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Plant Cell. 2007 Oct; 19(10): 3170–3193.
PMCID: PMC2174697

Proteome Analysis of Arabidopsis Leaf Peroxisomes Reveals Novel Targeting Peptides, Metabolic Pathways, and Defense Mechanisms[W]

Abstract

We have established a protocol for the isolation of highly purified peroxisomes from mature Arabidopsis thaliana leaves and analyzed the proteome by complementary gel-based and gel-free approaches. Seventy-eight nonredundant proteins were identified, of which 42 novel proteins had previously not been associated with plant peroxisomes. Seventeen novel proteins carried predicted peroxisomal targeting signals (PTS) type 1 or type 2; 11 proteins contained PTS-related peptides. Peroxisome targeting was supported for many novel proteins by in silico analyses and confirmed for 11 representative full-length fusion proteins by fluorescence microscopy. The targeting function of predicted and unpredicted signals was investigated and SSL>, SSI>, and ASL> were established as novel functional PTS1 peptides. In contrast with the generally accepted confinement of PTS2 peptides to the N-terminal domain, the bifunctional transthyretin-like protein was demonstrated to carry internally a functional PTS2. The novel enzymes include numerous enoyl-CoA hydratases, short-chain dehydrogenases, and several enzymes involved in NADP and glutathione metabolism. Seven proteins, including β-glucosidases and myrosinases, support the currently emerging evidence for an important role of leaf peroxisomes in defense against pathogens and herbivores. The data provide new insights into the biology of plant peroxisomes and improve the prediction accuracy of peroxisome-targeted proteins from genome sequences.

INTRODUCTION

Peroxisomes are ubiquitous cell organelles that compartmentalize primarily oxidative metabolic reactions. A characteristic of peroxisomes is their metabolic flexibility because their enzymatic constituents vary depending on the organism, the type of tissue, and the environmental conditions (for review, see Beevers, 1979; Hayashi and Nishimura, 2006). Plant peroxisomes differentiate into tissue- and developmental-specific variants, such as leaf peroxisomes in mature leaves and glyoxysomes in germinating seeds, and typically participate in metabolic pathways and networks that are spread over multiple subcellular compartments. For instance, the recycling of 2-phosphoglycolate during photorespiration requires orchestration of 15 chloroplastic, peroxisomal, and mitochondrial enzymes and numerous transport proteins to transfer the intermediates between cell compartments (for review, see Reumann, 2002; Reumann and Weber, 2006). Similarly, fatty acids are released from lipid bodies during seed germination and transferred to glyoxysomes, where the carbon chain is transformed into sucrose in coordination with mitochondrial and cytosolic enzymes. Compartmentalization of hydrogen peroxide–producing oxidases in peroxisomes offers the advantage that H2O2 and other reactive oxygen species (ROS) can immediately be detoxified at the site of production by catalase (CAT) and auxiliary antioxidative enzymes (for review, see del Rio et al., 2006). In addition to these basic metabolic functions, a role for peroxisomes in the biosynthesis of the plant hormone auxin and the signaling molecule jasmonic acid (JA) and in sulfur and nitrogen metabolism has been established (Zolman et al., 2001; Strassner et al., 2002; Nowak et al., 2004; Koo et al., 2006). Moreover, evidence is emerging from recent studies that peroxisomes not only have important functions in metabolism but also play an essential role in specific defense mechanisms conferring resistance against pathogen attack (Taler et al., 2004; Koh et al., 2005; Lipka et al., 2005).

Thanks to rapid advances in genome sequencing, the potential of computational approaches is increasingly being realized to predict peroxisomal proteins and explore the biology of plant peroxisomes. In silico analyses take advantage of conserved targeting sequences in peroxisomal proteins (i.e., the C- terminal tripeptide constituting the peroxisomal targeting signal type 1 [PTS1] of the prototype SKL> [> designates the extreme C terminus] or the cleavable PTS2 nonapeptide located in the N-terminal domain [prototype RLx5HL]) to predict targeting of unknown proteins to peroxisomes (Emanuelsson et al., 2003; Kamada et al., 2003; Neuberger et al., 2003a, 2003b; Reumann, 2004; Hawkins and Boden, 2006). Except for few proteins (e.g., plant CAT and Arabidopsis thaliana sarcosine oxidase; Kamigaki et al., 2003; Goyer et al., 2004), all known matrix proteins from plant peroxisomes carry either a C-terminal PTS1 or an N-terminal PTS2 (Reumann, 2004). Plant PTS motifs have been defined by experimental and bioinformatics approaches and yielded partially overlapping results but differed in specificity and peptide identity. A relatively large number (24 to 120) of PTS1 peptides have been suggested to target proteins to peroxisomes in higher plants (Hayashi et al., 1997; Mullen et al., 1997; Kragler et al., 1998), but experimental limitations required data extrapolation from amino acid substitutions to all possible residue combinations within the three-residue motif and thereby reduced motif specificity. By in silico analysis of plant EST databases (Reumann, 2004), nine major and 11 minor PTS1 peptides were defined according to stringent criteria and thus considered as rather specific, though certainly yet incomplete. A search of the Arabidopsis genome for proteins carrying these peptides revealed 280 putative PTS1/2 proteins listed in the database AraPerox (Reumann et al., 2004; www.araperox.uni-goettingen.de). Despite considerable progress, prediction by PTSs is still limited by the low number of PTS-containing proteins that have been characterized at the molecular level, at least in one plant species (18 PTS1 and 14 PTS2 proteins; Reumann, 2004). However, the predictive algorithms for PTS1 proteins in particular can steadily be improved, as 12 additional proteins with putative PTS1s have been shown to be targeted to peroxisomes in the past 3 years and, at least in some cases, have been demonstrated to follow the PTS1 targeting pathway. The recently cloned cDNAs encode, for instance, auxiliary enzymes of fatty acid β-oxidation, ROS metabolism, and JA activation, and one of two small heat shock proteins (sHsps); the second sHsp is the only additional PTS2 protein (Edqvist et al., 2004; Tilton et al., 2004; Goepfert et al., 2005, 2006; Leterrier et al., 2005; Lisenbee et al., 2005; Schneider et al., 2005; Turner et al., 2005; Koo et al., 2006; Ma et al., 2006; Helm et al., 2007; Zolman et al., 2007).

Taken together, the low number of well-characterized plant peroxisomal matrix proteins leads to four principal constraints limiting in silico predictions of peroxisomal PTS1/2 proteins: (1) insufficient coverage of functional targeting peptides, (2) limited knowledge of the identity and function of auxiliary targeting elements surrounding the short PTS peptides, (3) possible dominance of N-terminal nonperoxisomal targeting signals over C-terminal PTS1s, and (4) unpredictable masking of PTS surface exposure by polypeptide conformation, homo- and hetero-oligomerization, or posttranslational regulatory mechanisms. Thus, the currently available prediction tools suffer from both insufficient sensitivity (i.e., an incomplete detection of true PTS1/2 proteins) and insufficient specificity (i.e., the identification of a significant number of false positives). Notably, many proteins of peroxisomes cannot currently be predicted from genome sequences at all, including peripheral and integral membrane proteins, subunits that are imported into the matrix in a piggybacked fashion, and matrix proteins with internal PTS1-like peptides (e.g., yeast acyl-CoA oxidase and plant CAT; Klein et al., 2002; Kamigaki et al., 2003).

In the postgenomic era of plant peroxisomal research, a major challenge is the identification and functional characterization of plant-specific proteins. Many of the recently cloned cDNAs of plant peroxisomal proteins have been identified by computational homology analysis using the protein sequence of peroxisomal enzymes from mammals or fungi as query (Edqvist et al., 2004; Tilton et al., 2004; Goyer et al., 2004; Goepfert et al., 2005, 2006). Even though proven to be successful, this approach fails by definition to unravel plant-specific proteins. Instead, systematic large-scale experimental studies of plant peroxisomes are required. Technical advances in two-dimensional gel electrophoresis (2-DE), liquid chromatography (LC), and mass spectrometry (MS) laid the foundation for the progress in organellar proteome studies (for review, see Taylor et al., 2003; Peck, 2005; Glinski and Weckwerth, 2006) and for cell-specific profiling (Wienkoop et al., 2004). The major bottleneck of organellar proteomics is the purity of the cell compartment of interest. With respect to their fragile nature, the isolation of intact peroxisomes in high purity is difficult irrespective of the source organism but is even more challenging in plants, mainly due to the predominance of plastids as another contaminating cell compartment and a myriad of secondary metabolites with organelle-destabilizing effects in vitro. For these reasons, the first proteome studies of peroxisomes have only recently been reported (for review, see Saleem et al., 2006). As the availability of complete genome information or large EST collections is a prerequisite for straightforward protein identification and because peroxisome isolation protocols are hardly interchangeable between plant species, such proteome studies were hampered by the fact that the isolation of plant peroxisomes in high purity was restricted to few plant species (e.g., Pisum sativum and Spinacia oleracea), which lacked genome or EST sequence information. Complete genome analysis of Arabidopsis (Arabidopsis Genome Initiative, 2000) rendered this tiny weed the species of choice for proteome studies of plant peroxisomes, provided that the organelles could be isolated in sufficient purity and quantity. Initial proteome analyses of leaf peroxisomes and glyoxysomes from greening and etiolated Arabidopsis cotyledons have been reported, but probably due to insufficient organelle purity and/or technical limitations in protein identification, only few known and novel PTS-containing proteins have been identified (Fukao et al., 2002, 2003). Leaf peroxisomes are considered the variant of choice for a comprehensive long-term project aimed at the identification and functional analysis of all plant-specific proteins from peroxisomes because mature leaves are (1) physiologically the most significant plant tissue (site of photosynthesis, photorespiration, numerous biosynthetic pathways, and nitrogen and sulfur assimilation); (2) the most distinctive tissue of plants compared with yeast and mammals; and (3) second only to roots, the major plant organ that interacts with the environment (i.e., that perceives and immediately reacts to stress, both abiotic [high light, extreme temperature, drought, and hail] and biotic [pathogen and herbivore attack]).

With the objective to verify and ultimately improve the prediction accuracy of plant peroxisomal proteins from genome sequences, we applied the unbiased large-scale approach of organellar proteomics to leaf peroxisomes. A purification method was developed for leaf peroxisomes from mature Arabidopsis leaves in high purity by Percoll and sucrose density gradient centrifugation. Protein separation by 2-DE in combination with peptide mass fingerprinting (PMF) and peptide fragment fingerprinting supplemented by a gel-free shotgun approach led to the identification of 36 known and 42 novel peroxisomal proteins. Peroxisome targeting of the novel PTS-containing proteins was corroborated by the conservation of PTSs in orthologs of higher plants as revealed by analysis of protein and EST databases. By fluorescence microscopy, peroxisome targeting of 11 representative proteins carrying predicted PTS or PTS-related peptides was verified for fusion proteins with enhanced yellow fluorescent protein (EYFP) in onion epidermal cells. Thereby, three novel PTS1 tripeptides (SSL>, SSI>, and ASL>) and an internal PTS2 were established, providing valuable input for further development of prediction tools. The novel plant peroxisomal proteins indicate that leaf peroxisomes play an important role in catabolism of complex metabolites, in biosynthetic pathways, and in defense mechanisms against pathogens and herbivores.

