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First published online March 27, 2009; 10.1104/pp.109.137703 Plant Physiology 150:125-143 (2009) © 2009 American Society of Plant Biologists OPEN ACCESS ARTICLE
In-Depth Proteome Analysis of Arabidopsis Leaf Peroxisomes Combined with in Vivo Subcellular Targeting Verification Indicates Novel Metabolic and Regulatory Functions of Peroxisomes1,[W],[OA]Michigan State University-Department of Energy Plant Research Laboratory (S.R., S.Q., K.A., P.Y., K.M.-S., R.S., J.H.), Plant Biology Department (K.A., N.L., C.G.W., A.P.M.W., J.H.), and Biochemistry and Molecular Biology Department (C.G.W.), Michigan State University, East Lansing, Michigan 48824; and Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, Michigan 48109 (S.R., D.H., L.J.O.)
Peroxisomes are metabolically diverse organelles with essential roles in plant development. The major protein constituents of plant peroxisomes are well characterized, whereas only a few low-abundance and regulatory proteins have been reported to date. We performed an in-depth proteome analysis of Arabidopsis (Arabidopsis thaliana) leaf peroxisomes using one-dimensional gel electrophoresis followed by liquid chromatography and tandem mass spectrometry. We detected 65 established plant peroxisomal proteins, 30 proteins whose association with Arabidopsis peroxisomes had been previously demonstrated only by proteomic data, and 55 putative novel proteins of peroxisomes. We subsequently tested the subcellular targeting of yellow fluorescent protein fusions for selected proteins and confirmed the peroxisomal localization for 12 proteins containing predicted peroxisome targeting signals type 1 or 2 (PTS1/2), three proteins carrying PTS-related peptides, and four proteins that lack conventional targeting signals. We thereby established the tripeptides SLM> and SKV> (where > indicates the stop codon) as new PTS1s and the nonapeptide RVx5HF as a putative new PTS2. The 19 peroxisomal proteins conclusively identified from this study potentially carry out novel metabolic and regulatory functions of peroxisomes. Thus, this study represents an important step toward defining the complete plant peroxisomal proteome.
Surrounded by single membranes, peroxisomes are small, ubiquitous eukaryotic organelles mediating a wide range of oxidative metabolic activities that vary by the species, cell type, and environmental conditions in which the organism lives (Beevers, 1979  ; Van den Bosch et al., 1992). Plant peroxisomes are essential to physiological processes such as lipid metabolism, photorespiration, and plant hormone biosynthesis and metabolism (Olsen and Harada, 1995; Zolman et al., 2000; Hayashi and Nishimura, 2003; Nyathi and Baker, 2006; Reumann and Weber, 2006). They are also essential for embryogenesis and play pivotal roles in plant responses to abiotic and biotic stresses (Lin et al., 1999; Hu et al., 2002; Schumann et al., 2003; Sparkes et al., 2003; Fan et al., 2005; Lipka et al., 2005; Desai and Hu, 2008). As additional functions are discovered for plant peroxisomes, a comprehensive inventory of peroxisomal proteins will be crucial to determine the underlying mechanisms for the new roles.
