Glutathione Peroxidase‐Like Enzymes Cover Five Distinct Cell Compartments

Plant, Cell and Environment (2017) 40,1281–1295 doi: 10.1111/pce.12919

Original Article Glutathione peroxidase-like enzymes cover five distinct cell compartments and membrane surfaces in Arabidopsis thaliana

Safira Attacha1, David Solbach1, Krisztina Bela1,2,AnnaMoseler1, Stephan Wagner1, Markus Schwarzländer1 , Isabel Aller1, Stefanie J. Müller1 & Andreas J. Meyer1

1INRES-Chemical Signalling, University of Bonn, Friedrich-Ebert-Allee 144, 53113 Bonn, Germany and 2Department of Plant Biology, Faculty of Science and Informatics, University of Szeged, Közép fasor 52, H-6726 Szeged, Hungary

ABSTRACT ROS may directly damage biological molecules such as nucleic acids, amino acids, proteins and lipids. A particularly damaging Glutathione peroxidase-like enzymes (GPXLs) constitute a effect results from the onset of autocatalytic lipid peroxidation family of eight peroxidases in Arabidopsis thaliana.Incon- leading to loss of membrane integrity. To protect themselves trast to the eponymous selenocysteine glutathione peroxidases from damage, plants have evolved multiple detoxification sys- in mammalian cells that use glutathione as electron donor, tems for efficient removal of H O and phospholipid hydroper- GPXLs rely on cysteine instead of selenocysteine for activity 2 2 oxides. Besides acting as damaging toxins, peroxides are also and depend on the thioredoxin system for reduction. Although considered essential elements of signalling pathways involved plant GPXLs have been implicated in important agronomic in stress sensing and of the coordinated activation of defence traits such as drought tolerance, photooxidative tolerance and pathways. Molecular understanding of ROS-dependent signal- immune responses, there remain major ambiguities regarding ling pathways is essential to evaluate whether and how they their subcellular localization. Because their site of action is a may be suitable targets for selection and breeding of more prerequisite for an understanding of their function, we investi- stress resilient crops. gated the localization of all eight GPXLs in stable Arabidopsis Detoxification of peroxides can be catalysed by catalase in lines expressing N-terminal and C-terminal fusions with redox- peroxisomes, by ascorbate peroxidases and the ascorbate- sensitive green fluorescent protein 2 (roGFP2) using confocal glutathione cycle in the cytosol, plastids, mitochondria and per- microscopy. GPXL1 and GPXL7 were found in plastids, while oxisomes, as well as by peroxiredoxins, several glutathione-S GPXL2 and GPXL8 are cytosolic nuclear. The N-terminal transferases and glutathione peroxidases (GPXs) (Dixon & target peptide of GPXL6 is sufficient to direct roGFP2 into Edwards 2010; Mittler 2002). While the mechanism of most mitochondria. Interestingly, GPXL3, GPXL4 and GPXL5 all of these enzymes has been studied in quite some detail, rela- appear to be membrane bound. GPXL3 was found exclusively tively little is known about GPXs in plants, despite their in the secretory pathway where it is anchored by a single occurrence in multiple isoforms that suggest important and N-terminal transmembrane domain. GPXL4 and GPXL5 are non-redundant functions. Selenocysteine (Sec) containing anchored to the plasma membrane. Presence of an N-terminal mammalian GPx1 is regarded as the prototype GPX that uses myristoylation motif and genetic disruption of membrane associ- reduced glutathione (GSH) as obligate electron donor for ation through targeted mutagenesis point to myristoylation as peroxide reduction (Lubos et al. 2011). However, not all GPXs essential for membrane localization. (defined by homology) use GSH, nor do they all contain Sec as catalytic residue. Rather, several homologues contain a redox- Key-words: endoplasmic reticulum; Golgi; H2O2; lipid hydro- active Cys at the position of the Sec and display highest activity peroxide; myristoylation; plasma membrane; roGFP2; subcel- towards phospholipid hydroperoxides with thioredoxin (TRX) lular compartmentation; transmembrane domain. as reductant (Lubos et al. 2011). Based on the substrates and the electron donor, these homologues have been referred to INTRODUCTION as phospholipid hydroperoxide GPX, TRX peroxidases or A common feature of plants being exposed to diverse forms of GPX-type enzymes (Bela et al. 2015; Maiorino et al. 2015; environmental stress is the risk of excessive formation of reac- Schlecker et al. 2005). In Synechocystis, two homologues have fi tive oxygen species (ROS) by photosynthesis and respiration. been identi ed that were not able to utilize GSH as an electron ROS production, however, is not restricted to the electron donor and hence were named GPX-like enzymes (Gaber et al. transport chains but occurs at significant rates at the plasma 2001). The genome of Arabidopsis thaliana encodes a family of membrane via NADPH oxidases, in peroxisomes from multi- eight GPX homologues all of which carry a Cys in their active ple metabolic pathways and in the endoplasmic reticulum site (Rodriguez Milla et al. 2003). The biochemical evidence (ER) as a result of oxidative protein folding. If not detoxified, that is available for plant GPX homologues overall indicates a strong preference for the TRX system instead of GSH as Correspondence: A. J. Meyer. e-mail: [email protected] electron donor (Iqbal et al. 2006; Navrot et al. 2006). To avoid