RESULTS

Isolation of Peroxisomes from Mature Arabidopsis Leaves in High Purity

To identify plant-specific peroxisomal proteins by a large-scale proteomics approach, a new isolation method for leaf peroxisomes from Arabidopsis was established with the objectives of (1) increasing the stability of peroxisomes in aqueous solution, (2) reducing the physical interaction of peroxisomes with mitochondria and chloroplasts, a pronounced phenomenon in Brassicaceae, and (3) increasing the specificity of peroxisome enrichment by a combination of Percoll and sucrose density gradient centrifugation. To this end, 3- to 4-week-old plants were harvested at the end of the dark period and gently ground in a buffer of high osmolarity. After separation of intact chloroplasts, but notably without peroxisome sedimentation by differential centrifugation, the supernatant was laid directly on top of a discontinuous Percoll density gradient (Figure 1A). Cosedimentation of peroxisomes along with mitochondria and thylakoid membranes by differential centrifugation prior to isopycnic organelle separation increased irreversibly interorganellar adhesion and peroxisome contamination. Whereas chloroplasts, thylakoid membranes, and mitochondria were largely retained in the 15 and 38% (v/v) Percoll fraction near the top of the gradient, intact leaf peroxisomes passed the Percoll layer and were recovered at the bottom, visible as a whitish diffuse organelle sediment (Figures 1B and 1C; see Supplemental Figure 1 online). Peroxisome fractions of several Percoll gradients were combined and washed in Tricine/EDTA buffer containing 36% (w/w) sucrose. Approximately 7% of total activity of the leaf peroxisomal marker enzyme hydroxypyruvate reductase (HPR) of the crude extract was recovered in the first fraction of leaf peroxisomes (LP-P1) with a yield of ∼90 nkat HPR per 60 g fresh weight (Table 1). The peroxisome fraction was carefully homogenized and laid on top of a discontinuous sucrose density gradient (Figure 1D; see Supplemental Figure 1 online). After ultracentrifugation, intact leaf peroxisomes were enriched close to the bottom of the gradient, visible as a sharp white band, at the typical density of plant peroxisomes of ∼50 to 53% (w/w) sucrose (Figures 1E and 1F; see Supplemental Figure 1 online). Even though only half of the peroxisomes could be recovered, the contamination by chloroplasts and mitochondria was further reduced by a factor of three (Table 1). On average, 115 ± 29 μg protein were obtained with a specific HPR activity of 293 ± 76 nkat/mg protein (Table 1). The purity of leaf peroxisomes was high, as determined by the activities of marker enzymes for leaf peroxisomes (HPR), mitochondria (fumarase), and chloroplasts (NADP-dependent glyceraldehyde-3-phosphate dehydrogenase [GAPDH]) and the content of chlorophyll (thylakoids); contaminating chloroplasts and mitochondria were estimated to comprise only 0.1 and 1.7% of the total, respectively (Table 1).

Figure 1.
Isolation of Peroxisomes from Mature Arabidopsis Leaves.
Table 1.
Biochemical Analysis of Peroxisome Purity

Identification of Proteins from Mature Leaf Peroxisomes

Peroxisome fractions of highest purity (∼400 nkat HPR/mg protein) were analyzed by 2-DE to display the proteinaceous constituents of Arabidopsis leaf peroxisomes as maps of intact proteins (Figure 2). Upon gel staining with colloidal Coomassie blue, ∼180 separated protein spots were visualized, out of which 135 were identified with high confidence by peptide mass and fragment fingerprinting, using an automated platform based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) (Jahn et al., 2006). Summarized over several two-dimensional gels, in addition to 28 known plant peroxisomal proteins, 36 proteins were identified that had previously not been associated experimentally with plant peroxisomes and are referred to as novel proteins throughout this study.

Figure 2.
Two-Dimensional Protein Map of Isolated Leaf Peroxisomes from Arabidopsis.

With respect to peroxisome purity, few minor spots derived from dominant proteins of chloroplasts and mitochondria. For instance, the presence of the large and small subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase, the most abundant plant protein, and small subunits of the photosystems (e.g., PSBP; Figure 2) indicated a low contamination with chloroplasts. Similarly, a few tiny protein spots represented mitochondrial proteins, including the P and H subunits of Gly decarboxylase (GDC-P and GDC-H), which is the most prominent mitochondrial protein; subunit β of the ATP synthase complex (ATPase β); and Ser-hydroxymethyltransferase (Figure 2). By contrast, no dominant proteins were detected of the endoplasmic reticulum (ER) (e.g., BiP), the nucleus (e.g., histones and DNA polymerase subunits), the plasma or tonoplast membranes, or the Golgi. Likewise, major cytosolic proteins were not found except for two spots identified as cytosolic glyceraldehyde phosphate dehydrogenase subunit C and aconitase. Based on the staining intensity of leaf peroxisomal versus nonperoxisomal protein spots (Figure 2), we concluded that leaf peroxisomes were isolated at ∼95% purity, with a minor contamination by dominant proteins from mitochondria and chloroplasts but not by other subcellular compartments.

Although two-dimensional gel-based proteomics approaches offer the advantage that protein integrity and information on protein abundance, isoform identity, and posttranslational modifications are retained, limitations with respect to the identification of small, basic, and hydrophobic proteins are reported. To complement the gel-based approach and improve coverage of the peroxisomal proteome, a gel-free technique was added that employs one-dimensional LC separation at the level of proteolytic peptides, commonly referred to as the shotgun approach (Link et al., 1999). This technique is very sensitive and requires only small amounts of total protein, as demonstrated recently by cell-specific protein profiling in pooled trichome and epidermis cells from Arabidopsis (Wienkoop et al., 2004). The proteins identified by this method largely overlapped with those of the gel-based approach; however, additionally, eight known and six novel proteins were detected only by the shotgun strategy. These proteins generally had either a basic isoelectric point (pI > 8.4) and/or were small proteins (<150 residues), underscoring the complementary nature of the two approaches applied. In total, we identified 78 nonredundant proteins, comprising 36 known and 42 novel proteins (Table 2; see Supplemental Table 1 online). Filtered mass spectra for protein identifications are stored in ProMEX, a mass spectral reference library for plant proteomics, that can be searched with peptide product ion spectra of unknown samples (Hummel et al., 2007).

Table 2.
Novel Proteins Identified in Arabidopsis Leaf Peroxisomes

Known Plant Peroxisomal Proteins

Identified proteins already known as constituents of leaf peroxisomes or glyoxysomes in any plant species are listed in Supplemental Table 1 online. As valuable information on protein abundance could be deduced from the two-dimensional gels (Figure 2), a few selected proteins are described in more detail here. Consistent with the major function of leaf peroxisomes in glycolate recycling, the most prominent protein spots on the two-dimensional gels represented the enzymes involved in the photorespiratory C2 cycle, all of which were identified (see Supplemental Table 1 online). On the basis of the MS data (see Supplemental Table 2 online), closely related isoforms of high sequence similarity could often be distinguished, allowing conclusions on isoform expression and predominance in mature leaves. For instance, both isoforms of glycolate oxidase (GOX), GOX1 (At3g14415; PRL>) and GOX2 (At3g14420; ARL>), were specifically identified. Of the two isoforms each of Glu-glyoxylate aminotransferase (GGT) and peroxisomal malate dehydrogenase (pMDH), GGT1 (At1g23310; SKM>) and pMDH2 (At5g09660; RIx5HL) dominated over the second isoform, both of which were identified as well and are thus not restricted to nonphotosynthetic tissue (see Supplemental Tables 1 and 2 online). Catalase isoform 3 (CAT3; At1g20620; 80% identical with CAT1 and 2) was more prominent on the two-dimensional gel than CAT2 (At4g35090), even though these isoforms could hardly be separated by isoelectric focusing. CAT1 (At1g20630) was below the detection limit of the gel-based method but was identified by the shotgun method. Three further antioxidative enzymes were identified as well (see Supplemental Table 1 online).

Even though fatty acid β-oxidation is primarily performed by glyoxysomes of germinating seeds and is thought to play only a minor role in leaves, we detected not only a complete set of the core β-oxidation enzymes but also several auxiliary enzymes in leaf peroxisomes. The core β-oxidation enzymes included long-chain acyl-CoA synthetase isoform 7 (LACS7; At5g27600; SKL>); acyl-CoA oxidase isoform 4 (ACX4; At3g51840; SRL>); both Arabidopsis isoforms of multifunctional protein (i.e., multifunctional protein isoform 2 [MFP2; At3g06860; SRL>]) and abnormal fluorescence meristem 1 (AIM1; At4g29010; SKL>); and all three thiolase isoforms. Peroxisome Defective 1 (PED1/KAT2; At2g33150; RQx5HL) and peroxisomal keto-thiolase isoform 2 (PKT2/KAT5; At5g48880; RQx5HL) predominated on two-dimensional gels, whereas PKT1/KAT1 (At1g04710; RQx5HL) was identified only by the shotgun approach.

The auxiliary enzymes of fatty acid β-oxidation included Δ3,52,4- dienoyl-CoA isomerase (DCI; At5g43280; AKL>; Goepfert et al., 2005), enoyl-CoA hydratase isoform 2 (ECH2; At1g76150; Goepfert et al., 2006), sterol carrier protein isoform 2 (SCP-2; At5g42890; SKL>; Edqvist et al., 2004), and acyl-CoA thioesterase (ACH2; At1g01710; SKL>; Tilton et al., 2004). With respect to hormone biosynthesis, not only OPR3 but also 3-oxo-2-(2′-pentenyl)-cyclopentane-1-octanoic acid (OPC-8:0) CoA ligase 1 (OPCL1; At1g20510; SKL>), which activates OPC-8:0 (Koo et al., 2006), was detected. OPCL1 was represented by a low-abundance protein spot restricted to peroxisomes of highest purity, consistent with its low expression level under standard conditions and its induction by wounding. Two further acyl-CoA acitivating enzymes (AAEs) included AAE7/ACN1 (At3g16910; SRL>; Turner et al., 2005) and 4-coumarate-CoA ligase-like protein 1 (4CLP1; At4g05160; SKM>; Schneider et al., 2005). In agreement with the heat inducibility of one of the two sHsps from plant peroxisomes (HSP15.7-Px; Ma et al., 2006), only the constitutively expressed isoform ACD31.2-Px (At1g06460; RLx5HF) was identified. As peroxisomal membranes were not further enriched prior to proteome analysis, the number of integral membrane proteins identified was low (APX3, PEX11d, and PEX14), which is consistent with the low content of membrane proteins in peroxisomes in general (Fujiki et al., 1982; S. Reumann, unpublished data).