Because peroxisomes lack DNA, all peroxisomal proteins are imported directly from the cytosol or via the endoplasmic reticulum (ER; Purdue and Lazarow, 2001
Novel plant PTS peptides are being discovered in the postgenomic era. The characterization of only three additional PTS1 tripeptides allowed the prediction of about 100 additional PTS1-containing proteins in Arabidopsis (Reumann et al., 2007
Mass spectrometry (MS) analysis of proteins from purified peroxisomes is a powerful alternative approach to discover peroxisomal proteins, especially those unidentifiable by computational strategies. To date, researchers have generally used two-dimensional gel electrophoresis (2-DE) of purified plant peroxisomes followed by MS analysis to identify proteins from peroxisomes taken from Arabidopsis green cotyledons and leaves (Fukao et al., 2002
Because 2-DE has limitations in displaying hydrophobic, basic, and low-abundance proteins (Heazlewood and Millar, 2006
High Coverage of Known Plant Peroxisomal Proteins
We isolated peroxisomes from 4-week-old Arabidopsis leaves using a previously established method (Reumann et al., 2007
Mitochondria and chloroplasts are known to partially copurify with leaf peroxisomes. The detection of plastidic proteins by available polyclonal antisera (e.g. those against the inner envelope membrane protein Tic110 and the stromal chaperone ClpC) was below detection limits when analytical protein quantities (5 µg) were used (data not shown). Mitochondrial contamination, however, could be detected by immunoblotting using a monoclonal antiserum against the 30-kD voltage-dependent anion-selective channel (VDAC) protein from maize (Zea mays; Fig. 1A
). In addition, the intensity of the Arabidopsis VDAC band correlated with the abundance of a 110-kD protein band on silver-stained analytical SDS gels among different peroxisome isolates. The size of the protein indicated its identity as the P-subunit of the mitochondrial Gly decarboxylase, a major protein of leaf mitochondria (Fig. 1B; Supplemental Table S1). Using both immunoblotting and silver staining of protein gels as selection criteria, we identified three of approximately 30 preparations as leaf peroxisome isolates of highest purity and pooled these samples for subsequent proteome analysis. To increase the chance of discovering low-abundance proteins in the sample, we separated a large amount of peroxisomal proteins (500 µg) on a 1-D gel and cut the gel lane into 16 slices after electrophoresis (Fig. 1C). Proteins in each gel slice were in-gel digested by trypsin; the resulting peptide mixtures were subjected to LC-electrospray ionization-MS/MS. Proteins were judged to be present in the sample if they had a 95% probability of being correctly identified, as assigned by ProteinProphet (Nesvizhskii et al., 2003
We identified 302 proteins, among which 280 had at least two matching peptides and 285 were detected by  99% probability (Table I
; Supplemental Tables S1–S3). Sixty-five proteins are referred to in this study as established peroxisomal proteins, because they have been functionally characterized from at least one plant species. Except for seven proteins (ACX6, DEG15 protease, malate synthase, isocitrate lyase, polyamine oxidase 4, Hsp15.7, and sarcosine oxidase), we identified most matrix proteins that had been functionally characterized in plant peroxisomes (Supplemental Table S2). The 89% coverage (57 of 64 proteins) of established peroxisomal matrix proteins in this study is quite high compared with previous proteome analyses of plant peroxisomes. The coverage of membrane proteins in this study was also improved over a former study of leaf peroxisomes (Reumann et al., 2007). For example, all five members of the plant PEX11 family (PEX11a–PEX11e) of peroxisome proliferation promoters, the peroxisome biogenesis factor PEX14, two known metabolite transporters, PMP36 and PXA1, and MDAR4 (for monodehydroascorbate reductase isoform 4) were identified. Four of these 65 established peroxisomal proteins (ACX2, 4CLP2, PEX11b, and MDAR4; Supplemental Table S2) were identified for the first time in a proteomic study of plant peroxisomes.
We also identified 30 additional proteins whose peroxisomal association had been demonstrated previously only by proteome data and remained to be verified by an independent line of evidence (Table I; Supplemental Table S3). Twenty-two of these proteins had first been identified by Reumann et al. (2007) , which included, for instance, nearly all of those with a proven or predicted role in pathogen and herbivore defense (e.g. BGL1, TGG1 and TGG2, and MIF; Table I), further supporting their putative peroxisomal associations. Eleven additional proteins first associated with peroxisomes by Reumann et al. (2007) were already confirmed to be peroxisomal by a subcellular targeting study of YFP fusion proteins (Reumann et al., 2007) and are now listed in the group of established plant peroxisomal proteins (Supplemental Table S2). Eight of the 30 proteins were independently identified by Eubel et al. (2008; ZnDH, AAE1, MCD, IndA, CuAO, AAE17, and PM-16) or Fukao et al. (2003; sT3).