©2017JohnWiley&SonsLtd 1281 1282 S. Attacha et al. confusion resulting from protein names that are based on development (Pagnussat et al. 2005). Expression studies on homology and thus misleadingly suggest a functional link to different crop species including citrus, tomato, sunflower, glutathione, we adopt the nomenclature GPX-like (GPXL) barley and soybean have shown pronounced induction of for the Arabidopsis isoforms described in this work. several GPXL genes in response to abiotic and biotic stress Glutathione peroxidase-like enzymes have been implicated factors (see Bela et al. 2015 for a comprehensive review). The in efficient reduction of H2O2, organic hydroperoxides and documented changes in expression patterns in time and tissue phospholipid hydroperoxides in human, yeast and plants suggest important roles of GPXLs in stress defence and (Bela et al. 2015; Maiorino et al. 2015). In addition, there is acclimation. ample evidence that GPXLs may also act as H2O2 sensors A major limitation in the interpretation of expression data enabling the transfer of the primary oxidation from the and stress-dependent phenotypes of mutants is the lack of peroxidatic Cys in the active site to specific target proteins with reliable information on the subcellular localization of GPXLs. a regulatory or signalling role. In Saccharomyces cerevisiae,the While all Arabidopsis isoforms except GPXL7 have been GPX-like enzyme Orp1 (syn. Gpx3) is first oxidized to a detected by proteomic approaches, localization results are sulfenic acid, which can in turn undergo the formation of a often inconsistent as exemplified in the previous discussion mixed disulfide with the transcription factor Yap1. In a second for GPXL3. In the absence of suitable antibodies for step, Yap1 releases Orp1 through nucleophilic attack by a sec- immunogold labelling and electron microscopy, the use of ond thiol and formation of an internal disulfide on Yap1 lead- fluorescent proteins has been developed as a suitable comple- ing to its activation (Delaunay et al. 2002). This relay mentary approach for protein localization studies (Nelson mechanism has been exploited for the development of geneti- et al. 2007). In Arabidopsis, localization data for expression of cally encoded H2O2 sensors (Gutscher et al. 2009). A role in GFP fusions are available only for GPXL8 that has been re- stress-related H2O2 signalling has also been implicated for ported to localize to the cytosol and the nucleus (Gaber et al. Arabidopsis GPXL3. Miao and colleagues reported gpxl3 null 2012). Without robust localization data for the entire GPXL mutants as drought sensitive and GPXL3 overexpressor lines family, however, the generation of suitable hypotheses as drought tolerant (Miao et al. 2006). Based on this observa- concerning isoform-specific functions and non-redundant roles tion, the authors hypothesized that GPXL3 might be involved specific to different subcellular compartments is hindered. in drought stress signalling in guard cells through interference Associated with this is the risk that inappropriate assumptions with the type 2C Ser/Thr phosphatases ABI1 and ABI2 during about the subcellular localization may mislead future research the abscisic acid response. To support their hypothesis, Miao by stimulating flawed hypotheses for further functional et al. provided data indicating physical interaction of GPXL3 analysis. with ABI1 and ABI2 in both yeast two-hybrid and pull-down as- The aim of this work is to address this shortcoming and to says as well as bimolecular fluorescence complementation for provide comprehensive information on the subcellular localiza- GPXL3 and ABI2 fused with yellow fluorescent protein tion of all eight GPXLs in Arabidopsis thaliana.GPXLswere (YFP)-fragments complementing each other in the cytosol. fused with redox-sensitive GFP2 (roGFP2) as a functional fluo- Based on these results and on transient expression of green fluorescent reporter. roGFP2 instead of GFP has the advantage of rescent protein (GFP) fusions in protoplasts, the authors pro- self-indicating reducing and oxidizing compartments, which posed that GPXL3 functions as both a cytosolic redox can enable topology analysis of membrane proteins transducer and a scavenger of H2O2 in abscisic acid and drought (Brach et al. 2009). Fusion of GFPs to proteins of interest and stress responses. The proposed localization of GPXL3 in the cy- heterologous expression of these proteins at high levels in tran- tosol, however, conflicts with annotations based on bioinformat- sient expression systems inherently bears the risk of creating ics that predict mitochondrial targeting, and with proteomic data new artefacts through mistargeting. To minimize this risk, all that indicate the presence of GPXL3 in the Golgi or in plastids fusion proteins were not only expressed heterologously in (Helm et al. 2014; Nikolovski et al. 2012; Rodriguez Milla et al., tobacco but also in stably transformed Arabidopsis plants. 2003). The proteomic approach, however, also remains incon- clusive as organellar proteomes assembled from mass spectrometry data frequently suffer from contaminations from other MATERIALS AND METHODS compartments and because the techniques used cannot easily Plant growth conditions distinguish between proteins on the surface or within a given organelle. The latter limitation is particularly challenging for Arabidopsis thaliana (L.) Heynh ecotype Col-0 seeds were membrane-associated proteins with yet unknown topology. germinated on a soil mixture (Floragard, www.floragard.de) Recently, it has been reported that several GPXL isoforms supplemented with perlite and quartz sand in a ratio of 10:1:1. mediate redox signalling in the control of root architecture Plants were kept in a controlled growth chamber under long and that plastid-localized GPXL1 and GPXL7 in Arabidopsis day condition with a diurnal cycle of 16 h light at 19 °C and are required for the hormone-mediated control of lateral root 8 h dark at 17 °C. Humidity of the growth chamber was around À À development (Passaia et al. 2014). In contrast to roots, the 40%, and the light intensity was 100–120 μEm 2 s 1.Tobacco shoots of T-DNA insertional mutants for most Arabidopsis plants (Nicotiana benthamiana Domin) were grown in a soil GPXLs do not show major deviations from wild-type plants mixture at 22 °C (night/day) under a 16 h photoperiod with a À À (Passaia et al. 2014). An exception is gpxl5 that has been iden- light intensity of 120 μEm 2 s 1. To grow plants under axenic tified in a screen for mutants affected in female gametophyte conditions on plates, seeds were surface sterilized with 70% © 2017 John Wiley & Sons Ltd, Plant, Cell and Environment, 40,1281–1295 Subcellular localization of GPXLs in Arabidopsis 1283

