Ero1 is required for disulfide bond formation in the rice endosperm. Proc. Nat. Acad. Sci. 106(33), 14156–14161. Orsi, A., Sparvoli, F. and Ceriotti, A. (2001) Role of individual disulfide bonds in the structural maturation of a low molecular weight glutenin subunit. J. Biol. Chem. 276(34), 32322–32329. Parisy, V., Poinssot, B., Owsianowski, L., Buchala, A., Glazebrook, J. and Mauch, F. (2007) Identification of PAD2 as a c-glutamylcysteine synthetase highlights the importance of glutathione in disease resistance of Arabidopsis. Plant J. 49(1), 159–172. Pasternak, M., Lim, B., Wirtz, M., Hell, R., Cobbett, C.S. and Meyer, A.J. (2008) Restricting glutathione biosynthesis to the cytosol is sufficient for normal plant development. Plant J. 53(6), 999–1012. Pinheiro, H., Samalova, M., Geldner, N., Chory, J., Martinez, A. and Moore, I. (2009) Genetic evidence that the higher plant Rab-D1 and Rab-D2 GTPases exhibit distinct but overlapping interactions in the early secretory pathway. J. Cell Sci. 122(20), 3749–3758. Pompa, A. and Vitale, A. (2006) Retention of a bean phaseolin/maize gammazein fusion in the endoplasmic reticulum depends on disulfide bond formation. Plant Cell, 18(10), 2608–2621. Reisen, D., Leborgne-Castel, N., Ozalp, C., Chaumont, F. and Marty, F. (2003) Expression of a cauliflower tonoplast aquaporin tagged with GFP in tobacco suspension cells correlates with an increase in cell size. Plant Mol. Biol. 52(2), 387–400. Richter, S., Geldner, N., Schrader, J., Wolters, H., Stierhof, Y., Rios, G., Koncz, C., Robinson, D.G. and Jurgens, G. (2007) Functional diversification of closely related ARF-GEFs in protein secretion and recycling. Nature, 448(7152), 488–492. Ridge, R.W., Uozumi, Y., Plazinski, J., Hurley, U.A. and Williamson, R.E. (1999) Developmental transitions and dynamics of the cortical ER of arabidopsis cells seen with green fluorescent protein. Plant Cell Physiol. 40(12), 1253– 1261. Rojo, E., Gillmor, C.S., Kovaleva, V., Somerville, C.R. and Raikhel, N.V. (2001) VACUOLELESS1 is an essential gene required for vacuole formation and morphogenesis in Arabidopsis. Dev. Cell 1(2), 303–310.

Samalova, M., Fricker, M. and Moore, I. (2006) Ratiometric fluorescenceimaging assays of plant membrane traffic using polyproteins. Traffic, 7(12), 1701–1723. Sanderfoot, A. (2007) Increases in the number of SNARE genes parallels the rise of multicellularity among the green plants. Plant Physiol. 144(1), 6–17. Schwarzlander, M., Fricker, M.D., Muller, C., Marty, L., Brach, T., Novak, J., Sweetlove, L.J., Hell, R. and Meyer, A.J. (2008) Confocal imaging of glutathione redox potential in living plant cells. J. Microsc. 231(2), 299–316. Shanmugam, V., Tsednee, M. and Yeh, K. (2012) ZINC TOLERANCE INDUCED BY IRON 1 reveals the importance of glutathione in the cross-homeostasis between zinc and iron in Arabidopsis thaliana. Plant J. 69(6), 1006–1017. Sparkes, I., Tolley, N., Aller, I., Svozil, J., Osterrieder, A., Botchway, S., Mueller, C., Frigerio, L. and Hawes, C. (2010) Five Arabidopsis reticulon isoforms share endoplasmic reticulum location, topology, and membraneshaping properties. Plant Cell, 22(4), 1333–1343. Surpin, M., Zheng, H., Morita, M.T. et al. (2003) The VTI family of SNARE proteins is necessary for plant viability and mediates different protein transport pathways. Plant Cell, 15(12), 2885–2899. Tanaka, H., Kitakura, S., De Rycke, R., De Groodt, R. and Friml, J. (2009) Fluorescence imaging-based screen identifies ARF GEF component of early endosomal trafficking. Curr. Biol. 19(5), 391–397. Teh, O. and Moore, I. (2007) An ARF-GEF acting at the golgi and in selective endocytosis in polarized plant cells. Nature, 448(7152), 493–496. Tolley, N., Sparkes, I.A., Hunter, P.R., Craddock, C.P., Nuttall, J., Roberts, L.M., Hawes, C., Pedrazzini, E. and Frigerio, L. (2008) Overexpression of a plant reticulon remodels the lumen of the cortical endoplasmic reticulum but does not perturb protein transport. Traffic, 9(1), 94–102. Toyooka, K., Okamoto, T. and Minamikawa, T. (2000) Mass transport of proform of a KDEL-tailed cysteine proteinase (sh-EP) to protein storage vacuoles by endoplasmic reticulum–derived vesicle is involved in protein mobilization in germinating seeds. The Journal of Cell Biology, 148(3), 453– 464. Ueda, T., Yamaguchi, M., Uchimiya, H. and Nakano, A. (2001) Ara6, a plantunique novel type Rab GTPase, functions in the endocytic pathway of arabidopsis thaliana. EMBO J. 20(17), 4730–4741. Vernoux, T., Wilson, R.C., Seeley, K.A. et al. (2000) The ROOT MERISTEMLESS1/CADMIUM SENSITIVE2 gene defines a glutathione-dependent pathway involved in initiation and maintenance of cell division during postembryonic root development. Plant Cell, 12(1), 97–110. Voeltz, G.K., Prinz, W.A., Shibata, Y., Rist, J.M. and Rapoport, T.A. (2006) A class of membrane proteins shaping the tubular endoplasmic reticulum. Cell, 124(3), 573–586. West, M., Zurek, N., Hoenger, A. and Voeltz, G.K. (2011) A 3D analysis of yeast ER structure reveals how ER domains are organized by membrane curvature. The Journal of Cell Biology, 193(2), 333–346. Woollard, A.A. and Moore, I. (2008) The functions of Rab GTPases in plant membrane traffic. Curr. Opin. Plant Biol. 11(6), 610–619. Yamada, K., Nagano, A.J., Nishina, M., Hara-Nishimura, I. and Nishimura, M. (2008) NAI2 is an endoplasmic reticulum body component that enables ER body formation in Arabidopsis thaliana. Plant Cell, 20(9), 2529– 2540. Zechmann, B., Mauch, F., Sticher, L. and Mu¨ller, M. (2008) Subcellular immunocytochemical analysis detects the highest concentrations of glutathione in mitochondria and not in plastids. J. Exp. Bot. 59(14), 4017– 4027. Zheng, H., Kunst, L., Hawes, C. and Moore, I. (2004) A GFP-based assay reveals a role for RHD3 in transport between the endoplasmic reticulum and Golgi apparatus. Plant J. 37(3), 398–414.