Protein levels within a subcellular compartment can span a wide dynamic range. To compare the abundance of proteins discovered from our shotgun proteomic experiment, we calculated normalized spectral abundance factors (NSAFs; i.e. spectral counts that were normalized after taking into consideration protein size and variation between sample runs; Paoletti et al., 2006
Assigning Putative Novel Proteins of Leaf Peroxisomes
In addition to the 95 peroxisomal proteins mentioned above, the higher sensitivity of our approach allowed the identification of a significant number of putative novel peroxisomal proteins. However, this achievement was inevitably accompanied by an increased identification rate of proteins from chloroplasts and mitochondria (Fig. 2, A and B), thus making the annotation of putative novel proteins of leaf peroxisomes more difficult. To differentiate between nonperoxisomal and putative novel peroxisomal proteins, we took advantage of the large number of organellar proteome studies published for Arabidopsis mitochondria and chloroplasts, summarized in the Arabidopsis Subcellular Proteomic Database (www.plantenergy.uwa.edu.au/applications/suba2/; Heazlewood et al., 2007
Based on the above analyses, we concluded that 55 proteins have a strong probability for peroxisomal localization; thus, we referred to these proteins as putative novel proteins of leaf peroxisomes (Table I; Supplemental Table S3). Since the prediction of protein targeting to peroxisomes is not available at The Arabidopsis Information Resource (TAIR), most of these proteins were tentatively annotated as being cytosolic by TAIR for lacking identifiable organelle targeting signals. In contrast to the highly to moderately abundant proteins identified previously in leaf peroxisomes (see above), most putative novel proteins of plant peroxisomes detected for the first time in this study are of low abundance, with an average NSAF value of 0.77 x 10–3 (Fig. 2A). Forty-eight of these 55 proteins were identified with high confidence (
Based on annotation, sequence homology, and predicted function, we classified the putative novel proteins into several categories: β-oxidation (auxiliary enzymes), ROS metabolism (including glutathione metabolism and defense), other metabolic enzymes, nucleotide and nucleic acid metabolism, chaperones and proteases, and other proteins such as those with unknown functions (Table I).
A major functional group is composed of seven proteins with predicted auxiliary functions in fatty acid β-oxidation, four of which carry predicted PTS1s (Table I). Our study identified two additional members of the functionally uncharacterized family of small thioesterases (sTs). The predicted PTS1 (AKL>) for sT1 was previously shown to be highly conserved in homologous genomic sequences and plant ESTs (Reumann et al., 2004
Enzymes with annotated functions related to ROS metabolism include four glutathione S-transferases (GSTs) that belong to the U and F subfamilies (GSTU19 and GSTU20, and GSTF7 and GSTF10). Dehydroascorbate reductase 1 (DHAR1) is probably the last missing protein of the peroxisomal ascorbate glutathione cycle (Jimenez et al., 1997 ), catalyzed by four matrix-targeted or membrane-associated enzymes (i.e. APX3, MDAR1/4, glutathione reductase, and DHAR). Peroxisomal DHAR had been characterized biochemically in pea (Pisum sativum) peroxisomes (Jimenez et al., 1997), but the corresponding cDNA had not been cloned from any plant species. Additional metabolic enzymes that are not obviously β-oxidation related include a thiol methyltransferase (At2g43940), a dephospho-CoA kinase homolog (COAE), UDP-Xyl synthase 6 (UXS6), nucleoside-diphosphate kinase type 1 (NDPK1), a catalytic/coenzyme-binding protein (At5g02240), and a quinone reductase (FQR1); three of these six proteins carry C-terminal PTS1-related tripeptides: STA> (FQR1; At5g54500), STL> (At2g43940), and SRF> (At5g02240).