(v/v) ethanol and placed on nutrient medium [5 mM KNO3, transformants making use of the roGFP2 fluorescence under 2.5 mM KH2PO4,2mM MgSO4,2mM Ca(NO3)2,50μM a Leica M165FC stereomicroscope equipped with a Leica Fe-EDTA, 0.1% (v/v) micronutrient mix (70 mM H3BO4, DCF425C camera for image recording (Leica Microsystems, 14 mM MnCl2, 0.5 mM CuSO4,1mM ZnSO4, 0.2 mM NaMoO4, Wetzlar, Germany). A GFP filter of 470 ± 40 nm excitation 10 mM NaCl and 0.01 mM CoCl2), 0.8% (w/v) agar and pH 5.8] and emission at 525 ± 50 nm was used. Arabidopsis plants trans- and antibiotics for selection when required. formed with constructs harbouring the Bar gene mediating Basta® resistance were grown on soil under long-day condi- À1 Construction of redox-sensitive GFP2 fusions tions. Plants in two-leaf stage were sprayed with a 240 mg L glufosinate ammonium solution (Basta; Bayer Crop Science). To obtain C-terminal and N-terminal fusions of full-length The treatment was repeated 1 week later. Resistance became GPXLs with roGFP2, roGFP2 was cloned in frame with obvious 5 days after Basta application; non-resistant plants GPXLs by Gateway Cloning (Invitrogen, Carlsbad, CA, died shortly afterwards. USA) using the vector pSS01 for C-terminal fusion and the vector pCM01 for N-terminal fusion (Brach et al. 2009). On both T-DNAs, expression of the fusion constructs is driven by Fluorescence imaging the CaMV 35S promoter. The primers for C-terminal and Ratiometric imaging of transgenic Arabidopsis lines expressing N-terminal fusions were designated GPXLx_F for the forward roGFP2 constructs was performed on a confocal laser scanning primer and GPXLx_C and N for the reverse primers for microscope (Zeiss LSM 780, attached to an Axio Observer.Z1; C-terminal and N-terminal roGFP2 fusions, respectively Carl Zeiss Microscopy, Jena, Germany). Images were collected (Table S1). Fusion of the truncated constructs of GPXL3, with a 40× (C-Apochromat 40×/1.2 W Korr) or a 63× lens GPXL4, GPXL5 and GPXL6 to the N-terminus of roGFP2 (Plan-Apochromat 63×/1.40 Oil DIC). roGFP2 was excited at was achieved through assembly PCR using gene specific 405 and 488 nm. Fluorescence emission was collected from primers(TableS1).Inthefirst round, the nucleotide sequence 505 to 530 nm, and intensities from four scans were averaged. fi of GPXL31–34 was ampli ed using primers P6 and P23, For co-localization of roGFP2 fusion proteins in mitochondria, GPXL3Δ1–34 using P27 and P26, GPXL4G2A using P28 and seedlings were incubated with 0.5 μM tetramethylrhodamine P29, GPXL5G2A using P31 and P32 and GPXL61–65 using P15 methyl ester (TMRM) for at least 15 min at RT. TMRM stain- and P34. The second primer of each pair overlaps with ing was recorded with excitation at 543 nm and emission roGFP2. roGFP2 was amplified in the second round using recorded from 560 to 615 nm. In case of GFP localization at primers P24 and P36 for GPXL31–34,P25andP37for the plasma membrane, seedlings were incubated with 5 μM GPXL3Δ1–34, P30 and P36 for GPXL4G2A, P33 and P36 for FM4-64 for 2 min on ice. FM4-64 was excited at 488 nm, while GPXL5G2A and P35 and P36 for GPXL61–65. The resulting emission was detected between 620 and 680 nm in root tissues. PCR products were mixed 1:1 and amplified using primers P6 Chlorophyll autofluorescence was excited with 488 nm and and P36 for GPXL31–34,P27andP37forGPXL3Δ1–34,P28 collected from 647 to 745 nm. and P36 for GPXL4 G2A, P31 and P36 for GPXL5G2A and Ratiometric analysis was performed using a custom-written P15 and P36 for GPXL61–65. The resulting fragments were MATLAB script (Fricker 2016). The ratio analysis was purified and mixed with pDONR201 for the BP reaction and performed on a pixel-by-pixel basis as 405/488 ratio following recombined in the LR reaction with the destination vectors averaging in (x,y) using a 3 × 3 kernel, correction of the pB7WG2 or pK7WG2 (Karimi et al. 2005). 405 nm channel for autofluorescence bleed through into the 488 nm channel and subtraction of background signals for each Transient and stable transformation channel. Transformation of tobacco leaf epidermal cells was performed as described previously (Sparkes et al. 2006) using RESULTS Agrobacterium tumefaciens strain AGL1 (Lazo et al. 1991) con- Sequence analysis and predicted subcellular taining binary vectors with DNA-fragments coding for roGFP2 localization fusion proteins. Transfected cells were imaged by confocal laser scanning microscopy 2–4 days after inoculation. Stable The Arabidopsis genome encodes eight GPXL isoforms, which transformation of Arabidopsis was achieved by the floral dip have been predicted to be localized in different subcellular method (Clough & Bent 1998). After electroporation of binary compartments. Different bioinformatics algorithms, however, vectors into AGL1, the bacteria were incubated in 30 mL selec- lead to different predictions, and, where available, experimen- À tive lysogeny broth medium (50 μgmL 1 rifampicin, tal evidence is frequently inconsistent with predictions (Fig. 1). À À 100 μgmL 1 ampicillin, 100 μgmL 1 spectinomycin and The amino acid alignment suggests the presence of N-terminal À 50 μgmL 1 kanamycin) for 24 h on a shaker at 28 °C and targeting signals for GPXL1, GPXL6 and GPXL7 (Fig. S1). 220 rpm as preculture. Subsequently, 300 mL of selective lysog- While the sequences of GPXL1 and 7 have been associated eny broth medium was inoculated with this preculture and with a strong probability of plastid targeting, the situation is incubated for a further 24 h under the same conditions. Seeds more ambiguous for GPXL6. The highest scoring prediction were sterilized and placed on nutrient medium solidified with is mitochondria, but some algorithms also predict the plasma

0.8% (w/v) micro agar. The T1 generation was screened for membrane, the plastids and the nucleus as putative targets ©2017JohnWiley&SonsLtd, Plant, Cell and Environment, 40,1281–1295 1284 S. Attacha et al.

Figure 1. Phylogenetic tree of the Arabidopsis GPXL protein family. The unrooted tree is based on a multiple sequence alignment of amino acids 77 to 160 of GPXL1 and the respective homologous sequences from other GPXLs using MAFFT (Katoh & Standley 2013) with default settings and was constructed using MrBayes (Ronquist & Huelsenbeck 2003). Node values correspond to posterior probabilities. The scale bar indicates the expected changes per site. The colour code displays subcellular localizations predicted by SUBAcon (Hooper et al. 2014). Additional localizations for which bioinformatics prediction as documented in SUBA3 (Tanz et al. 2013) exist are indicated in parentheses and compartments for which experimental evidence is available are underlined. V, vacuole; CW, cell wall; G, Golgi; N, nucleus; PM, plasma membrane; ER, endoplasmic reticulum. GPXL1, At2g25080; GPXL2, At2g31570; GPXL3, At2g43350; GPXL4, At2g48150; GPXL5, At3g63080; GPXL6, At4g11600; GPXL7, At4g31870; GPXL8, At1g63460.

(Fig. 1). GPXL3 contains a short 39 amino acids long was C-terminally tagged with roGFP2. The roGFP2-tag was N-terminal extension compared with GPXL2 and GPXL8 exploited as a simple GFP and imaged together with chloro- (Fig. S1). Most bioinformatics algorithms interpret the first 12 phyll autofluorescence. All images from parenchyma cells amino acids of this extension as a mitochondrial targeting revealed co-localization of GPXL1-roGFP2 with chlorophyll signal to guide the mature protein to the mitochondrial import autofluorescence depicted as a yellow merge colour machinery (Fig. 1). This, however, contrasts with experimental (Fig. 2e–h). Epidermal pavement cell chloroplasts with low evidence for the cytosol, the Golgi and plastids (Helm et al. chlorophyll content appear green in the merge image (Fig. 2h). 2014; Miao et al. 2006; Nikolovski et al. 2012). All other GPXLs Even distribution of fluorescence suggests localization of the are predicted to be localized at the plasma membrane or the fusion protein in the stroma, rather than to the thylakoids. This cytosol, but again, experimental evidence from proteome conclusion is further supported by the visualization of analyses and protein–protein interaction studies is not always roGFP2-labelled stromules protruding from chloroplasts in consistent with the predictions (Fig. 1). tobacco leaf epidermal cells (Fig. S2). To investigate the subcellular targeting of GPXLs, fusion Glutathione peroxidase-like enzyme 7 is also predicted to be proteins with roGFP2 were generated and initially expressed targeted to plastids (Fig. 1), but this localization has not been transiently in tobacco leaves. roGFP2 can be imaged as a explored experimentally. C-terminal fusions of GPXL7 with conventional GFP, but it has the additional feature of self- roGFP2 resulted in plastidic localization again with identifica- indicating reducing and oxidizing subcellular compartments, tion of individual epidermal plastids that displayed a strong which can allow fine-determination of membrane protein roGFP2 signal in the background of low chlorophyll signal topology (Brach et al. 2009) (Fig. 3a). (Fig. 2i–l).