In summary, the 36 known plant peroxisomal proteins identified in isolated leaf peroxisomes included all soluble enzymes involved in photorespiration, ROS metabolism, and JA biosynthesis. The detection of many core and auxiliary enzymes involved in fatty acid β-oxidation and sulfur and nitrogen metabolism (see Supplemental Table 1 online) indicated that (1) the dynamic range of protein identification was sufficiently high to cover low-abundance enzymes that are predominantly expressed in peroxisome variants other than leaf peroxisomes and that (2) β-oxidation–related enzymes may play a yet underestimated role in leaf peroxisomal metabolism.

Several matrix enzymes, although encoded by single Arabidopsis genes, often appeared on the two-dimensional gels as multiple protein spots arranged in a characteristic horizontal pattern. This pattern was most obvious for highly abundant matrix enzymes, namely GGT1, Ser-glyoxylate aminotransferase, HPR, and pMDH2, but also observed for minor enzymes, such as ascorbate peroxidase (APX3) and OPCL1 (Figure 2; see Supplemental Figures 2A to 2C online). The horizontal spot series of CAT3 differed slightly in that the differences in pI between spots were smaller (see Supplemental Figure 2D online). Although horizontal spot trains are often seen as artifacts of 2-DE, the uniform and regular patterns observed here might be indicative of the presence of charge-modifying posttranslational modifications, such as phosphorylation and acetylation.

Novel Plant Peroxisomal Proteins

Among the 42 novel proteins, a surprisingly large number had annotated functions related to fatty acid β-oxidation (Table 2). One protein is a yet uncharacterized isoform of the extended superfamily of acyl-activating enzymes, namely AAE5 (At5g16370; SRM>), belonging to clade VI of largely unknown PTS1-carrying AAEs, as does AAE7/ACN1 (Shockey et al., 2003; Reumann et al., 2004; Turner et al., 2005). Regarding the superfamily of enoyl-CoA hydratases/isomerases (ECHIs; Reumann et al., 2004), with MFP and AIM1 being the most prominent enzymes, we now identified monofunctional members of plant peroxisomes. The first ECHI, referred to as isoform a (ECHIa; At4g16210; SKL>), belongs to clade VI of putative plant enoyl-CoA hydratases. Two further monofunctional ECHIs identified, namely, ECHIb (At4g14430; PKL>) and ECHIc (At1g65520; SKL>), have been grouped in clade III and are the closest Arabidopsis homologs of peroxisomal monofunctional Δ32-enoyl-CoA isomerase (PECI) from fungi and mammals (Geisbrecht et al., 1999; Reumann et al., 2004). Most intriguing of this enzyme family is ECHId, annotated as putative naphthoate synthase (NS/ECHId; At1g60550; RLx5HL) because the enzyme is plant specific among eukaryotes and represents the Arabidopsis homolog of cyanobacterial MenB (clade V), an enoyl-CoA hydratase involved in phylloquinone biosynthesis (Johnson et al., 2001; Gross et al., 2006, see also references therein). This provides experimental evidence for peroxisome targeting of the single Arabidopsis homolog of MenB, further supported by conservation of the predicted PTS2 in homologous ESTs (see Supplemental Figure 3O online), and indicates that phylloquinone biosynthesis is partly compartmentalized in plant peroxisomes.

One protein spot was identified as a monofunctional hydroxyacyl-CoA dehydrogenase, annotated as hydroxybutyryl-CoA dehydrogenase (HBCDH; At3g15290) and carrying the predicted major PTS tripeptide PRL> (Reumann et al., 2004). Of the experimentally uncharacterized Arabidopsis family of small thioesterases (STs) with six PTS1-possessing members (Reumann et al., 2004), the second isoform of clade I (ST2; At1g04290; SNL>) is now experimentally associated with plant peroxisomes. Five novel proteins belong to the superfamily of short (polypeptide)-chain dehydrogenases/reductases (SDRs) and include four monofunctional SDRs (SDRa, At4g05530, SRL>; SDRb, At3g12800, SKL>; SDRc, At3g01980, SYM>; and SDRd, At3g55290, SSL>) and a bifunctional short- and medium-chain dehydrogenase/reductase (BSMDR; At1g49670; SRL>) with an N-terminal SDR and a C-terminal medium-chain dehydrogenase/reductase domain of the subgroup of NADPH:quinone reductases (COG0604, Qor). The latter was identified specifically by the gel-free approach.

Further novel enzymes with predicted catalytic activities related to fatty acid β-oxidation included epoxide hydrolase isoform 3 (EH3; At4g02340; ASL>) and one of two Arabidopsis homologs of acetoacetyl-CoA thiolase (ACAT2, EC 2.3.1.9, At5g48230, which is 80% identical with ACAT1, At5g47720; Carrie et al., 2007). GOX3 (At4g18360; AKL>) is a yet unknown isoform that shares 85% identity at the amino acid level with GOX1/2 but is predominantly expressed in nonphotosynthetic tissue and presumably plays a role outside of photorespiration (Kamada et al., 2003; Reumann et al., 2004). Enzymes associated with peroxisomal NADP metabolism included isoforms of NADP-dependent isocitrate dehydrogenase (IDH; At1g54340; SRL>) and 6-phosphogluconate dehydrogenase (6PGDH; At3g02360; SKI>), one of three enzymes of the oxidative pentose phosphate pathway (OPPP). Several novel plant peroxisomal enzymes are predicted to catalyze GSH-dependent reactions. These identified proteins included an isoform of glutathione reductase (GR; At3g24170; TNL>), isoform θ 1 of the extended Arabidopsis family of glutathione S-transferases (GSTT1, formerly GST10, At5g41210, SKI>, EC 2.5.1.18; Dixon et al., 2002), and the single Arabidopsis homolog of S-(hydroxymethyl)glutathione dehydrogenase (HMGDH; At5g43940; EC 1.1.1.284; Dolferus et al., 1997).

Seven identified proteins are functionally associated with plant defense mechanisms, which was rather unexpected. Four enzymes are β-glucosidases of the superfamily of glycoside hydrolase family 1, many of which are reported to mediate plant defense against herbivores (Xu et al., 2004; Table 2). The β-glucosidases included β-glucosidase 1 (BGL1; At1g52400; Stotz et al., 2000), PYK10 (At3g09260), and the myrosinase homolog thioglucoside glucohydrolase 2 (TGG2; At5g25980; Barth and Jander, 2006). All three defense-related enzymes were apparently upregulated in leaf peroxisomes isolated from senescent plants (see Supplemental Figures 2E and 2F online); TGG1 (At5g26000; Barth and Jander, 2006) was specifically identified in the latter (data not shown). The myrosinase-associated protein EPITHIOSPECIFIER MODIFIER1 (ESM1; At3g14210), which alters glucosinolate hydrolysis and insect resistance (Zhang et al., 2006), was identified in highly purified leaf peroxisomes as well. Two other defense proteins identified comprised one of three Arabidopsis homologs of vertebrate macrophage migration inhibitory factor (MIF; At3g51660; SKL>) and ozone-induced protein 1 (OZI1; At4g00860), a plant-specific small polypeptide of only 80 residues that is induced by ozone and pathogen attack (Sharma and Davis, 1995).

Likewise unexpected was the identification of proteins with annotated functions related to nucleic acid metabolism. These six proteins included two Gly-rich RNA binding proteins (GRP7 and 8; At2g21660 and At4g39260; Staiger et al., 2003), two DEAD-box RNA helicases (RH11, At3g58510; RH37, At2g42520), an unknown RNA binding protein (RBP; At4g17520), and a nucleotide binding protein (NBP; At4g16566). Except for NBP carrying the PTS1-like C-terminal tripeptide SKV>, all proteins related to nucleic acid metabolism lack predicted PTSs.

Proteins with other annotated functions included two enzymes reported to be involved in Cys and Met biosynthesis. O-acetylserine sulfhydrylase (OASS A1; At4g14880) is a presumably cytosolic isoform lacking an obvious PTS. Isoform 1 of cobalamin (vitamin B12)-independent Met synthase (METE1; At5g17920; EC 2.1.1.14; Ravanel et al., 2004) carries the PTS1-related C-terminal tripeptide SAK>. One of two Arabidopsis homologs of betaine aldehyde dehydrogenase (BADH; At3g48170; SKL>) was identified as well, homologs of which had previously been localized to the peroxisome matrix in monocotyledons (Nakamura et al., 1997). Another protein spot (ATF1; At1g21770; SSI>) was identified as a closely related homolog of a recently crystallized minimal acetyl transferase (ATF2; At1g77540; SSI>; Tyler et al., 2006). The single Arabidopsis transthyretin-like protein (TLP; At5g58220) is homologous to mammalian transthyretin, a transport protein for vertebrate hormones (Eneqvist et al., 2003; Hennebry et al., 2006a, 2006b). Interestingly, the full-length protein contains a predicted PTS2 (RLx5HL) outside of the N-terminal domain in a linker region that connects two enzymatic domains, whereas two alternative splice variants from Arabidopsis lack the putative targeting signal (Figure 3A; see Supplemental Figure 3P online; Hennebry et al., 2006a; Ramazzina et al., 2006). By MS sequencing of three tryptic peptides covering major parts of the linker region, including the putative PTS2, the TLP homolog identified in Arabidopsis peroxisomes was demonstrated to represent the full-length protein, strengthening the idea that the internal PTS2 peptide targets full-length TLP to peroxisomes (Figure 3). Lastly, four novel proteins lacked annotated domains and sequence similarity with known proteins deposited in the protein databases; three of these unknown proteins (UPs) carried PTS-like tripeptides (UP1, At1g52410, SSL>; UP2, At3g15950, SLN>; and UP3, At2g31670, SSL>; Table 2).

Figure 3.
Domain Structure of Arabidopsis Transthyretin-Like Protein and Its Splice Variants.

In Silico Analysis of the Targeting Function of Putative PTS Peptides

The majority of the novel proteins found to be associated with leaf peroxisomes (64%) carried predicted well-known PTSs (16 proteins; Reumann, 2004) or PTS-related peptides, for instance, SSL>, SSI>, ASL>, SAK>, or SYM> (11 proteins; Table 2), strongly supporting their localization in the peroxisome matrix. Because the sole presence of a putative PTS peptide is an insufficient criterion to definitely conclude peroxisome targeting (see Introduction), we further investigated the predicted targeting function of PTS peptides using both computational and experimental analyses. As part of the computational analyses, predicted orthologs of the novel proteins were retrieved from protein and EST databases of higher plants and analyzed for conservation of the predicted targeting peptides as outlined previously (Reumann, 2004; Reumann et al., 2004). Most proteins fulfilled the initial requirements for subsequent analysis except for a few proteins that either appeared to be specific to Brassicaceae (e.g., EH3 and UP1-3) or shared high sequence similarity with other Arabidopsis homologs (e.g., SDRd, BADH, GOX3, and METE1), preventing the specific detection of apparent orthologs.