In fulfillment of our intention to identify regulatory proteins, more than 20 of the putative novel proteins can be categorized as nonmetabolic proteins that most likely have regulatory roles in metabolism, signal transduction, or protein processing. Three proteins are likely involved in protein/peptide processing or turnover. Lon protease homolog 2 (Lon2) is orthologous to proteases from mammalian and yeast peroxisomes (Kikuchi et al., 2004
In the category of nucleotide and nucleic acid metabolism, we uncovered two isoforms of the Gly-rich RNA-binding family of proteins (GRPs) that added to the two GRPs previously identified by Reumann et al. (2007) Additional proteins with predicted regulatory functions include an acid phosphatase class B homolog, four isoforms of the 14-3-3 protein family of phosphorylated protein-binding factors (general regulatory factors), two thioredoxin homologs (TRX-H-3/5), two GTP-binding proteins, and a second homolog of the peroxisomal acetyl transferase (ATF2). Furthermore, a gibberellin-responsive GAST1 protein homolog, a cold-regulated protein, a putative cytochrome b5 homolog, and a senescence-associated protein (B12D1) were also assigned to this group. Homologs of actin and tubulin were also detected but were not associated with specific gene models, due to high sequence identity among the paralogs. Lastly, a few proteins lacking functional annotations are referred to as unknown proteins (UPs), among which three proteins carry predicted PTSs (UP5 [SRL>] and UP6 [SKL>]) or PTS1-related tripeptides (UP7 [SLM>]). Contrary to PTS1 conservation in UP5 and UP6 homologs across diverse plant families, peroxisome targeting of UP7 may be restricted to the Brassicaceae (Fig. 3, G–I).
To validate peroxisomal targeting of the putative novel proteins identified in this proteome analysis, we tested the subcellular localization of a subset of these proteins utilizing in vivo targeting analysis of YFP fusions. During the course of this work, HAOX1 and Lon2 were independently confirmed by alternative methods to be peroxisome targeted (C. Mayer and S. Reumann, unpublished data; T. Johnson and L.J. Olsen, unpublished data; Table II ) and therefore were excluded from further analysis. We divided the proteins into three categories based on the detection of predicted PTSs, PTS-related peptides, or no recognizable PTSs in these proteins and focused on representatives from each category.
The first group contained all seven putative novel proteins with predicted PTS1/2 (sT1, sT5, ELT1, ATF2, HIT2, UP5, and UP6; Table I), as well as eight PTS-containing proteins also identified independently by Eubel et al. (2008 ; AAE1, AAE17, MCD, ZnDH, CuAO, IndA, and P-M16) or Fukao et al. (2003; sT3) but whose peroxisomal localization had not been verified by alternative means (Table I). In fact, these eight proteins are also highly likely to be peroxisomal, because their homologs in some or all other plant species contain PTS or PTS-related sequences (see Reumann et al., 2007, for sT3; see Supplemental Fig. S1 for the other seven). The second group included four putative novel proteins with C-terminal PTS1-related sequences or N-terminal PTS2-related sequences, namely, the putative copper chaperone CCH (SQV>), a putative coenzyme-binding protein (At5g02240; SRF>), UP7 (SLM>), and HIT3 (RVx5HF). We also added to this group HIT1/NBP (SKV>), another His triad family protein besides HIT2 and HIT3, because this protein had been identified previously through proteomics (Reumann et al., 2007) but had not been confirmed for its peroxisomal localization by alternative methods. The third group consisted of 16 putative novel proteins lacking predicted PTSs (Table II), most of which have annotated functions not associated with peroxisomes previously. For medium-throughput cloning of candidate genes, we first created two Gateway-compatible destination vectors for fusion of the coding regions of candidate cDNAs to the N terminus (for PTS2-containing proteins) or the C terminus (for PTS1-containing proteins) of the enhanced YFP and for driving gene expression by the cauliflower mosaic virus 35S promoter. Genes encoding proteins without predicted PTSs were cloned into both vectors or first into the vector for PTS2-containing proteins, because PTS2-related sequences are more difficult to detect than PTS1-like sequences. We were unable to clone AAE17, P-M16, and UP6 into destination vectors. As a result, YFP fusions for 33 genes (Table II) were transiently coexpressed with the peroxisomal marker gene CFP-PTS1 in tobacco (Nicotiana tabacum) leaves. Subcellular protein targeting was analyzed by confocal laser scanning microscopy (CLSM) 2 to 3 d after Agrobacterium tumefaciens-mediated infiltration of the constructs. All 12 proteins with predicted PTS1 or PTS2 peptides were verified to be targeted to peroxisomes, largely with one-on-one colocalization between their YFP fusions and the CFP-PTS1 peroxisome marker (Fig. 4 ). Notably, seven of these proteins (AAE1, ELT1, MCD, sT1, sT3, sT5, and ZnDH) have annotated functions in lipid metabolism (Table II).