GPXL1-roGFP2 and GPXL7-roGFP2 are targeted to GPXL2 and GPXL8 are soluble cytosolic-nuclear plastids proteins Glutathione peroxidase-like enzyme 1 has been identiﬁed as a Glutathione peroxidase-like enzymes 2 and 8 both lack obvious plastidic protein more than a decade ago (Kleffmann et al. target peptides (Fig. S1) and were predicted to localize in the 2004). Ambiguity results from proteome studies that have cytosol. For experimental validation, both proteins were reported GPXL1 to reside at the chloroplast envelope (Ferro C-terminally tagged with roGFP2 and stably expressed in et al. 2003) or on thylakoid membranes (Peltier et al. 2004). Arabidopsis. Ratiometric analysis of a cytosolic roGFP2 con- To experimentally resolve the subcellular localization, GPXL1 trol results in low 405/488 nm ﬂuorescence excitation ratios

Figure 2. Glutathione peroxidase-like enzyme 1-roGFP2 and GPXL7-roGFP2 fusion proteins are targeted to plastids. Confocal images represent untransformed Arabidopsis plants (a–d) and plants expressing either GPXL1-roGFP2 (e–h) or GPXL7-roGFP2 (i–l) fusion protein, respectively. The nominally ratiometric roGFP2 was excited at 488 nm only and imaged simultaneously with chlorophyll autofluorescence excited at 633 nm. Epidermal pavement cell chloroplasts with low chlorophyll content appear green in the merge image (arrow head). Scale bars = 20 μm.

indicating a reduced roGFP2 sensor false-coloured in blue the N-terminal fusion preferentially labelled the ER network (Fig. 3a). A roGFP2 ER lumen control shows high 405/488 nm with the ring-like appearance of the nuclear envelope as a fluorescence excitation ratios indicating an oxidized roGFP2 characteristic structure (Fig. S4a). The roGFP2 tag, however, false coloured in red (Fig. 3a). Given the pronounced ratio remained reduced indicating that the fusion protein decorated difference resulting in a binary readout, no further quantitative the cytosolic face of the endomembranes. In contrast to the analysis is necessary, although the spatial pattern of fluores- complete absence of mitochondrial targeting in tobacco, stable cence signal may be used for independent validation. For both, expression of GPXL6-roGFP2 inArabidopsisresulted inpartial GPXL2 and GPXL8, the C-terminally fused roGFP2 displayed targeting to the mitochondrial matrix as visualized through low fluorescence ratios confirming localization in the cytosol co-localization of roGFP2 with the mitochondrial marker and the nucleus (Fig. 3b). To further exclude the possibility of TMRM (Fig. 4a). In addition, GPXL6-roGFP2 was found in cryptic C-terminal targeting signals for peroxisomes, both the cytosol and the nucleus (Fig. 4b) and occasionally, in highly proteins were also fused to the C-terminus of roGFP2 and expressing lines again on the cytosolic face of the ER membrane transiently expressed in tobacco leaves. These fusion constructs (Fig. S4b). To further test for the function of the putative were also found in the cytosol and the nucleus (Fig. S3). targeting signal, the first 65 amino acids were fused to the

N-terminus of roGFP2. Stable expression of GPXL61–65- GPXL6-roGFP2 fusions are dual targeted to roGFP2 in Arabidopsis resulted in clear mitochondrial localization, as indicated by strict co-localization with the mitochondria and cytosol TMRM signal (Fig. 4c). The protein sequence of GPXL6 indicates an N-terminal 65 amino acid stretch that is interpreted as a mitochondrial GPXL4 and GPXL5 are anchored to the plasma targeting signal by most bioinformatics algorithms (Fig. S1 and membrane Fig. 1). GPXL6, N-terminal and C-terminal fusions with roGFP2 were initially expressed in tobacco leaves. While the To determine the subcellular localization of GPXL4 and C-terminal fusion was found in the cytosol and the nucleoplasm, GPXL5, roGFP2 was fused to the C-terminus of both proteins.

Figure 3. Ratiometric imaging of roGFP2 fusion proteins shows GPXL2 and GPXL8 in the cytosol and the nucleus. (a) Ratiometric analysis of SEC22-roGFP2 and roGFP2-SEC22 transiently expressed in tobacco leaf epidermal cells as controls for fluorescence ratio readouts for roGFP2 fusions of proteins in membranes of ER and Golgi. Scale bars = 20 μm. (b) Arabidopsis wild-type plants stably transformed with GPXL2-roGFP2 (top) and GPXL8-roGFP2 (bottom) reporter gene fusion constructs. The merge of the two roGFP raw images and the blue false colour of the roGFP ratio images show the reduced status of roGFP2 confirming localization of GPXL2 and GPXL8 in the cytosol and the nucleus. Scale bars = 20 μm.

While transient expression of these proteins in tobacco resulted phylogenetic tree and found five distinct clades based on four in cytosolic and nuclear localization (Fig. S5a,b), stable putative gene duplications during early land plant evolution. expression in Arabidopsis consistently resulted in exclusive AtGPXL4 and 5 were generated by an additional gene duplica- localization of the fusion proteins to the plasma membrane tion during angiosperm evolution and both belong to the same (Fig. 5a,b). The C-terminally fused roGFP2 remained reduced cluster that was annotated as cytosolic (Margis et al. 2008). in all cases indicating localization on the cytosolic face of the More detailed sequence analysis of the GPXL4/5 clade membrane. Membrane localization of both constructs was highlighted that all 28 representatives of this clade contained confirmed through co-localization with the membrane marker the N-terminal myristoylation motif (Fig. S6). As a result, Gly FM4-64 (Fig. 5c,d). The N-termini of both GPXL4 and GPXL5 may get myristoylated during translation after cleavage of the resemble the classical myristoylation motif MGxxxSxx N-terminal Met enabling anchoring to the plasma membrane. (Johnson et al. 1994; Resh 2016) (Fig. S1). Based on 166 GPXL To test the hypothesis of the N-terminus playing a role in sequences from land plants, Margis et al. generated a plasma membrane localization, we first masked the putative © 2017 John Wiley & Sons Ltd, Plant, Cell and Environment, 40,1281–1295 Subcellular localization of GPXLs in Arabidopsis 1287

Figure 4. Glutathione peroxidase-like enzyme 6-roGFP2 is dual targeted to mitochondria and cytosol. Arabidopsis wild-type plants were stably transformed with the reporter gene fusions. (a) Partial co-localization of GPXL6-roGFP2 with the mitochondrial marker tetramethylrhodamine methyl ester (TMRM). (b) Ratiometric imaging of roGFP2. The blue false colour of the ratio image indicates reducing conditions in the cytosol (for colour coding, see Fig. 3). Scale bars = 20 μm. (c) Co-localization of GPXL61–65-roGFP2 with the mitochondrial marker TMRM. Scale bar = 10 μm.