On average, 30 plant ESTs were retrieved for each novel protein. Except for one EST homologous to SDRa terminating with FRL>, the orthologs of nine novel proteins with predicted PTS1 (ECHIa/b/c, ST2, SDRa/c, BSMDR, GSTT1, and MIF) carried approximately four different C-terminal tripeptides each, all of which represented major or minor PTS1s (see Supplemental Figure 3 online). Five other novel proteins had previously been reported to carry conserved PTS1 (SDRb/DECR, HBCDH, IDH, 6PGL, and 6PGDH; Reumann et al., 2004). Likewise, the postulated peroxisome-targeting function of the PTS1-related tripeptide SSI> present in ATF1 was supported by the detection of several homologous ESTs possessing either minor noncanonical PTS1s (SSM> or SNM>) or closely related tripeptides, such as SHI> and SNI> (see Supplemental Figure 3N online). In addition, most plant homologs of ATF1 carried a basic residue at position −4, which is reported to enhance peroxisome targeting by weak PTS1 tripeptides (Mullen et al., 1997; Maynard et al., 2004). Regarding SDRc (SYM>), only PTS1-like peptides were present in homologous ESTs (e.g., SF[ML]>, AYM>; see Supplemental Figure 3H online). However, the high content of basic amino acid residues and Pro in front and the lack of acidic residues in the C-terminal tripeptides themselves were consistent with protein targeting by the PTS1 pathway. Although a dissection of plant homologs of ACAT2 into apparent orthologs of ACAT1 or ACAT2 was difficult, many EST sequences were found to carry C-terminal 10–amino acid extensions either terminating with predicted (SKL>) or novel PTS1s (SSL>; see below) or PTS1-like tripeptides (SAL> or SLL>; see Supplemental Figure 3J online).

Regarding novel proteins carrying predicted PTS2, the peroxisome targeting function of the predicted PTS2 in naphthoate synthase (NS/ECHId; At1g60550; RLx5HL) was well supported by conservation of the nonapeptide as major or minor PTS2 (R[LIMV]x5HL) in plant orthologs. Moreover, characteristic amino acid residues predicted to act as targeting-enhancing elements (Arg and Pro) were present in close proximity to the PTS2 (Reumann, 2004; see Supplemental Figure 3O online). Likewise, the predicted internal PTS2 (RLx5HL) of TLP was conserved as putative major or minor PTS2s in all homologous plant ESTs spanning this region (e.g., R[LIMV]x5HL and RIx5HM) except for one slightly divergent nonapeptide (RVx5HV) present in Medicago (see Supplemental Figure 3P online). Even though full-length TLP variants appear to predominate among plant ESTs, some plant species, such as Oryza and Triticum, were found to express alternative splice variants similar to Arabidopsis that exactly lack this nonapeptide (Figure 3B; see Supplemental Figure 3P online; Hennebry et al., 2006a).

Experimental Verification of Predicted PTSs

To experimentally verify protein targeting to peroxisomes and the peroxisome-targeting function of predicted PTSs, subcellular targeting of seven representative fusion proteins with EYFP was analyzed by fluorescence microscopy upon transient cDNA expression in onion epidermal cells. Four proteins carried predicted major PTS1 peptides (SRL>, SKL>, or PRL>), whereas three proteins possessed predicted minor PTS1s (SKI> or PKL>) of lower peroxisome-targeting probability (Reumann, 2004). For all proteins, subcellular targeting of two constructs was analyzed (i.e., EYFP fusions of the full-length proteins of interest and fusions attaching only the predicted PTS1 targeting domain [PTD], comprising the C-terminal 10–amino acid residues, including the putative PTS1 tripeptide, to EYFP).

NADP-dependent IDH (At1g54340; SRL>) was chosen as one of the proteins carrying predicted major PTS1 peptides. NADP-dependent IDH, fused at its N terminus with EYFP (EYFP-IDH), was indeed detected in punctate structures in onion epidermal cells (Figure 4A1). In double transformants expressing simultaneously a peroxisome-targeted cyan fluorescent protein (CspMDH-CFP; Fulda et al., 2002; Ma et al., 2006), the yellow fluorescent subcellular structures were identified conclusively as peroxisomes (Figures 4A1 and 4A2). EYFP extended by the C-terminal 10–amino acid residues of IDH, including the predicted PTS1 SRL> (EYFP-PTDIDH), was directed from the cytosol to punctate structures, thereby identifying SRL> as the PTS of peroxisomal IDH (Figure 4B). Two further proteins, namely, the full-length fusion proteins of the predicted DECR homolog SDRb (At3g12800; SKL>) and the monofunctional hydroxyacyl-CoA dehydrogenase HBCDH (At3g15290; PRL>), were targeted to peroxisomes as well (Figures 4C1, 4C2, 4E1, and 4E2). EYFP extended by the PTDs of these proteins showed a punctate fluorescence pattern, characterizing the C-terminal tripeptides SKL> and PRL> as the targeting signals of SDRb/DECR and HBCDH, respectively (Figures 4D and 4F).

Figure 4.
Verification of Peroxisome Targeting of Novel Peroxisomal Proteins Carrying Predicted Major or Minor PTS1s.

Regarding proteins carrying predicted minor PTS1 peptides, the fusion protein between EYFP and ECHIb (At4g14430; PKL>), one of two predicted homologs of mammalian PECI (Geisbrecht et al., 1999), was targeted to punctate structures in onion epidermal cells that coincided with CFP-labeled peroxisomes (Figures 4I1 and 4I2). EYFP, extended by the C-terminal 10–amino acid residues of ECHIb, including the predicted PTS1 PKL> (EYFP-PTDECHIb), was directed from the cytosol to punctate structures, thereby identifying the minor PTS1 PKL> as the PTS of peroxisomal ECHIb (Figure 4J). Likewise, the isoform GSTT1 (At5g41210) with the putative PTS1 SKI> was shown to be located in peroxisomes, as the yellow fluorescent spots coincided with cyan fluorescent peroxisomes upon expression of EYFP-GSTT1 (Figures 4K1 and 4K2). The transferase was imported into peroxisomes via the PTS1 pathway because attachment of the C-terminal 10–amino acid domain of GSTT1 to EYFP showed a punctate pattern of fluorescence (Figure 4L). The full-length protein of the NADPH-producing dehydrogenase 6PGDH (At3g02360; SKI>) fused to the C-terminal end of EYFP was the only full-length fusion protein that remained cytosolic, for yet unknown reasons (Figure 4M). The C-terminal 10–amino acid residues of 6PGDH, however, directed EYFP to punctate subcellular structures that moved along cytoplasmic strands and coincided with peroxisomes (Figures 4N1 and 4N2). Although not identified within the proteome analysis, subcellular targeting of 6P-gluconolactonase (6PGL), the precedent enzyme of 6PGDH in the peroxisomal OPPP (Corpas et al., 1998, 1999), was also investigated using one specific isoform (At5g24400) out of five Arabidopsis homologs. This enzyme carries both a predicted N-terminal transit peptide and a conserved PTS1 (SKL>; Reumann et al., 2004). Whereas 6PGL-EYFP was targeted to plastid-like structures consistent with the presence of a functional N-terminal transit peptide (see Supplemental Figure 4 online), the inversely arranged fusion protein, EYFP-6PGL, was targeted to peroxisomes (Figures 4G1 and 4G2), as was EYFP-PTD6PGL with the PTS1 SKL> (Figure 4H). These data further strengthen the idea that this 6PGL isoform is targeted to both plastids and peroxisomes in vivo, probably regulated by alternative translation (Reumann et al., 2004).

Taken together, predicted peroxisome targeting of novel proteins identified in isolated leaf peroxisomes was experimentally confirmed for five of the six full-length candidate proteins and additionally for 6PGL as a metabolically related enzyme; all predicted PTS1s were characterized as the functional targeting signals of the respective proteins. Peroxisome targeting of full-length 6PGDH may have been disturbed by the N-terminal EYFP tag or depend on yet unknown auxiliary import factors missing in the standard expression system of onion epidermal cells.

Characterization of Novel Targeting Peptides

We next investigated whether representative novel proteins carrying PTS1-related peptides at the extreme C terminus or PTS2 peptides outside of the defined N-terminal domain are indeed imported into peroxisomes and whether these peptides function as targeting signals. The three PTS1-related tripeptides chosen for these experiments were SSL>, SSI>, and ASL>. The full-length fusion protein composed of EYFP and Arabidopsis enoyl-CoA hydratase ECH2 (At1g76150; SSL>) was targeted to peroxisomes (Figures 5A1 and 5A2). Extension of EYFP by the C-terminal 10–amino acid residues of ECH2 directed the reporter protein to punctate structures that coincided with peroxisomes (Figures 5B1 and 5B2); thus, the C-terminal PTS1-like tripeptide SSL> is the PTS1 of ECH2 and a novel functional PTS1 tripeptide in higher plants. Likewise, EYFP-ATF1 (At1g21770) terminating with SSI> was detected in peroxisomes (Figures 5C1 and 5C2). The putative PTS1 domain of this protein directed EYFP to punctate structures that coincided with peroxisomes, establishing SSI> as a second novel plant PTS1 (Figures 5D1 and 5D2). For epoxide hydrolase isoform 3 (EH3; At4g02340) carrying the C-terminal PTS1-like tripeptide ASL>, organelle targeting was difficult to demonstrate owing to a pronounced cytosolic background staining. Nevertheless, protein targeting to peroxisomes could be demonstrated by double labeling in a few cells (Figures 5E1 and 5E2). Attachment of the C-terminal 10–amino acid residues of EH3 to EYFP directed the reporter protein from the cytosol to punctate structures that coincided with peroxisomes (Figured 5F1 and 5F2). With these results, we conclusively demonstrated targeting of another three proteins to peroxisomes (ECH2, ATF1, and EH3) and established the PTS1-like peptides SSL>, SSI>, and ASL> as novel functional PTS1 peptides for higher plants.

Figure 5.
Subcellular Targeting Analysis of Novel Peroxisomal Proteins Carrying Unknown PTSs.

By current definition, PTS2 nonapeptides are confined to the N-terminal domain comprising ∼40 amino acid residues. To determine whether the bifunctional TLP identified in Arabidopsis leaf peroxisomes (Figures 2 and and3,3, Table 2) was targeted to peroxisomes by the internally located putative PTS2 (RLx5HL), the full-length cDNA of TLP was cloned downstream of EYFP and transiently expressed in onion epidermal cells. The fusion protein EYFP-TLP was detected mainly in the cytosol. However, despite the strong background fluorescence, targeting of EYFP-TLP to punctate structures suggestive of peroxisomes was observed in some cells (Figure 5G). The low contrast between the yellow fluorescent punctate structures and the background, however, allowed conclusive organelle identification as peroxisomes by double labeling only in a single cell (data not shown). In an alternative approach, the His residue known to be essential for the targeting function of PTS2 (Kato et al., 1998; Reumann, 2004; Ma et al., 2006) was mutated to Asp (RLx5HL→RLx5DL). Indeed, the mutated fusion protein EYFP-TLPΔPTS2 was no longer targeted to peroxisomes but exclusively detected in the cytosol and the nucleus, as was EYFP alone (Figure 5H). Thus, these data demonstrate targeting of a eukaryotic TLP homolog to peroxisomes and an internal location of a functional PTS2 in an endogenous eukaryotic protein.