Peroxisomal targeting for three of the five proteins with PTS-relative peptides was also confirmed. Of the four proteins with PTS1-related sequences, HIT1/NBP (SKV>) and UP7 (SLM>) localized to peroxisomes (Fig. 5, A and D ), whereas CCH (SQV>) and At5g02240 (SRF>) were found to localize to the cytosol and nucleus, respectively (Table II). Peroxisome targeting of HIT1/NBP and UP7 suggested that SKV> and SLM> may be new PTS1 tripeptides. To further test this hypothesis, we analyzed the localization of two fusion proteins lacking the respective tripeptides. As expected, the truncated proteins failed to target to peroxisomes (Fig. 5, B and E). We also examined the subcellular localization of YFP with SKV> or SLM> fused to its C terminus (YFP-SKV or YFP-SLM) and observed strong colocalization of the respective fusion proteins with CFP-PTS1; in both cases, targeting to the nucleus and cytosol was also observed (Fig. 5, C and F). Besides UP7 and HIT1, the Arabidopsis genome contains 10 more proteins terminating with SLM> and 13 additional proteins containing C-terminal SKV>; these proteins thereby emerge as putative peroxisomal matrix proteins (Supplemental Table S4). Collectively, our results identified both SKV> and SLM> as new PTS1 tripeptides and suggested that some Arabidopsis proteins containing these two tripeptides may potentially target to the peroxisome as well.
HIT3, which has a PTS2-related nonapeptide (RVx5HF) in its N-terminal domain, targeted to spherical structures labeled by CFP-PTS1 (Fig. 6A ). Interestingly, this protein seems to be dual targeted, as reticulate structures mostly surrounding the chloroplasts were also labeled by yellow fluorescence (Fig. 6B). Thus far, three of the five Arabidopsis HIT proteins have been found to be associated with peroxisomes, revealing the unique involvement of this class of nucleotide hydroxylases/transferases in plant peroxisome functions.
We also tested the localization of 16 proteins that lack recognizable PTSs. Consistent with its predicted involvement in the peroxisomal ascorbate glutathione cycle, DHAR1 localized strongly to peroxisomes (Fig. 7A ). COAE localized to the periphery of peroxisomes, suggesting its possible association with the membrane of these organelles (Fig. 7B). B12D1, a senescence-associated small protein of 88 amino acids, targeted to peroxisomes and led to peroxisome aggregation (Fig. 7C). This peroxisomal phenotype indicates that B12D1 may have a role in peroxisome distribution; alternatively, the phenotype may represent a dominant negative effect caused by attaching the 27-kD YFP to a small protein. Finally, NDPK1 was highly enriched in peroxisomes, paralleled by its weak targeting to the nucleus and cytosol (Fig. 7D). These results collectively demonstrated that an increasing number of peroxisomal proteins lacking predicted or closely related PTS1/2 tripeptides are being identified. These proteins may be either surface associated with or imported into peroxisomes via alternative yet unknown import mechanisms. However, peroxisome targeting of the remaining 12 proteins lacking obvious PTS-related sequences was not supported by our transient expression assays, as they were found to be associated with the nucleus, plasma membrane, or cytosol (Table II). These data suggest that (1) some proteins from this group could be contaminants from other compartments and (2) our transient expression system may have limitations in validating some of the true peroxisomal proteins.
Increasing the Dynamic Range of Protein Identification A major goal of current plant peroxisomal proteome research is to detect low-abundance proteins by increasing the dynamic range of protein identification. This goal is challenging for leaf peroxisomes, because enzymes for the photorespiratory C2 cycle and ROS detoxification predominate in leaf peroxisomes, constituting 63% of the total NSAF values of leaf peroxisomal proteins (Fig. 2C). These enzymes, together with the numerous enzymes related to fatty acid β-oxidation (another 23%), constitute nearly 90% of the leaf peroxisomal proteome (Fig. 2C). The predominance of these matrix enzymes makes it difficult to identify by gel-based approaches novel peroxisomal proteins, especially those that have similar subunit molecular masses (30–60 kD), as the abundant proteins. In addition, the moderate abundance of leaf peroxisomes in mesophyll cells, their high fragility in aqueous solution, and their tight association in vivo with chloroplasts and mitochondria further limit the sensitivity of peroxisomal protein identifications and lead to minor, unavoidable copurification of mitochondria and chloroplasts with leaf peroxisomes. Using anti-VDAC immunoblotting and silver-stained gels for purity assessment, 1-DE, high-resolution HPLC separation of peptides, and high-sensitivity MS/MS, our study improved the dynamic range of protein identification and overcame these limitations to some degree.