myristoylaton site by fusing roGFP2 to the N-termini of To minimize the risk of possible mistargeting resulting from GPXL4 and GPXL5, respectively. In tobacco, these constructs heterologous expression, both N-terminal and C-terminal strictly labelled endomembranes including the nuclear fusion proteins of GPXL3 with roGFP2 were stably expressed envelope with a reduced roGFP2 (Fig. S5c,d). When in Arabidopsis. As in tobacco leaves, expression of roGFP2-GPXL4 and roGFP2-GPXL5 were stably trans- GPXL3-roGFP2 in Arabidopsis resulted in a punctate labelling formed into Arabidopsis, expression of the fusion proteins pattern, but surprisingly, ratiometric analysis of the roGFP2 was very low, but still, they appeared predominantly on tag indicated oxidizing conditions in the respective compart- endomembranes including the nuclear envelope with roGFP2 ment (Fig. 7a top row, red false colour). Expression of facing the cytosol indicated by the blue/greenish false colour roGFP2-GPXL3 resulted in labelling of endomembranes of the ratio images (Fig. S7). To further assess the role for including the nuclear envelope. In this case, however, the myristoylation, G2A mutant variants of GPXL4 and GPXL5 roGFP2 tag appeared to be largely reduced (Fig. 7a bottom were generated and fused with C-terminal roGFP2. In row, blue false colour). Taken together, the ratiometric analysis Arabidopsis, both of these fusion proteins were not found at of N-terminal and C-terminal fusions of GPXL3 with roGFP2 the plasma membrane anymore. Instead, they remained strongly suggests that GPXL3 is targeted to the ER and/or soluble in the cytosol and the nucleus (Fig. 6). the Golgi and that the protein contains a transmembrane domain (TMD), which anchors the protein to the membrane. GPXL3 is a type II transmembrane protein in the Localization of GPXL3 fusion proteins in the Golgi apparatus was conﬁrmed by co-localization with the Golgi marker secretory pathway mannosidase I-red ﬂuorescent protein (Fig. S8). Indeed, the Expression of GPXL3 with roGFP2 fused to its C-terminus in protein sequence of GPXL3 contains an N-terminal extension tobacco leaves resulted in the observation of punctate struc- of 39 amino acids that includes a highly hydrophobic domain tures. Despite prediction of GPXL3 as a mitochondrial protein between amino acid 19 and 32. Together with few neighbouring (Fig. 1), no co-localization with TMRM could be found. amino acids, this domain may constitute a TMD that anchors ©2017JohnWiley&SonsLtd, Plant, Cell and Environment, 40,1281–1295 1288 S. Attacha et al.

Figure 5. Expression of GPXL4-roGFP2 and GPXL5-roGFP2 in Arabidopsis. (a,b) Ratiometric imaging of roGFP2 in leaves expressing GPXL4- roGFP2 (a) and GPXL5-roGFP2 (b). The blue false colour of the ratio images indicates reducing conditions in the cytosol (for colour coding, see Fig. 3). (c,d) Co-localization of GPXL4-roGFP2 (c) and GPXL5-roGFP2 (d) with the membrane marker FM4-64 in Arabidopsis roots. roGFP2 and FM4-64 were excited simultaneously at 488 nm, and fluorescence was recorded at 505–530 nm for roGFP2 and 620–680 nm for FM4-64 2 min after incubation with 5 μM FM4-64. Scale bars = 20 μm.

GPXL3 to theER membrane (Fig.7b). A single-spanning TMD and phospholipid hydroperoxides in mammalian cells and would be consistent with the binary response of N-terminally plants (Bela et al. 2015; Brigelius-Flohe & Maiorino 2013; fused roGFP2 being reduced and C-terminally fused roGFP2 Rouhier & Jacquot 2005). While in vertebrates four distinct being oxidized(Fig.7c).Thefirst 34 aminoacids ofGPXL3 were clusters of GPx exist, the plant homologues appear to be evolu- found to be sufficienttotargetroGFP2tothesecretorypathway tionary more uniform and show the highest similarity in amino where it labelled the ER and the Golgi (Fig. 8a). Deletion of the acid sequence with human GPx4 (Kim et al. 2014; Margis et al. first 34 amino acids from GPXL3 resulted in a soluble protein 2008). Nevertheless, all plant GPXLs can be clustered in five localized in the cytosol and the nucleus (Fig. 8b). well-separated groups represented by GPXL proteins localized or predicted to be localized to different subcellular compartments (Margis et al. 2008). The eight GPXLs in Arabidopsis DISCUSSION are clearly distinguishable by their primary sequence, and Glutathione peroxidases and GPXLs are proposed to fulfil attempts have been made to group the proteins into subgroups essential functions in detoxification of H2O2, organic peroxides based on their predicted localization (Navrot et al. 2006). In the © 2017 John Wiley & Sons Ltd, Plant, Cell and Environment, 40,1281–1295 Subcellular localization of GPXLs in Arabidopsis 1289

Figure 6. Ratiometric imaging of G2A mutant variants of GPXL4 and GPXL5. Both mutated proteins were fused with roGFP2 at their C-termini and stably transformed into Arabidopsis wild-type plants. (a) GPXL4G2A-roGFP2. (b) GPXL5G2A-roGFP2. The blue false colour of the ratio images indicates reducing conditions in the cytosol (for colour coding, see Fig. 3). Arrow heads point at nuclei in which the signal fills the nucleoplasm and decorates the nucleoli indicating that plasma membrane association is overcome in mutated protein variants. Scale bars = 20 μm. present study, we investigated the subcellular localization by hydroperoxides as putative in vivo substrates, this may indicate expressing translational fusions of all eight Arabidopsis GPXLs an involvement of plastidic GPXLs in defence responses. with roGFP2 as a reporter gene. The advantage of roGFP2 While the even distribution of ﬂuorescence of fusion proteins compared with a standard single-wavelength ﬂuorescent pro- in chloroplasts and labelling of stromules suggest localization tein becomes apparent for proteins targeted to the secretory in the stroma, the exact sub-organellar localization of GPXL1 pathway that roGFP2 validates by a distinct ratiometric and GPXL7 remains unclear and may require complementary response. Linked to this, roGFP2 offers a second major high-resolution approaches like, for example, immunogold- advantage of indicating the orientation of integral membrane labelling in future work. proteins along the secretory pathway (Brach et al. 2009). N-terminal and C-terminal fusions of single spanning proteins The GPXL6 presequence is sufficient to target hence also self-indicate their orientation within the membrane. roGFP2 to the mitochondrial matrix