Analysis of Artificial Copurification of Large Protein Complexes with Leaf Peroxisomes

The defense- and nucleic acid metabolism-related proteins identified in isolated leaf peroxisomes were represented by a relatively high number of proteins lacking known PTSs. Some of these defense-related proteins, such as BGL1, PYK10, and Brassica myrosinases, are reported to form polymers (Eriksson et al., 2002; Nagano et al., 2005; Lee et al., 2006). Likewise, nucleic acid metabolism-related proteins may be components of large protein complexes, such as ribosomes, exosomes, spliceosomes, or P-bodies (for review, see Eulalio et al., 2007). Even though unlikely, it was conceivable that these proteins were accidentally copurified with leaf peroxisomes through large complexes of similar sedimentation behavior in density gradient centrifugation. To distinguish between a physiologically relevant or artificial association of defense- and nucleic acid metabolism-related proteins with leaf peroxisomes, the isolation protocol was modified in such a way that intact leaf peroxisomes were lysed in hypoosmotic media by conditions that are not expected to reduce the sedimentation rate of protein complexes. To this end, Arabidopsis leaves were worked up in grinding and washing buffer lacking sucrose under otherwise identical conditions. Indeed, the fractions of the sucrose density gradient corresponding to those of intact leaf peroxisomes (fractions 11 and 12, Figure 1) contained substantially less total protein and largely lacked HPR activity, indicating that intact leaf peroxisomes were efficiently lysed and no longer recovered (data not shown). When such a fraction volume corresponding to ∼200 μg leaf peroxisomal protein in the presence of osmoticum was subjected to 2-DE, none of the defense-related proteins (BGL1, TGG2, PYK10, or ESM1) were detected by Coomassie staining (see Supplemental Figure 5 online). Similarly, the nucleic acid metabolism-related proteins GRP7 and 8 and RBP were not visible. Although two very faint spots may coincide with the RNA helicases RH11 and RH37 (see Supplemental Figure 5 online), these data as a whole strongly argue against an artificial copurification of high molecular weight complexes formed by the defense- or nucleic acid metabolism-related proteins with leaf peroxisomes and support a physiologically relevant association of most if not all of these PTS-deficient proteins with leaf peroxisomes.

DISCUSSION

Only ∼50 plant peroxisomal matrix proteins have been established for Arabidopsis either experimentally or by homology to peroxisomal proteins from other plant species (see summary at www.araperox.uni-goettingen.de). The low number of known peroxisomal enzymes in general and of plant-specific proteins in particular severely restricts our current knowledge of the organelle's physiological functions and alternative protein targeting mechanisms. The two most promising large-scale approaches to ultimately characterize the proteome of plant peroxisomes in its entity are (1) bioinformatics analyses predicting PTS1 and PTS2 proteins from genome sequences in combination with subcellular targeting studies of candidate fluorescent fusion proteins, and (2) proteome analyses. As in silico predictions are currently limited by several parameters (see Introduction) and because highly pure peroxisomes can finally be isolated from Arabidopsis, proteome analysis is the method of choice. This study demonstrates that proteome data yield important information not only directly on the identity of novel proteins and the recognition of newfound metabolic pathways but also indirectly on protein targeting. Leaf peroxisomal proteome analysis enables us to (1) recognize novel targeting peptides, (2) extend the location of PTS2 domains, and (3) posit the existence of novel PTS1/2-independent targeting pathways.

Plant-specific peroxisome functions, such as photosynthesis-related pathways, hormone biosynthesis, and pathogen defense mechanisms, are expected to be most comprehensively depicted by proteome analyses of Arabidopsis peroxisomes from mature rosette leaves rather than heterotrophic suspension-cultured cells or germinating seeds, even though these tissues offer significant technical advantages. The challenges in isolating highly pure Arabidopsis leaf peroxisomes include not only obvious traits (e.g., low leaf fresh weight and predominance of chloroplasts) but also unexpected parameters, such as the extreme fragility of Arabidopsis leaf peroxisomes in aqueous extracts, probably owing to the high concentrations of secondary metabolites, and their pronounced physical association with chloroplasts and mitochondria in Brassicaceae. For in-depth proteome analysis, we optimized our previous method (Ma et al., 2006) in several aspects, such as plant growth and time of harvest, differential centrifugation, and by the use of both Percoll and sucrose density gradients in particular (see Results and Methods). Contaminating proteins of chloroplasts and mitochondria, the major contaminants of plant peroxisomes, were hardly detectable in the final fraction by sensitive biochemical methods. The higher apparent content of contaminants estimated from the spot representation on two-dimensional gels (∼5%) compared with the activities of compartment-specific marker enzymes (0.1% plastids and 1.7% mitochondria; Table 1) may reflect facilitated solubilization of the rather acidic nonperoxisomal proteins compared with the neutral or basic peroxisomal proteins.

The Proteome Map of Arabidopsis Leaf Peroxisomes

The success of organellar proteome studies is determined by the number of novel true positives that indeed reside in the compartment of interest. The gel-based and shotgun approaches applied as complementary protein identification strategies maximized proteome coverage. The 78 proteins identified represent approximately twice as many proteins as reported previously in peroxisomes from rat liver (Kikuchi et al., 2004) or plants (Fukao et al., 2002, 2003). The 33 known matrix proteins include 65% of all currently known matrix proteins from plant peroxisomes (51 Arabidopsis proteins; Table 3). As this ratio has been obtained from only one of several plant peroxisomal variants and from standard plants, the coverage is considered high. Comprehensive proteome analyses of other tissue-specific and developmental peroxisome variants (e.g., glyoxysomes and gerontosomes) and from plants subjected to abiotic or biotic stresses are expected to promote the detection of the remaining known and further predicted proteins. A comprehensive membrane proteome analysis will be made possible by the availability of larger organelle quantities and the selective enrichment of peroxisomal membranes.

Table 3.
Proteins Identified in Arabidopsis Leaf Peroxisomes

Forty-three novel proteins (42 proteins by the proteome approach and 6PGL as an EYFP fusion protein) had not previously been localized to plant peroxisomes in any species and had not, for the most part, been experimentally analyzed. Seventeen of these novel proteins carry previously defined PTSs (16 PTS1 and one PTS2) and had been predicted to be peroxisomal proteins from the Arabidopsis genome (Reumann et al., 2004). Together with six predicted PTS1 proteins localized experimentally to peroxisomes in the meantime (4CLP1, AAE7/ACN1, ACH2, DCI, OPCL1, and SCP-2), ∼40% of the nonhypothetical Arabidopsis proteins (23 out of 50) predicted to be peroxisome targeted with high probability have been covered by our proteome study, confirming reciprocally the in silico approach. The high number of PTS-carrying proteins of this study (30 of 36 known and 22 of 42 novel proteins) contrasts with the low numbers of two previous two-dimensional gel-based peroxisomal proteome studies from greening and etiolated cotyledons (one novel PTS protein out of 20; Fukao et al., 2002; three out of 13; Fukao et al., 2003; see Supplemental Figures 6A and 6B online). With respect to novel protein identity, the overlap between the three proteome studies is marginal (only one protein, i.e., NADP-dependent IDH, At1g54340, SRL>) and difficult to rationalize in light of the large number of β-oxidation–related enzymes reported here for leaf peroxisomes but not previously for glyoxysomes, the peroxisome variant most specialized on fatty acid degradation (Fukao et al., 2003; see Supplemental Figure 6A online). Compared with the methods for peroxisome enrichment (single Percoll gradient only) and protein identification (PMF alone) applied in the two earlier studies, our sophisticated protocol for peroxisome purification and the highly confident protein identifications are considered the major improvements toward reliable and comprehensive coverage of the plant peroxisomal proteome.

The horizontal spot pattern of several matrix enzymes is suggestive of charge-modifying posttranslational modifications, such as phosphorylation and acetylation, both of which are currently being investigated in greater detail. Some peroxisomal protein kinases are reported and several are predicted, along with phosphatases, to be matrix targeted (Fukao et al., 2002, 2003; Dammann et al., 2003; Reumann et al., 2004). Acetylation of internal Lys residues also decreases the pI of a polypeptide and has recently been recognized as an important regulatory signal in many cellular processes (for reviews, see Polevoda and Sherman, 2002; Fuchs et al., 2006). ATF2 (At1g77540; SSI>), whose x-ray structure has recently been resolved, is a minimal acetyl transferase of unknown subcellular localization (Tyler et al., 2006). We have now established its closely related homolog, ATF1 (At1g21770; SSI>; 80% identical with ATF1) as a novel constituent of plant peroxisomes by multiple lines of evidence, including in vivo EYFP targeting studies (Figures 5C and 5D). Conservation of the novel PTS1 SSI> of ATF1 in ATF2 strongly suggests that the entire two-member protein family is peroxisomal in Arabidopsis. Protein acetylation may thus be an as yet unrecognized regulatory mechanism of plant peroxisomal enzymes. Proteins showing multiple horizontal spot patterns (see Supplemental Figure 2 online) are reasonable candidate substrates of ATF1.

Targeting Verification for Improving Targeting Prediction

By in silico analysis for conservation of the predicted PTS in homologous plant ESTs (Reumann, 2004), the peroxisome targeting function of the predicted PTS peptides was supported for 17 of 28 novel proteins (Table 2; see Supplemental Figure 3 online; Reumann et al., 2004). Exceptions included novel proteins, whose PTS1-like peptides were unaltered in homologous ESTs, and Brassicaceae-specific proteins. Notably, no evidence against peroxisomal targeting, for instance, acidic amino acid residues at any position of the tripeptide, was obtained for any of the proteins. By subcellular analysis of reporter protein fusions, peroxisome targeting of six out of seven representative novel proteins with predicted PTS1s was confirmed. Artificial localization data possibly caused by cDNA expression from the strong 35S cauliflower mosaic virus promoter were excluded by colocalization of the proteins of interest with a cyan fluorescent peroxisome marker, as applied in previous studies (Fulda et al., 2002; Goepfert et al., 2006; Ma et al., 2006). Three enzymes with β-oxidation–related annotations (SDRb, ECHIb, and HBCDH), a glutathione S-transferase (GSTT1), and three enzymes involved in NADP metabolism (IDH, 6PGL, and 6PGDH; see below) were conclusively established for plant peroxisomes. Only full-length EYFP-6PGDH remained cytosolic, possibly owing to interference of the EYFP tag with PTS1 exposure or posttranslational regulatory mechanisms. Nonetheless, this 6PGDH isoform is considered peroxisomal owing to its conserved PTS1 and functional PTS1 domain (SKI>; Figure 4N; Reumann et al., 2004), the lack of predicted N-terminal targeting signals, and its apparent absence in the proteomes of chloroplasts and mitochondria (Kruft et al., 2001; Millar et al., 2001; Peltier et al., 2002).