The number of known and putative novel proteins identified as well as experimentally verified novel proteins of Arabidopsis peroxisomes in our study also surpasses those published for peroxisomes from fungi and mammals (for review, see Saleem et al., 2006 Among the five tested proteins with 98% or less probability of protein identification, two were confirmed to be peroxisomal (ATF2 and B12D1). These data, together with the fact that several known peroxisomal proteins were also identified by lower probability (Supplemental Table S2), demonstrate that some true positives could be identified from the group of proteins with slightly lower probability of protein identification.
The improved representation of hydrophobic and basic proteins by 1-DE, as opposed to 2-DE, enabled us to now detect several peroxisomal membrane proteins (Supplemental Table S2), the number of which is comparable to that identified by Eubel et al. (2008)
In summary, our in-depth proteome analysis represents a significant extension of protein identification from former studies (Fukao et al., 2002
It was previously shown that NSAF quantification of proteins correlates well with gel-based quantification (Zybailov et al., 2008 ). However, it was also found that spectral counting tends to underestimate the amount of the most abundant proteins, such as catalase in the case of peroxisomes, whereas gel-based quantification tends to be less accurate for low-abundance proteins (Zybailov et al., 2008). In addition, hydrophobic membrane proteins on average have fewer tryptic cleavage sites than hydrophilic soluble proteins. As a result, fewer peptides per protein are expected to be detected for membrane proteins than for soluble proteins, thus leading to a systematic underestimation of membrane protein abundance. Since the focus of our study was predominantly on the discovery of low-abundance peroxisomal proteins, we overloaded the protein gel before generating slices for tryptic digests, which may have also compromised NSAF-based quantification. Finally, our results were obtained from a single shotgun proteomic experiment with no replications, yet significant variations in spectral counts could be observed between independent experiments. Nevertheless, the NSAF-based protein quantification data presented in this study do represent a tentative indication of protein abundance in peroxisomes. Further targeted studies, including larger numbers of replicates, will be required for a detailed quantification of the peroxisomal proteome.
The increased detection of nonperoxisomal proteins made our annotation of potential novel proteins more difficult. Notably, many of the proteins stringently classified here as plastidic or mitochondrial contaminants were identified only in the most recent and comprehensive proteome studies of chloroplasts and mitochondria (Kleffmann et al., 2004
Intriguingly, most proteins designated as potential novel peroxisomal proteins lack computationally predictable targeting signals and have been tentatively annotated as cytosolic. The lack of predictable PTS1/2s in many novel proteins of peroxisomes is largely consistent with our current knowledge of the mechanisms and signals for protein targeting to peroxisomes. First, hydrophobic proteins, such as integral and peripheral proteins of the peroxisomal membrane, which are better covered by the 1-DE shotgun proteomic approach chosen here, lack PTS1/2 sequences. Second, accumulating evidence suggests that low-abundance matrix proteins preferentially carry unusual and mostly unknown variants of PTS peptides (Reumann et al., 2007
Using in vivo targeting analysis, we confirmed the peroxisomal association of 19 proteins identified by proteomics. We consider the terminal placement of the reporter protein well suited for conclusive subcellular targeting analyses. For instance, in YFP-X constructs, predicted PTS1 domains are in their native conformation with regard to the full-length protein (i.e. surface exposed or internally hidden). Creating "internal" fusions by inserting GFP 10 amino acids upstream from the C terminus (Eubel et al., 2008
Our data have defined SLM> and SKV> as two novel plant PTS1s and suggested that the PTS2-related N-terminal nonapeptide RLx5HF is a functional PTS2. SLM> is an unusual PTS1, as it contains a Leu residue at position –2, which is absent from all plant PTS1 tripeptides characterized to date. Plant PTS1 tripeptides have been shown previously to tolerate significant structural deviations from the general requirement of a positively charged residue at this position (Mullen et al., 1997