GPXL1 and GPXL7 are plastidic proteins The exact localization of GPXL6 has been ambiguous with contradicting data from mass spectrometry-based proteome Glutathione peroxidase-like enzymes 1 and 7 have long been analysis experiments. Published results include localization at considered as plastid-targeted isoforms based on bioinformat- the plasma membrane (Marmagne et al. 2007; Marmagne ics sequence analysis and predictions (Mullineaux et al. 1998, et al. 2004), in the cytosol (Ito et al. 2011; Zargar et al. 2015) Rodriguez Milla et al., 2003). While GPXL1 was found in and in mitochondria (Brugiere et al. 2004; Yoshida et al. chloroplast proteomes multiple times (Ferro et al. 2003; 2013). Localization at the plasma membrane or the cytosol is Kleffmann et al. 2004; Peltier et al. 2004), the properties of supported by an interaction of GPXL6 with a plasma GPXL7 at the protein level remained enigmatic. Availability membrane-localized tetratricopeptide protein in an unbiased of only very few expressed sequence tags may indicate low ex- large-scale protein–protein interaction screen (Arabidopsis pression level that may hamper detection of GPXL7 in plastid Interactome Mapping Consortium 2011). Brugiere et al. proteomes. Our results clearly show that full-length GPXL1 (2004) reported GPXL6 as a mitochondrial protein based on and GPXL7 tagged with roGFP2 at their C-termini can be analysis of the hydrophobic proteome of mitochondrial mem- expressed and are targeted to plastids. This result is consistent branes. Integration into the membrane, however, is doubtful with partially overlapping functions of both proteins in photo- because GPXL6 does not contain a distinctive hydrophobic oxidative stress and immune responses deduced from analysis domain that could serve as a TMD. Yoshida et al. (2013) found of gpxl1 gpxl7 double null mutants (Chang et al. 2009). The GPXL6 by TRX affinity chromatography of mitochondrial function in immune response is particularly intriguing as it extracts from Arabidopsis with mitochondrial TRXo1 as bait. has been reported that lipid peroxidation after infection of Despite the fact that TRX-based affinity chromatography Arabidopsis by Pseudomonas syringae is predominantly con- bears the risk of identifying false positives due to promiscuity finedtoplastids(Zoelleret al. 2012). With phospholipid of the mutated bait TRX, this is currently the best hint for ©2017JohnWiley&SonsLtd, Plant, Cell and Environment, 40,1281–1295 1290 S. Attacha et al.

Figure 7. Stable expression of GPXL3-roGFP2 and roGFP2-GPXL3 in Arabidopsis. (a) GPXL3-roGFP2 is localized in the secretory pathway, predominantly in the Golgi. The red false colour of the ratiometric image indicates that the roGFP2 reporter was fully oxidized. roGFP2-GPXL3 is localized in the secretory pathway highlighted by the nuclear ring (arrow heads). Blue in the false-colour ratiometric image indicates that the roGFP2 reporter was highly reduced. Scale bars = 20 μm. (b) Protein structure model of GPXL3 with a α-helical N-terminal extension. The AtGPXL3 protein core (residues 46–206) was modelled using the MODELLER tool and oxidized GPXL5 from poplar (Protein Data Bank code 2P5R) as a template and the unordered N-terminal extension added manually to the model. Hydrophobicity of amino acids is indicated by different shades of red colour. The indicated transmembrane domain (TMD) is the consensus TMD predicted by ARAMEMNON (Schwacke et al. 2003). (c) Cartoon displaying the topology of GPXL3 fused either N-terminally or C-terminally with roGFP2. localization of GPXL6 in mitochondria, where reduction of sufficient to target roGFP2 to the mitochondrial matrix GPXL6 by TRXo as the exclusive mitochondrial TRX class strongly suggests that native GPXL6 is indeed a mitochondrial would be expected (Iqbal et al. 2006; Navrot et al. 2006). protein. This interpretation is further supported by the identifi- Because N-terminal fusion of a putative mitochondrial protein cation of a GPXL6 homolog in the mitochondrial proteome of with roGFP2 is expected to mask the mitochondrial target pep- potato (Salvato et al. 2014). Additional presence of GPXL6 tide, cytosolic localization of the respective fusion proteins fur- resulting from translation being initiated at Met64 as a putative ther supports mitochondrial localization of the native protein. second start codon cannot be excluded at this point. The observed binding to membranes of the secretory pathway fi most likely resulted from arti cial interaction of the protein GPXL2 and GPXL8 are soluble proteins in the with unknown components of the endomembrane system. cytosol Partial targeting of GPXL6-roGFP2 to mitochondria indicates the general ability of the GPXL6 propeptide to target mito- Glutathione peroxidase-like enzymes 2 and 8 have strong chondria. The fusion of GPXL6 with roGFP2, however, may predictions for the cytosol, but both enzymes have also been interfere with the transfer to the matrix through obstruction suggested to be secreted or ER proteins (Fig. 1) (Margis et al. of the import machinery. Similar observations have been made 2008; Navrot et al. 2006). Our data show that Arabidopsis earlier with GRX1-roGFP2 fusions where residual cytosolic GPXL2 and GPXL8 are soluble cytosolic and nuclear proteins. localization could not be fully avoided despite the use of a The localization of roGFP2 fusion proteins is consistent with well-characterized presequence for matrix targeting (Albrecht lack of obvious target peptides. Our results from stable homol- et al. 2014). The fact that the prepeptide GPXL61–65 was ogous expression in Arabidopsis are consistent with the

Figure 8. The first 34 amino acids of GPXL3 are sufficient for ER targeting. (a) Stable expression of GPXL31–34-roGFP2 in Arabidopsis. (b) Stable expression of GPXL3Δ1–34-roGFP2 in Arabidopsis.Scalebars=20μm. observation that GPXL8-GFP fusions are cytosolic nuclear homologous expression system. GPXL4 and GPXL5 are when expressed heterologously and transiently in onion members of a distinct clade indicated as cytosolic on the basis epidermal cells (Gaber et al. 2012). GPXL2 has been shown of individual representatives being localized in the cytosol to be linked to superoxide dismutase 1 via the linker protein (Margis et al. 2008). The conservation of the typical DJ-1, both of which reside in the cytosol (Xu et al. 2010). Based myristoylation motif MGxxxSxx in analysed members of this on this interaction, a function in channelling H2O2 generated GPXL4/5 clade suggests that N-terminal myristoylation of by superoxide dismutase 1 to GPXL2 for further reduction GPXL is an evolutionary ancient and highly conserved feature. has been proposed. Characterization of null mutants and While exclusive localization at the plasma membrane may overexpression lines suggests that GPXL8 protects cellular suggest that GPXL4 and 5 are involved in metabolism of lipid components such as nuclear DNA against oxidative stress components released from the membrane, the exact function (Gaber et al. 2012). Further analysis of the two cytosolic of both enzymes remains unclear. It is intriguing that mamma- GPXLs is currently hampered by the lack of null mutants for lian GPx4 as the closest relative to plant GPXLs is preferen- GPXL2 and the lack of interaction partners for GPXL8. tially membrane associated (Lubos et al. 2011). In addition, overexpression of tryparedoxin-dependent non-selenium GPXL4 and GPXL5 reside at the plasma membrane GPX (Px) closely related to mammalian GPx4 renders African trypanosomes less sensitive towards linoleic acid hydroperox-