The large number of 11 proteins with PTS1-related peptides suggested their role as functional yet unknown PTS1s. The PTS1-like peptides SSL>, SSI>, and ASL> were previously absent from or below threshold in plant PTS1 sequences (Reumann, 2004), and experimental data argued against a targeting function of SSI> and SSL>. Conclusive experimental support for an allowance of Ser at position −2 in general had not been reported ([SACGT][RKHLN][LMIY]>, Mullen et al., 1997; [SACP][RK][LMI]>, Hayashi et al., 1997; [SACGTP][RKHLN][LMIY]>, Kragler et al., 1998). Peroxisome targeting of the three full-length fusion proteins and the corresponding PTS domain constructs (Figure 5) identified ECH2, EH3, and ATF1 conclusively as plant peroxisomal proteins and SSL>, SSI>, and ASL> as novel noncanonical plant PTS1 tripeptides. Full-length ECH2 had previously been shown to be peroxisomal (Goepfert et al., 2006). Our results on EH3 support earlier biochemical localization data on Ricinus epoxide hydrolase in glyoxysomes (Stark et al., 1995) and point to a physiological role of EH3 distinct from lipid mobilization in germinating seeds.

The primary structure of the novel PTS1 peptides and their remarkable frequency in the proteome of leaf peroxisomes (e.g., four SSL> proteins), in contrast with their rare abundance in previously known plant PTS1 proteins, raises further biological questions. For instance, which structural feature specifically allows Ser to replace the generally essential basic residue at position −2 in plants? The emerging abundance of SSL>, SSI>, or ASL> in leaf peroxisomal proteins may be partly attributed to our ability to now detect weakly expressed peroxisomal matrix proteins. We propose that the newfound noncanonical PTS1s occur preferentially in low-abundance matrix proteins because the tripeptides have a reduced affinity to the cytosolic PTS1 receptor PEX5 and lower the speed of cargo import into the matrix. The previously known major plant peroxisomal matrix proteins, by contrast, rely on highly efficient canonical PTS peptides to adjust the rate of protein import to the intensity of gene expression (G.E. Antonicelli and S. Reumann, unpublished data). Thus, the identification of low-abundance proteins appears to be of major importance to fully cover the natural variety of plant peroxisomal proteins and the range of functional PTS1/2 peptides in higher plants.

Even though our isolation method paves the way for analysis of other abundant peroxisome variants from Arabidopsis, it remains unrealistic to envision proteome studies of all plant microbody forms in the near future (e.g., from flowers, trichomes, stems, and roots). Prediction-based in silico studies will therefore remain the second major large-scale approach to fully describe the plant peroxisomal proteome. The prediction of plant peroxisomal proteins benefits significantly from the proteome data generated in the course of this study, in terms of both increased sensitivity and specificity. First, the three novel PTS1 peptides allow extended genome screens for putative PTS1 proteins and a significant enlargement of the database AraPerox (www.araperox.uni-goettingen.de; Table 3). The 100 additional new entries are predicted to include numerous interesting true peroxisomal proteins (for example, see Supplemental Figure 8 online) but may also comprise some false positives, if peroxisome targeting of some proteins depends on yet unknown enhancing elements. Experimental analysis of the predicted peroxisome targeting function of further PTS1-related peptides (e.g., GR, TNL>; METE1, SAK>; and NBP, SKV>) is expected to reveal further functional PTS1s.

Second, upon experimental verification of the targeting function of predicted PTS peptides, novel peroxisomal matrix proteins can be applied as input sequences to improve in silico predictions of PTS1/2 proteins. EYFP extended by the C-terminal domains of any of the 11 representative novel proteins showed a punctate distribution, conclusively demonstrating that the proteins carry functional PTS1s and use the PTS1 targeting pathway (Figure 4). In addition, peroxisome targeting of three other proteins identified in both Arabidopsis and spinach leaf peroxisomes has been confirmed for the corresponding full-length Arabidopsis homologs as EYFP fusion proteins (ECHIa, ECHId/NS, and SDRa) and their predicted targeting domains (SKL>, RLx5HL, and SRL>, respectively; L. Babujee, V. Wurtz, C. Ma, F. Lueder, A. van Dorselaer, and S. Reumann, unpublished data). In summary, the characterization of 26 novel PTS1/2 proteins at the molecular level, combined with experimental definitions of their targeting signals as reported in this and previous studies of the past 3 years (12 additional proven or putative PTS1/2 proteins; see Introduction), represents a significant extension of the previous number of known matrix proteins applicable as input sequences for EST database searches and discriminative analyses of PTS1 domains. The data will make possible a more comprehensive definition of functional PTS peptides, the characterization of auxiliary targeting elements, and an investigation of any interdependence of these parameters in higher plants.

An Internal PTS2 in TLP

The characterization of a functional PTS2 in the Arabidopsis TLP is biologically significant because (1) eukaryotic transthyretin homologs previously have not been localized to peroxisomes and (2) eukaryotic proteins have not been demonstrated to bear functional PTS2s at an internal position. TLPs are homologous to the mammalian thyroid-transport protein transthyretin, but they lack hormone binding activity and are generally considered cytosolic. The proteome data and the subcellular reporter protein studies of native and modified EYFP constructs provide conclusive evidence for peroxisome targeting of full-length Arabidopsis TLP (Figures 2, ,3,3, 5G, and 5H). The low import efficiency of EYFP-TLP may reflect conformation-limited accessibility of the internal PTS2 for PEX7 and/or its competition with another targeting signal, for instance, the yet unknown peptide that directed the inversely arranged full-length fusion protein, TLP-GFP, to the plasma membrane (Nam and Li, 2004).

Genome screens for PTS2 proteins should thus be extended beyond the N-terminal 40–amino acid domain (Table 3). Internal PTS2s, however, are thought to remain rare in nature and to occur preferentially in bifunctional proteins that evolved by gene fusion, as deduced for plant TLP, a predicted bifunctional enzyme of purine metabolism. The two catalytic domains of plant TLP were apparently fused shortly after the divergence of Dictyostelium in the plant lineage, to facilitate stoichiometric protein synthesis, peroxisome targeting, protein–protein interaction, and possibly metabolite channeling. Uricase, in fact, produces 5-hydroxyisourate (5-HIU) rather than the presumed pathway end product, allantoin. The enzyme that succeeds uricase in purine catabolism and opens the ring (i.e., 5-HIU hydrolase [COG2351]) turned out to be encoded by tlp (Figure 3A; Lee et al., 2005; Jung et al., 2006). The reaction product is converted to (S)-allantoin by a decarboxylase (COG3195; Ramazzina et al., 2006) that is homologous to the additional N-terminal domain of bifunctional Arabidopsis TLP (Figure 3). The likely role of Arabidopsis TLP in purine metabolism matches peroxisome targeting of full-length TLP324 to the same compartment as uricase. The shorter splice variants of plants lacking the PTS2 are probably cytosolic (e.g., AtTLP311 and AtTLP286; Figure 3A; see Supplemental Figure 3P online) and are expected to conduct distinct yet unknown physiological roles, possibly including hormone transport-related functions (e.g., in brassinosteroid signaling) (Nam and Li, 2004).

Peroxisomal Proteins Lacking Obvious PTSs Indicate Alternative Targeting Pathways

A significant number of the novel leaf peroxisomal proteins were expected to lack predicted PTS1/2 peptides (see Introduction). Some of these proteins had previously been predicted (e.g., GR and OASS A1) or experimentally demonstrated to be cytosolic (METE1, Ravanel et al., 2004; ACAT2, Carrie et al., 2007). Biochemically isolated cytosolic fractions, however, generally contain a significant portion of soluble proteins deriving from lysed peroxisomes. Additionally, owing to an insufficient consideration of plant PTS1 peptides in most subcellular prediction programs, peroxisome targeting is often overlooked and excluded at the outset from common reporter constructs with C-terminal tags. Peroxisome targeting of a few proteins remains ambiguous. OZI1 has also been detected in isolated mitochondria, but the corresponding organelle fraction contained several plant peroxisomal enzymes (Heazlewood et al., 2004). As OZI1 was restricted to leaf peroxisomes of high purity in our study and lacks a predicted mitochondrial presequence, the protein is considered to be peroxisome targeted. By contrast, Mn-dependent superoxide dismutase (At3g10920) has been identified from a minor two-dimensional gel spot, whose intensity appeared to negatively correlate with leaf peroxisome purity. The predicted mitochondrial presequence and its reported targeting to mitochondria (del Rio et al., 2003; Heazlewood et al., 2004) require quantitative proteomics methods to conclusively address dual subcellular targeting.

Among the proteins lacking PTS-like peptides, the majority strikingly belong to two functional groups, defense-related proteins (the myrosinase and β-glucosidase system), or proteins associated with nucleic acid metabolism. The negative result of a control experiment investigating an artificial copurification of high molecular weight complexes strongly supports a physiologically relevant association of the proteins with leaf peroxisomes; the two RNA helicases RH11 and RH37 are potential exceptions (see Supplemental Figure 5 online). An artificial adherence of defense- and nucleic acid metabolism-related proteins to the peroxisomal membrane induced upon cell lysis is considered unlikely but is an inherent option of organellar proteome studies in general that cannot be excluded by any controls.

At first glance, the prediction of ER signal peptides in five out of seven defense-related proteins suggests an ER contamination; the lack of housekeeping ER proteins in the same fraction, however, excludes this option. Likewise, copurification of ER bodies, known to contain PYK10 as a major constituent, with leaf peroxisomes is unlikely because these wound-inducible organelles are hardly detectable in rosette leaves of standard plants (Matsushima et al., 2002). Also, leaf wounding by plant harvest was reduced to a minimum. We therefore propose an alternative pathway for targeting of these defense proteins to peroxisomes. Recent phylogenetic analyses suggest that peroxisomes stem from the ER (Gabaldon et al., 2006; Schluter et al., 2006), and few peroxisomal membrane proteins (e.g., plant APX and a viral protein) are delivered to peroxisomes via distinct ER subdomains (Lisenbee et al., 2003; McCartney et al., 2005). Specific soluble proteins, for instance, those that are glycosylated as they pass the ER, as are PYK10 and myrosinases (Halkier and Gershenzon, 2006), may thus enter the peroxisomal matrix also via the ER and possibly the Golgi. Two glycosylated plastidic proteins have likewise recently been shown to pass the ER (Villarejo et al., 2005; Nanjo et al., 2006). Alternatively, some glycoside hydrolases may be peripheral peroxisomal membrane proteins, as is PEN2/BGLU26 (Lipka et al., 2005).