While transient protein expression in tobacco leaves is an ideal methodology for testing subcellular protein targeting in a high-throughput fashion, it may have some limitations. Although the 14 proteins that failed to show peroxisome targeting in our transient assay are possible contaminants from other cell compartments, some of them may be truly associated with peroxisomes in plant cells. For example, members of the 14-3-3 protein family (general growth factors [GRFs]) generally bind to phosphorylated proteins and regulate signal transduction by regulating the localization of the bound proteins (DeLille et al., 2001
The many proteins identified in this study and conclusively demonstrated to be peroxisome targeted in vivo suggest that leaf peroxisomes perform a wider range of metabolic functions than previously anticipated. One example is fatty acid β-oxidation-related metabolism. We identified a number of, to our knowledge, new auxiliary enzymes presumably involved in leaf peroxisomal β-oxidation. Besides the six known peroxisomal acyl-activating enzymes (AAEs), AAE1 and AAE17 belong to distinct clades of the AAE superfamily (Shockey et al., 2003
Malonyl-coenzyme A decarboxylase (MCD) catalyzes the breakdown of malonyl-CoA, an intermediate in fatty acid biosynthesis, to acetyl-CoA and carbon dioxide and is encoded by a single gene in Arabidopsis. The mammalian MCD is located in mitochondria, peroxisomes, and the cytoplasm (Sacksteder et al., 1999
Copper amine oxidases (CuAOs; EC 1.4.3.6) and flavin-containing amine oxidases catalyze the oxidative deamination of polyamines, ubiquitous compounds essential for cell growth and proliferation (Cona et al., 2006
HIT proteins constitute a superfamily of nucleotide-binding, -hydrolyzing, and -transfering enzymes, using the common motif His-x-His-x-His-xx (x being a hydrophobic residue) as the active catalytic site (Brenner, 2002
In the Arabidopsis genome, we have found five HIT proteins encoded by At5g16566 (HIT1), At5g48545 (HIT2), At3g56490 (HIT3), At1g31160, and At5g58240. Our study conclusively identified three of them to be peroxisomal, all of which carry PTSs. Interestingly, HIT2 was identified as one of the few genes up-regulated in Arabidopsis stem explants treated with indole 3-butyric acid (IBA), an indole-3-acetic acid precursor that specifically promotes adventitious root formation (Ludwig-Müller et al., 2005
Our confocal microscopic analysis suggests that the putative dephospho-CoA kinase, COAE, is possibly associated with the membrane of peroxisomes despite its lack of predicted transmembrane domains (Fig. 7B; Table II). Dephospho-CoA kinases catalyze the final step of CoA biosynthesis by phosphorylating the 3'-hydroxyl group of Rib, using ATP as a phosphate donor (Obmolova et al., 2001
Nucleoside diphosphate kinase catalyzes the transfer of a phosphate group from a nucleoside triphosphate, such as ATP, onto a nucleoside diphosphate, such as GDP, thereby converting one molecule of each GDP and ATP into GTP and ADP. By this reaction, NDPK controls the level of GTP, which is, for example, required for the conversion of inactive GDP-bound GTPases to the active GTP-bound form. NDPK2, another isoform of NDPK in Arabidopsis, was shown to bind and stimulate the GTPase activity of small G proteins and serve as a signal transducer in phytochrome signaling (Choi et al., 1999
We have taken an important step toward defining a comprehensive protein map of plant peroxisomes. Next to the predominating enzymes involved in photorespiration and ROS metabolism, we can now detect a large number of peroxisomal proteins of moderate or even low abundance, among which are some regulatory proteins with unexpected peroxisome-related functions that had not been revealed by previous proteome studies of Arabidopsis peroxisomes. Confirmation of the peroxisome association of all of the putative novel proteins with in vivo targeting analysis or alternative methods is crucial before we can unequivocally further expand the current map of the peroxisome proteome. Mapping of the targeting signals on the novel proteins lacking predicted PTSs will provide important insights into yet unknown import pathways and transport mechanisms of matrix and membrane proteins of low abundance. Functional analyses of these novel proteins by reverse genetics should shed light on new metabolic and regulatory mechanisms and signal transduction cascades in peroxisomes.