Formation of phospholipid hydroperoxides occurs frequently ides but not H2O2 (Diechtierow & Krauth-Siegel 2011). The in the plasma membrane, particularly under stress situations high degree of sequence identity between GPXL4 and GPXL5 leading to increased activity of superoxide-generating NADPH resulting from relatively recent gene duplication may suggest oxidases (Gupta et al. 2016). N-terminal myristoylation is a overlapping functions. On the other hand, gpxl5 null mutants common mechanism of associating proteins with the plasma appeared in a screen for mutants arrested in maternal gameto- membrane. Based on the presence of a recognition motif with phyte development (Pagnussat et al. 2005), while gpxl4 mutants Gly in position 2 of the nascent peptide that becomes exposed have been reported as viable (Passaia et al. 2014). This differ- after co-translational Met cleavage, myristoyl-CoA:protein ence may possibly be explained by differential expression N-myristoyl transferase catalyses the co-translational transfer rather than different biochemical properties. of myristate from the myristoyl-CoA donor onto the N-terminal Gly via an amide bond (Resh 2016). Myristoylation GPXL3 is a type II transmembrane protein in the on its own is, however, not sufficient to anchor a protein to a secretory pathway phospholipid bilayer, and hence, a second signal is required for stable membrane binding (Resh 2006). The differential In contrast to bioinformatics predictions for mitochondrial expression patterns for GPXL4-roGFP2 and GPXL5-roGFP2 targeting, GPXL3 was found exclusively in the secretory path- in tobacco and Arabidopsis may suggest that this second signal way of Arabidopsis, irrespective of whether the protein was is based on a specificprotein–protein interaction for which the fused with roGFP2 reporter proteins at its N-terminus or second interaction partner may be available only in the C-terminus. This result is in contrast with reports of GPXL3 ©2017JohnWiley&SonsLtd, Plant, Cell and Environment, 40,1281–1295 1292 S. Attacha et al. in the plastid proteome (Helm et al. 2014) and localization of C-terminal fusion with GFP, LjGPXL3 has recently been pro- GPXL3-GFP fusions in the cytosol after transient expression posed to be present in the ER and the cytosol (Matamoros in Arabidopsis protoplasts (Miao et al. 2006). While proteome et al. 2015). The image presented by Matamoros et al. as analysis of subcellular compartments bears a risk of contamina- evidence for localization of LjGPXL3-GFP in the cytosol, tions, our results suggest that also transient expression systems however, shows a clearly defined nuclear ring that is a classical are prone to localization artefacts. Careful inspection of images characteristic for ER localization. Bioinformatics analysis of presented by Miao et al. (2006) for fluorescence complementa- LjGPXL3 also indicates the presence of an N-terminal exten- tion of YFP and expression of GPXL3-GFP in both cases indi- sion with a hydrophobic domain and a high probability for cates a fluorescence distribution consistent with plastids and targeting to the secretory pathway (Fig. S9). may result from a bleed through of chlorophyll autofluores- Protein folding in the ER typically coincides with the forma- cence into the YFP or GFP channel, respectively. tion of intramolecular disulfides. Formation of disulfides from Our results consistently show that GPXL3 is a type II thiols of cysteine residues in nascent peptides entering the membrane-bound protein that is resident in the secretory path- secretory pathway is catalysed by ER thiol oxidases and way. The protein contains a short N-terminal cytosolic domain quiescin sulfhydryl oxidases (Aller & Meyer 2013). Both followed by a single hydrophobic domain of 14 highly hydro- enzymes use molecular oxygen as the terminal electron phobic amino acids (residues 19–32) and the catalytic domain acceptor and produce H2O2 as a by-product that needs to be starting with amino acid 40. Generally, a stretch of 17–25 detoxified. While in mammalian cells, the ER harbours three mainly hydrophobic amino acids is considered sufficient to H2O2-reducing peroxidases, peroxiredoxin IV (PrxIV) and form a membrane spanning α-helix (Cosson et al. 2013). It glutathione peroxidases 7 and 8 (GPx7/8) (Ramming et al. has been shown by electron microscopy that membrane thick- 2014), no such H2O2-reducing systems have been identified in ness increases along the secretory pathway towards the plasma the ER of plants yet. As such, the presumed peroxidase membrane (Sandelius et al. 1986), and relatively short TMDs function of GPXL3 offers a valuable perspective to fill this have been considered a mechanism for retention of membrane gap. Interestingly, the membrane localization that we have proteins in the ER and the Golgi (Cosson et al. 2013; Sharpe found for GPXL3 is mirrored by mammalian GPx8 that also et al. 2010). Only 14 hydrophobic residues in the core of the contains a TMD and interacts directly with membrane- putative TMD of GPXL3 may, however, still be too short for associated Ero1 (Ramming et al. 2014). a membrane spanning helix. The consensus prediction from Reduction of H2O2 and possibly phospholipid hydroperox- ARAMEMNON thus includes some further amino acids at ides requires efficient re-reduction of GPXL3. Plant GPXLs both ends of the hydrophobic patch to reach 21 amino acids are now generally regarded as TRX-dependent phospholipid- as the average length of a TMD. This, however, would include hydroperoxide peroxidases (Bela et al. 2015), but in the ER a dilysine motif immediately adjacent to the hydrophobic patch lumen, no TRXs would be available for reduction of GPXL3. plus an additional Lys in position À4 of the presumed TMD on Protein disulfide isomerases may provide electrons instead, the cytosolic side. The resulting overall positive charge in this which has recently been shown for mammalian GPx7 in vitro domain would be consistent with the ‘positive-inside’ rule that (Bosello-Travain et al. 2013; Wang et al. 2014). If H2O2 pro- predicts a surplus of positive charges on the cytosolic face of duced by the oxidative folding machinery would be reused the membrane and contributes to the apparent free energy of for oxidation of protein disulfide isomerases, this would membrane insertion (Lerch-Bader et al. 2008; von Heijne increase the efficiency of the entire machinery by 100% based 1992). N-terminal fragments of 34 amino acids that include on the consumption of molecular oxygen per disulfide formed. the hydrophobic domain were found sufficient to integrate As such, this work opens the door for detailed investigation of the protein into the ER and Golgi membranes with consistent the exact physiological function of GPXL3 in the future. Such orientation resulting in a cytosolic N-terminus and a lumenal analyses will also need to reinvestigate whether and how the C-terminus. Taken together, these results suggest that GPXL3 lumenal GPXL3 may be mechanistically involved in drought contains a minimal length TMD that is sufficient to prevent the stress responses as shown phenotypically by Miao et al. (2006). protein from leaving the secretory pathway behind the Golgi. The fact that N-terminally fused roGFP2 leads to predominant CONCLUSION labelling of the ER rather than the Golgi may suggest that the cytosolic tail of 18 amino acids has a function in ER–Golgi This work provides a comprehensive localization study of the transfer. Analysing the exact molecular mechanisms control- subcellular localization and membrane topology of the whole ling this transfer as a more general principle of cellular traffick- GPXL family. As such, it resolves contradicting observations ing will make a challenging task for future studies, and GPXL3 that have been hampering mechanistic studies of individual will make a suitable model. family members. Our findings validate GPXL1 and GPXL7 Localization of GPXL3 in the Golgi is consistent with results as plastidic, and we show that GPXL2 and GPXL8 are both from quantitative proteomics for localization of organelle pro- cytosolic and nuclear localized. GPXL6 is mitochondrial, while teins by isotope tagging that also indicated the presence of the possibility of cytosolic localization by a second splicing var- GPXL3 in the Golgi apparatus (Nikolovski et al. 2012). Phylo- iant or differential initiation of translation cannot be ruled out. genetic analysis of GPXLs from Arabidopsis and Lotus Our data have revealed unexpected localizations for GPXL3 in japonicus identifies LjGPXL3 as the closest homolog of the secretory pathway, predominantly the Golgi, while GPXL4 AtGPXL3 (Fig. S9). Based on immunogold labelling and and GPXL5 are anchored to the plasma membrane. 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(2004) New functions of the Arrows indicate the three conserved cysteines present in thylakoid membrane proteome of Arabidopsis thaliana revealed by a simple, Arabidopsis GPXLs. Sequences were aligned by MAFFT with fast, and versatile fractionation strategy. Journal of Biological Chemistry 279, default settings using JalView. Gaps within the signal peptides 49367–49383. Ramming T., Hansen H.G., Nagata K., Ellgaard L. & Appenzeller-Herzog C. until position 70 were removed manually. Highly similar resi- > (2014) GPx8 peroxidase prevents leakage of H2O2 from the endoplasmic retic- dues (Score 0.8) are framed and coloured in red. Identical ulum. Free Radical Biology and Medicine 70,106–116. residues are marked in white letters on red background.