The association of nucleic acid metabolism-related enzymes with peroxisomes, which are well known to lack DNA, RNA, and ribosomes, is first puzzling. Nevertheless, peroxisome targeting is supported by the considerable number of six proteins, their specific detection in leaf peroxisomes of highest purity, and the absence of cytosolic and nuclear contaminants from isolated leaf peroxisomes. In light of the specialization of peroxisomes in purine (i.e., adenine, guanosine) catabolism and our limited knowledge of the physiological role of most of these proteins, their participation in DNA and/or RNA degradation may be proposed. The lack of both recognizable PTSs and ER signal peptides suggests a second alternative targeting mechanism. In conclusion, numerous novel proteins identified in this proteome study and lacking conventional targeting signals suggest, apart from intriguing and often unexpected physiological functions, the existence of two different yet unknown targeting pathways for soluble proteins to peroxisomes and make possible straightforward follow-up studies.

Extended Physiological Functions of Leaf Peroxisomes in Fatty Acid β-Oxidation, Glutathione Metabolism, and Herbivore Defense

Consistent with our initial hypothesis, many novel proteins are indeed plant specific and afford valuable insights into novel metabolic and defense pathways of plant peroxisomes, three examples of which will be outlined in more detail. The superfamily of SDRs comprises important enzymes participating in metabolism of complex secondary metabolites (Kallberg and Persson, 2006). As plant peroxisomal family members had previously not been characterized, the five SDRs reported here indicate a major yet unexplored role of SDRs in leaf metabolism and fatty acid degradation. SDRa (At4g05530; SRL>) is predicted to be orthologous to the vertebrate peroxisomal NADP-dependent carbonyl reductase involved in retinol reduction in the framework of arachidonic acid metabolism (see Supplemental Table 3 online; Fransen et al., 1999). Developmental coexpression analysis suggests that peroxisomal NADP-dependent IDH (Figure 4) rather than the peroxisomal OPPP provides SDRa, for instance, during senescence, with NADPH (Zimmermann et al., 2004). SDRb (At3g12800; SKL>) is most likely the Arabidopsis ortholog of yeast and mammalian peroxisomal 2,4-dienoyl-CoA reductase (DECR), an auxiliary enzyme required for β-oxidation of unsaturated fatty acids (see Supplemental Table 3 online; Gurvitz et al., 1997; Fransen et al., 1999; Reumann et al., 2004). Even though sharing previously unpredicted atypical PTS1s and similar expression patterns (e.g., in flowers, during seed development), SDRc (At3g01980; SYM>) and SDRd (At3g55290; SSL>) are predicted to differ in cofactor specificity (see Supplemental Table 3 online). SDRd is related to a potato (Solanum tuberosum) SDR implicated in metabolism of a steroid-like compound that affects gibberellin homeostasis and tuberization (Bachem et al., 2001). Because both enzymatic domains of bifunctional BSMDR (At1g49670; SRL>) are homologous to monofunctional enzymes from mammals, the enzyme is proposed to catalyze two subsequent oxidations of a hydroxyl group and a double bond of a steroid, phenylpropanoid derivative or lipid peroxidation product.

Pea plant peroxisomes contain GSH, whose primary function presumably is in ROS detoxification by the ascorbate-glutathione cycle (Jimenez et al., 1997, 1998). GR is the only GSH-dependent plant peroxisomal enzyme characterized biochemically. Pea GR has been partially purified, but peptide sequences have not been reported (del Rio et al., 2006; Romero-Puertas et al., 2006). With our identification of one peroxisomal GR isoform (At3g24170), dehydroascorbate reductase remains the last unknown cycle enzyme. Beyond GR, we identified two further GSH-dependent enzymes and are thus proposing multiple physiological roles of GSH in the peroxisomal matrix (see Supplemental Figure 7 online). Peroxisome targeting of GSTT1 (At5g41210; SKI>) has been demonstrated by three independent lines of evidence (Table 2, Figures 4K and 4L). The constitutive expression of GSTT1 and the high glutathione peroxidase activity of the recombinant enzyme (Wagner et al., 2002) suggest a physiological function in endogenous rather than stress metabolism, for instance, in detoxification of lipid peroxidation products. Alternatively, and in the context of the identification of two enzymes of Cys and Met biosynthesis in leaf peroxisomes (OASS A1 and METE1; Table 2), GSTT1 may also relate to sulfur amino acid metabolism, similar to yeast peroxisomal GST ω (Barreto et al., 2006). The single Arabidopsis HMGDH (At5g43940) associates plant peroxisomes with formaldehyde detoxification. The enzyme is central to the two- to three-step pathway, the major physiological and presumably cytosolic route for detoxification of the cytotoxic agent in plants. Interestingly, recombinant HMGDH also reveals high activity with S-nitrosoglutathione (Sakamoto et al., 2002; Diaz et al., 2003), possibly representing the missing link between plant peroxisomes and NO signaling (del Rio et al., 2002).

Few but comprehensive recent studies indicate that peroxisomes are substantially involved in plant defense against pathogen attack (Taler et al., 2004; Koh et al., 2005; Lipka et al., 2005). These mechanisms currently lack counterparts in yeast and mammals and point to plant-specific peroxisomal functions that evolved in sessile organisms. The Arabidopsis homolog of human macrophage MIF (At3g51660; SKL>) is most likely another player in the self-protection of plants. Human MIF reveals a wide array of cellular defensive effects, for instance, in immune responses (Sun et al., 1996; for review, see Calandra and Roger, 2003; Thiele and Bernhagen, 2005). As eukaryotic MIF homologs have previously not been localized to peroxisomes, Arabidopsis will be ideal for studying functional specification of MIF paralogs in peroxisomal defense mechanisms.

Lipka et al. (2005) identified PEN2 as a peroxisomal member of the superfamily of glycoside hydrolases (family 1; pfam00232; see Supplemental Figure 8 online) and characterized the protein as part of an inducible preinvasion resistance system. β-d-glucosidases are involved in a wide array of biological functions; apart from lignification and hormone level homeostasis, the enzymes play a major role in chemical defense against pathogens and herbivores (Xu et al., 2004), as best exemplified by myrosinases, also referred to as TGGs. Myrosinases hydrolyze glucosinolates (β-thioglucoside-N-hydroxysulfates) (i.e., sulfur-rich glucoside derivatives) and are among the most prominent defense compounds to liberate bioactive isothiocyanates, nitriles, or epithionitriles with a wide range of biological activities in plant–herbivore and plant–pathogen interactions (Kliebenstein et al., 2005; Grubb and Abel, 2006; Halkier and Gershenzon, 2006). TGG1 (At5g26000) and TGG2 (At5g25980), identified here along with their regulatory subunit ESM1, are the two major myrosinases in above-ground tissue with major redundant function in glucosinolate breakdown and insect defense, all of which had previously not been linked to peroxisomes (Barth and Jander, 2006). Myrosinase activity is regulated by ESM1, which represses nitrile formation relative to isothiocyanate production (Zhang et al., 2006). In addition to two further glycoside hydrolases identified in leaf peroxisomes (BGL1, At1g52400; and PYK10, see Results), five glycoside hydrolases are predicted to be targeted to peroxisomes (Xu et al., 2004). The characterization of SSL> as a functional PTS1 (Figures 5A and 5B) enables recognition of another entire clade of putative peroxisomal isoforms (see Supplemental Figure 8 online). In summary, our proteome data further support the emerging defense function of plant peroxisomes against pathogens and indicate a protective role of the organelle against herbivory by insects. Future studies need to conclusively demonstrate targeting of these defense-related proteins to peroxisomes, unravel the proposed ER-mediated biogenesis pathway, identify the glucoside substrates, and reveal their precise defense role.

METHODS

Plant Growth

Standard Arabidopsis thaliana ecotype Columbia-0 plants were grown for 3.5 to 4.5 weeks in a 16-h-light/8-h-dark cycle (light period 10 to 2 pm) at 22°C under a light intensity of 100 to 150 μE m−2 s−1. For the isolation of leaf peroxisomes from senescent leaves, 4-week-old plants were subjected for 4 d to dark-induced senescence. Plants were harvested at the end of an extended dark period (18 h dark).

Isolation of Leaf Peroxisomes

Arabidopsis leaves were ground in grinding buffer (170 mM Tricine-KOH, pH 7.5, 1.0 M sucrose, 1% [w/v] BSA, 2 mM EDTA, 5 mM DTT, 10 mM KCl, and 1 mM MgCl2) in the presence of protease inhibitors using a mortar and a pestle. The suspension was filtered, and chloroplasts were sedimented at 5000g (1 min, SS34 rotor). Approximately 20 mL of supernatant was loaded onto a Percoll density gradient prepared in TE buffer (20 mM Tricine-KOH, pH 7.5, and 1 mM EDTA) supplemented with 0.75 M sucrose and 0.2% (w/v) BSA underlaid by 36% sucrose (w/w) in TE buffer (top to bottom: 3 mL of 15% Percoll, 9 mL of 38% Percoll, 2 mL mixture of 38% Percoll, and 36% [w/w] sucrose at a ratio of 2:1 and 1:2, and 3 mL of 36% (w/w) sucrose in TE buffer). The Percoll gradients were centrifuged for 12 min at 10,500 rpm and 10 to 20 min at 15,000 rpm (SS34 rotor). For analytical purposes, the gradient was fractionated in 2-mL fractions and small aliquots frozen for enzymatic analyses. For preparative purposes, the peroxisome fractions at the bottom of the gradient of several Percoll gradients were combined, diluted in 36% (w/w) sucrose in TE buffer and sedimented by centrifugation (30 min, 15,000 rpm, SS34 rotor), yielding the leaf peroxisomal fraction LP-P1. The organelles were gently homogenized and loaded on top of a discontinuous sucrose density gradient (1 mL 41% [w/w], 1 mL 44% [w/w], 1 mL 46% [w/w], 2 mL 49% [w/w], 0.5 mL 51% [w/w], 1 mL 55% [w/w], and 1 mL 60% [w/w] in TE buffer) and ultracentrifuged (30 min to overnight, 25,000 rpm; Beckman SW40). For analytical purposes, the gradient was fractionated in 1-mL fractions and small aliquots frozen for enzymatic analyses. For preparative purposes, the leaf peroxisome fraction LP-P2, visible as a sharp white band above 55% (w/w) sucrose, was harvested, supplemented with protease inhibitors, and stored in appropriate aliquots. For the analysis of an artificial copurification of high molecular weight complexes with leaf peroxisomes, sucrose was omitted from the grinding and washing buffer and leaves and the fraction corresponding to that of intact leaf peroxisomes worked up in an identical manner. The activities of marker enzymes and protein concentration were determined as described (Ma et al., 2006) or by the 2-D Quant Kit (GE Healthcare) using BSA as standard. The contamination C of leaf peroxisomes by mitochondria and chloroplasts was calculated based on the relative activities of the corresponding marker enzyme activities (e.g., Afum. [% of CE]) that were recovered in the leaf peroxisome fraction LP-P2 (or LP − P1) compared with the crude extract (CE) according to the following formulae (Table 1):

equation M1
equation M2

2-DE

Improved protein solubilization was achieved in the presence of urea, thiourea, and zwitterionic detergents. The limited solubilization capabilities of 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) were effectively compensated by the addition of amidosulfobetaine-14, though accompanied with a slight decrease in resolution in the first dimension as indicated by vertical smearing of high abundant proteins with basic pI, such as GOX (Figure 2). The proteins (200 to 500 μg) were precipitated (Wessel and Flügge, 1984), dissolved in IEF buffer (7 M urea, 2 M thiourea, 2% [w/v] CHAPS, 2% [w/v] amidosulfobetaine-14, and 0.5% IPG buffer; Destreak), the solution clarified by centrifugation (20 min, 16,000 xg), and the proteins subjected for ∼60 kVh to isoelectric focusing (nonlinear Immobiline 18-cm pH gradient, pH 3.0 to 10). The proteins were then reduced and alkylated by equilibration for 12 min each in buffer (6 M urea, 50 mM Tris-HCl, pH 8.8, 30% [v/v] glycerol, 2% [w/v] SDS, and 0.001% [w/v] bromophenol blue) containing either 10 mg/mL DTT or 25 mg/mL iodoacetamide, respectively. For SDS-PAGE and the second dimension, the proteins were separated on large gradient gels (10 to 15% acrylamide) under denaturing conditions and stained with colloidal Coomassie Brilliant Blue.