Plant Growth and Isolation of Leaf Peroxisomes
Arabidopsis (Arabidopsis thaliana ecotype Columbia) plants were grown for 4 to 6 weeks in a 16-h-light/8-h-dark cycle under 100 to 150 µE m–2 s–1 light. Rosette leaves were harvested and leaf peroxisomes isolated as described previously (Reumann et al., 2007
Proteins were precipitated by chloroform/methanol (Wessel and Flugge, 1984
The proteins of highly pure Arabidopsis leaf peroxisomes (500 µg) were precipitated (Wessel and Flugge, 1984
The extracted peptides were automatically injected by a Waters nanoAcquity Sample Manager (www.waters.com) and loaded for 5 min onto a Waters Symmetry C18 peptide trap (5 µm, 180 µm x 20 mm) at 4 µL min–1 in 2% acetonitrile/0.1% formic acid. The bound peptides were then eluted onto a Waters BEH C18 nanoAcquity column (1.7 µm, 100 µm x 100 mm) and eluted over 120 min with a gradient of 5% B to 90% B in 103 min using a Waters nanoAcquity UPLC system (buffer A = 99.9% water/0.1% formic acid, buffer B = 99.9% acetonitrile/0.1% formic acid) into a ThermoFisher LTQ-FTICR mass spectrometer (www.thermo.com) at a flow rate of 300 nL min–1. Survey scans were taken in the Fourier transformation at 25,000 resolution at a mass-to-charge ratio of 400, and the top 10 ions in each survey scan were then subjected to automatic low-energy collision-induced dissociation in the LTQ-FTICR mass spectrometer. The resulting MS/MS spectra were converted to peak lists using BioWorks Browser version 3.2 (ThermoFisher) with default parameters and searched using the Mascot searching algorithm version 2.2 (www.matrixscience.com) against the TAIR8 genome database downloaded from TAIR (http://www.Arabidopsis.org/). The Mascot output was then analyzed using Scaffold (www.proteomesoftware.com) to probabilistically validate protein identifications using the ProteinProphet (Nesvizhskii et al., 2003
Two destination vectors were created using Gateway cloning technology (Invitrogen). Using the pPZP212 binary vector (Hajdukiewicz et al., 1994
Four-week-old tobacco (Nicotiana tabacum) plants were used for the Agrobacterium tumefaciens-mediated transient expression assays. A. tumefaciens strains C85C1 and GV3101 (pMP90) containing the plasmid of interest were incubated at 28°C overnight, washed, and resuspended in water to an optical density at 600 nm of 0.5. Cells transformed with plasmids harboring either a YFP fusion or the CFP-PTS1 peroxisomal marker were mixed and infiltrated into tobacco leaves using 1-mL needleless syringes. Leaves of infiltrated plants were analyzed after 2 d by CLSM (Zeiss LSM 510 META). We used 458-, 514-, and 633-nm lasers to excite CFP, YFP, and chlorophyll, respectively. Fluorescence was detected using an emission filter of a 460- to 510-nm band pass for CFP, a 520- to 555-nm band pass for YFP, and a 650-nm long pass for chlorophyll. All images were acquired from single optical sections.
The following materials are available in the online version of this article.
We thank the Arabidopsis Biological Resource Center for providing cDNA clones, Ethan Dawson-Baglien for technical assistance, and Karen Bird for editorial assistance. Received February 25, 2009; accepted March 23, 2009; published March 27, 2009.
1 This work was supported by the National Science Foundation Arabidopsis 2010 Program (grant no. MCB 0618335 to J.H. and grant no. MCB 0618279 L.J.O.). 
2 Present address: Centre for Organelle Research, Faculty of Science and Technology, University of Stavanger, N–4036 Stavanger, Norway.
3 Present address: Department of Plant Biochemistry, Heinrich-Heine-University, D–40225 Duesseldorf, Germany. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Jianping Hu (huji{at}msu.edu).
[W] The online version of this article contains Web-only data.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.109.137703 * Corresponding author; e-mail huji{at}msu.edu.
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