Figure S2 GPXL1-roGFP2 fusion proteins are localized in the GhGPx: Gossypium hirsutum; GrGPx: Gossypium raimondii; stroma of tobacco leaf chloroplasts. The GPXL1-roGFP2 fu- HaGPx: Helianthus annuus; HvGPx: Hordeum vulgare; sion protein co-localizes with chlorophyll auto fluorescence in LeGPx: Lycopersicon esculentum;MtGPx:Medicago the main body of the chloroplasts. Localization in the stroma truncatula;NtGPx:Nicotiana tabacum; OsGPx: Oryza sativa is visible by labelling of stromules. Scale bar = 20 μm. ssp. japonica cv. Nipponbare; PpGPx: Pinus pinaster;PtaGPx: Figure S3 Transient expression of GPXL2 and GPXL8 with Pinus tadea;PvGPx:Phaseolus vulgaris;SbGPx:Sorghum bi- roGFP2 fused at their N-terminus in tobacco. roGFP2-GPXL2 color;StGPx:Solanum tuberosum; ZmGPx: Zea mays. and roGFP2-GPXL8 are both localized in the cytosol and the Figure S7 Stable expression of N-terminal roGFP2 fused to nucleus. 405/488 nm ratio showed roGFP2 in the reduced state GPXL4 and GPXL5 in Arabidopsis. roGFP2-GPXL4 (a) and indicated by a blue false colour. Scale bars = 20 μm. roGFP2-GPXL5 (b) both appear attached to the cytosolic face Figure S4 Transient expression of N- and C-terminal fusion of of the ER membrane. The nuclear ring typical for ER seen for roGFP2 to GPXL6 in tobacco and Arabidopsis. (a) GPXL6- both fusion proteins is indicated by arrow heads. Scale roGFP2 (top) localized in the cytosol while roGFP2-GPXL6 bars = 20 μm. (bottom) appears to be attached to the surface of ER indicated Figure S8 Co-localization of GPXL3-roGFP2 with the Golgi by the nuclear ring (arrow head) in tobacco. (b) Confocal im- marker ManI-RFP in tobacco. GPXL3-roGFP2 co-localize ages of GPXL6-roGFP2 fusion protein which is localized to with ManI-RFP represented by yellow punctate structures in the surface of the ER in Arabidopsis. The ratiometric analysis the merge image. Scale bar = 20 μm. showed that roGFP2 is reduced in all cases. Scale bars = 20 μm. Figure S9 Sequence analysis of Lotus japonicus GPX3. (a) Figure S5 C- and N-terminal fusions of GPXL4 and GPXL5 Unrooted phylogenetic tree of Lotus japonicus GPXs and with roGFP2 expressed in tobacco leaves. GPXL4-roGFP2 Arabidopsis thaliana GPXLs. Full length protein sequences (a) and GPXL5-roGFP2 (b) fusion proteins are localized in were aligned using the MUSCLE algorithm. Calculation of the cytosol and the nucleus. roGFP2-GPXL4 (c) and the phylogenetic tree was done with MrBayes using the Mar- roGFP2-GPXL5 (d) are both attached to the ER membrane. kov-Chain-Monte-Carlo (MCMC) algorithm. L. japonicus se- The nuclear ring typical for the ER can be seen for both fusion quences except LjGPX3 were retrieved from www.kazusa.or. proteins (arrow heads). Scale bars = 20 μm. jp/lotus. The LjGPX3 sequence was obtained from Matamoros Figure S6 A clade of nominally cytosolic GPXLs from different et al. (2015). A. thaliana sequences were retrieved from TAIR. species containing a conserved putative myristoylation motif at LjGPX3 is closely related to AtGPXL3. (b) Constrained Con- their N-termini. The alignment shows N-terminal fragments of sensus TOPology Prediction server (CCTOP) results for 40-42 amino acids of all GPXL sequences that cluster in a dis- LjGPX3. The figure shows the comparison of the results from tinct clade described as cytosolic GPXLs (Margis et al. 2008, various trans-membrane prediction programs. The figure FEBS J., 275, 3959-3970). All sequences contain the classical shows predicted trans-membrane domains (yellow) and the pu- myristoylation motif MGxxxSxx (Johnson et al. 1994, Annu. tative orientation of the protein (red = cytosolic, blue = non-cy- Rev. Biochem., 63, 869-914) at their N-termini. Visualization tosolic). LjGPX3 contains one putative transmembrane of the alignment was done with ESPript 3.0 (Robert & Gouet, domain close to the N-terminus. (c) TargetP prediction results 2014, Nucleic Acids Res., 42, W320–W324). Sequences and no- for LjGPX3. cTP = Chloroplast transit peptide, menclature, except Arabidopsis GPXLs, were retrieved from mTP = mitochondrial targeting peptide, SP = secretory path- ‘PeroxiBase - The peroxidase database’ (Fawal et al., 2013, way signal peptide. Reliability class (RC) from 1–5where1in- Nucleic Acids Res., 41, D441–D444; http://peroxibase.tou- dicates the strongest prediction. 1 = x > 0.800, 5 = 0.200 > x. louse.inra.fr). AoGPx: Asparagus officinalis; AtGPXL: The length of the putative signal peptide is 36 AA and is pre- Arabidopsis thaliana; BnGPx: Brassica napus;BrGPx:Brassica dicted to target LjGPX3 to the secretory pathway with very rapa;CsGPx:Citrus sinensis; GaGPx: Gossypium arboreum; high probability.