Protein Identification from Two-Dimensional Gels

Manually excised gel plugs were processed as described in another recent large-scale proteome analysis (Werner et al., 2007). Briefly, an automated platform for the identification of gel-separated proteins (Jahn et al., 2006) was used to digest the proteins in gel with trypsin and to prepare the proteolytic peptides for MALDI-TOF-MS according to the thin layer affinity method. For each sample, a PMF spectrum and fragment ion spectra of up to four selected precursor ions were acquired within the same automated analysis loop using an Ultraflex I mass spectrometer (Bruker Daltonics). Database searches in the NCBI nonredundant primary sequence database restricted to the taxonomy of Arabidopsis were performed using the Mascot Software 2.0 (Matrix Science) licensed in house. Carboxamidomethylation of Cys residues was specified as fixed and oxidation of methionines as variable modification. One trypsin missed cleavage was allowed. Mass tolerances were set to 100 ppm for PMF searches and to 100 ppm (precursor ions) and 0.7 D (fragment ions) for MS/MS ion searches. Only proteins represented by at least one peptide sequence match above identity threshold in combination with the presence of at least four peptide masses assigned in the PMF were considered as identified.

LC-MS Shotgun Protein Analysis

Protein analysis was performed according to Wienkoop et al. (2004; see also detailed protocol in Morgenthal et al., 2007). Protein pellets (100 to 200 μg) were dissolved in 100 to 200 μL Lys-C solubilization buffer (50 mM Tris-HCl and 8 M Urea, pH 7.5) and digested with Endoproteinase Lys-C (Roche Applied Science) with an enzyme:substrate ratio of 1:1000 for 5 h at 37°C with gentle shaking. The digests were subsequently diluted fourfold with trypsin digestion buffer (50 mM Tris-HCl, 10% acetonitrile, and 10 mM CaCl2, pH 7.5), resulting in a final concentration of 2 M urea. After addition of immobilized trypsin beads (Applied Biosystems) (enzyme:substrate ratio of 1:20), the samples were further incubated for 10 h at 37°C with gentle shaking. The digests were dissolved in 0.1% formic acid. Samples were concentrated on a precolumn and subsequently loaded onto a 15-cm silica-based C18 RP monolithic column (Merck Bioscience). Elution of the peptides was performed using a 2-h gradient from 100% solvent A (5% acetonitrile and 0.1% formic acid in water) to 100% solvent B (90% acetonitrile and 0.1% formic acid in water) using the Agilent nano HPLC system with a flow rate of 400 nL per min. Eluting peptides were analyzed with an LTQ mass spectrometer (Thermo Electron) operated in a data-dependent mode. Each full MS scan was followed by three MS/MS scans, in which the three most abundant peptide molecular ions were dynamically selected for collision-induced dissociation using a normalized collision energy of 35%. The temperature of the heated capillary and electrospray voltage was 150°C and 1.9 kV, respectively. After MS analysis, DTA files were created from raw files and searched against a database (http://www.arabidopsis.org/) using Bioworks 3.1 software (Thermo Fisher Scientific) using a precursor ion mass tolerance of 500 ppm, oxidized Met as dynamic modification, and fully tryptic peptides with one allowed miscleavage. With DTASelect, a list of identified proteins was obtained using the following criteria: Xcorr: −1 2.0, −2 2.2, and −3 3.5 for hits with at least two different peptides. Selected peroxisomal proteins found by a single peptide were postanalyzed manually and protein identification confirmed for 3 of 13 proteins (GOX3, BSMAR, and PYK10; Table 2). All the filtered and manually validated spectra are stored in a mass spectral reference library for plant proteomics called ProMEX (Hummel et al., 2007), which is accessible online (http://promex.mpimp-golm.mpg.de/). Using text query searches, for instance the Arabidopsis Genome Initiative code of the protein, it is possible to list all the corresponding product ion spectra. Unknown samples can be searched against the ProMEX library using dta, mfg, or mz data format files.

Subcellular Localization Analysis in Allium cepa

The full-length cDNA clones of IDH (At1g54340; SRL>) and 6PGL (At5g24400; SKL>) were cloned by RT-PCR using appropriate oligonucleotide primers (see Supplemental Table 4 online) as described previously for other cDNAs (Ma et al., 2006). ECHIb (At4g14430; PKL>), SDRb (At3g12800, previously At3g12790; SKL>), HBCDH (At3g15290; PRL>), 6PGDH (At3g02360; SKI>), GSTT1 (At5g41210; SKI>), ECH2 (At1g76150; SSL>), EH3 (At4g02340; ASL>), and TLP (At5g58220; internal RLx5HL) were ordered from the ABRC. The cDNA of the minimal acetyltransferase 1 (ATF1; At1g21770; SSI>) was ordered from the RIKEN BioResource Center, and the missing N-terminal 12–amino acid residues were added by PCR (see Supplemental Table 4 online). The cDNAs were subcloned using appropriate oligonucleotide primers (see Supplemental Table 4 online) into pGEMT using the pGEM-T Easy vector system (Promega) or directly into pCAT-YFP-N-fus to fuse their 5′ end in frame with EYFP, under control of a double 35S cauliflower mosaic virus promoter (Fulda et al., 2002; Ma et al., 2006). For 6PGL, the predicted transit peptide was omitted when fused 5′-terminally with EYFP (EYFP-6PGL). Site-directed mutagenesis was performed using PfuUltra high-fidelity (HF) DNA polymerase for mutagenic primer-directed replication of both plasmid strands of EYFP-TLP in pCAT-EYFP-Nfus using the Quick-Change II site-directed mutagenesis kit (Stratagene; see Supplemental Table 4 online). Thereby, the internal PTS2 (RLx5HF) was mutated to the loss-of-function variant RLx5DF. For analysis of the peroxisome targeting function of putative PTS domains, the C-terminal 10 residues of ECHIb, SDRb/DECR, HBCDH, IDH, 6PGDH, 6PGL, GSTT1, ECH2, EH3, and ATF1 were fused to the C terminus of EYFP by PCR using an extended reverse primer and subcloned likewise into pCAT, replacing the EYFP insert. In double transformants, peroxisomes were labeled with CFP as described previously (Fulda et al., 2002; Ma et al., 2006). Onion epidermal cells were transformed biolistically and the onion slices placed on wet paper in Petri dishes and stored on a benchtop for 16 to 24 h. For analysis by fluorescence microscopy, the onion skin epidermal cell layer was peeled and transferred to a glass slide. All subcellular analyses were reproduced at least three times in independent experiments. Fluorescence microscopy was performed as described (Ma et al., 2006).

Supplemental Data

The following materials are available in the online version of this article.

  • Supplemental Figure 1. Work Flow of the Isolation of Leaf Peroxisomes from Mature Leaves of Arabidopsis by Percoll and Sucrose Density Gradient Centrifugation.
  • Supplemental Figure 2. Detection of Specific Leaf Peroxisomal Matrix Enzymes in Multiple Spots and Upregulation of Defense-Related Proteins in Leaf Peroxisomes from Senescent Plants.
  • Supplemental Figure 3. PTS Conservation of Novel Arabidopsis Proteins Identified in Isolated Leaf Peroxisomes.
  • Supplemental Figure 4. Subcellular Targeting Analysis of 6PGL-EYFP in Onion Epidermal Cells.
  • Supplemental Figure 5. Analysis of a Copurification of High Molecular Weight Complexes with Arabidopsis Leaf Peroxisomes.
  • Supplemental Figure 6. Venn Diagram Comparing the Number of Proteins Identified in This and Previous Proteome Studies on Arabidopsis Peroxisomes.
  • Supplemental Figure 7. Model of Plant Peroxisomal Glutathione Metabolism.
  • Supplemental Figure 8. Phylogenetic Analysis of the Arabidopsis Glycoside Hydrolase Family 1.
  • Supplemental Table 1. Known Proteins of Plant Peroxisomes Identified in Isolated Leaf Peroxisomes from Arabidopsis.
  • Supplemental Table 2. MS Data of Proteins Identified.
  • Supplemental Table 3. Sequence Conservation of SDR Domains in Peroxisomal SDRs.
  • Supplemental Table 4. Cloning, Subcloning, and Mutagenesis Primers.
  • Supplemental Data Set 1. Amino Acid Sequence Alignments Used in Construction of the Arabidopsis Glycoside Hydrolase Family 1 Phylogenetic Tree in Supplemental Figure 8.

Supplementary Material

[Supplemental Data]

Acknowledgments

We thank G. Gottschalk (Göttingen Genomics Center, Germany) and T. Pieler of the medical faculty for cDNA sequencing, I. Feuβner for the support of our research, M. Fulda for stimulating discussions, J.W. Kellmann (Max-Planck-Institute of Ecological Chemistry, Jena, Germany) for reading the manuscript, and A. Liavonchanka and G. Wieser for some cDNA subcloning work. We appreciate the editorial corrections and the help in SDR analysis provided by K. Bird (Plant Research Laboratory, Lansing, MI) and B. Persson (Karolinska Institutet, Stockholm, Sweden), respectively. cDNA provision by the ABRC and RIKEN is acknowledged. The research has been supported by grants from the Deutsche Forschungsgemeinschaft (RE1304/2 and RE1304/4) and by a Dorothea-Erxleben stipend from the government of Lower Saxony (to S.R.).

Notes

The author responsible for the distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Sigrun Reumann (ed.gdwg@namuers).

[W]Online version contains Web-only data.

www.plantcell.org/cgi/doi/10.1105/tpc.107.050989

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