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First published online September 11, 2009; 10.1104/pp.109.142505 Plant Physiology 151:1354-1365 (2009) © 2009 American Society of Plant Biologists OPEN ACCESS ARTICLE
Arabidopsis LON2 Is Necessary for Peroxisomal Function and Sustained Matrix Protein Import1,[C],[W],[OA]Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 77005
Relatively little is known about the small subset of peroxisomal proteins with predicted protease activity. Here, we report that the peroxisomal LON2 (At5g47040) protease facilitates matrix protein import into Arabidopsis (Arabidopsis thaliana) peroxisomes. We identified T-DNA insertion alleles disrupted in five of the nine confirmed or predicted peroxisomal proteases and found only two—lon2 and deg15, a mutant defective in the previously described PTS2-processing protease (DEG15/At1g28320)—with phenotypes suggestive of peroxisome metabolism defects. Both lon2 and deg15 mutants were mildly resistant to the inhibitory effects of indole-3-butyric acid (IBA) on root elongation, but only lon2 mutants were resistant to the stimulatory effects of IBA on lateral root production or displayed Suc dependence during seedling growth. lon2 mutants displayed defects in removing the type 2 peroxisome targeting signal (PTS2) from peroxisomal malate dehydrogenase and reduced accumulation of 3-ketoacyl-CoA thiolase, another PTS2-containing protein; both defects were not apparent upon germination but appeared in 5- to 8-d-old seedlings. In lon2 cotyledon cells, matrix proteins were localized to peroxisomes in 4-d-old seedlings but mislocalized to the cytosol in 8-d-old seedlings. Moreover, a PTS2-GFP reporter sorted to peroxisomes in lon2 root tip cells but was largely cytosolic in more mature root cells. Our results indicate that LON2 is needed for sustained matrix protein import into peroxisomes. The delayed onset of matrix protein sorting defects may account for the relatively weak Suc dependence following germination, moderate IBA-resistant primary root elongation, and severe defects in IBA-induced lateral root formation observed in lon2 mutants.
Peroxisomes are single-membrane-bound organelles found in most eukaryotes. Peroxin (PEX) proteins are necessary for various aspects of peroxisome biogenesis, including matrix protein import (for review, see Distel et al., 1996  ; Schrader and Fahimi, 2008). Most matrix proteins are imported into peroxisomes from the cytosol using one of two targeting signals, a C-terminal type 1 peroxisome-targeting signal (PTS1) or a cleavable N-terminal type 2 peroxisome-targeting signal (PTS2) (Reumann, 2004). PTS1- and PTS2-containing proteins are bound in the cytosol by soluble matrix protein receptors, escorted to the peroxisome membrane docking complex, and translocated into the peroxisome matrix (for review, see Platta and Erdmann, 2007). Once in the peroxisome, many matrix proteins participate in metabolic pathways, such as β-oxidation, hydrogen peroxide decomposition, and photorespiration (for review, see Gabaldon et al., 2006; Poirier et al., 2006).
In addition to metabolic enzymes, several proteases are found in the peroxisome matrix. Only one protease, DEG15/Tysnd1, has a well-defined role in peroxisome biology. The rat Tysnd1 protease removes the targeting signal after PTS2-containing proteins enter the peroxisome and also processes certain PTS1-containing β-oxidation enzymes (Kurochkin et al., 2007
In contrast with DEG15, little is known about the other eight Arabidopsis proteins that are annotated as proteases in the AraPerox database of putative peroxisomal proteins (Reumann et al., 2004
PXM16 is the only one of the nine Arabidopsis M16 (pitrilysin family) metalloproteases (García-Lorenzo et al., 2006 ; Rawlings et al., 2008) containing a predicted PTS. M16 subfamilies B and C contain the plastid and mitochondrial processing peptidases (for review, see Schaller, 2004), whereas PXM16 belongs to M16 subfamily A, which includes insulin-degrading peptidases (Schaller, 2004). A tomato (Solanum lycopersicum) M16 subfamily A protease similar to insulin-degrading enzymes with a putative PTS1 was identified in a screen for proteases that cleave the wound response peptide hormone systemin (Strassner et al., 2002), but the role of Arabidopsis PXM16 is unknown.
Arabidopsis LON2 is a typical Lon protease with three conserved domains: an N-terminal domain, a central ATPase domain in the AAA family, and a C-terminal protease domain with a Ser-Lys catalytic dyad (Fig. 1A
; Lee and Suzuki, 2008
In this work, we examined the roles of several putative peroxisomal proteases in Arabidopsis. We found that lon2 mutants displayed peroxisome-deficient phenotypes, including resistance to the protoauxin indole-3-butyric acid (IBA) and age-dependent defects in peroxisomal import of PTS1- and PTS2-targeted matrix proteins. Our results indicate that LON2 contributes to matrix protein import into Arabidopsis peroxisomes.
LON2 Is Necessary for IBA-Stimulated Lateral Root Formation
We analyzed mutants defective in predicted peroxisomal proteases to assess contributions of the proteases to peroxisome functions. pxm16-1 and pxm16-2 have T-DNA insertions in exons 1 and 21 of PXM16, respectively; lon2-1 and lon2-2 carry independent insertions in the final LON2 exon (Fig. 1B). The lon2-1 and lon2-2 T-DNAs may allow LON2 expression; however, the lon2-1 and lon2-2 products would lack the C-terminal PTS1 and likely mislocalize to the cytosol. The previously described T-DNA insertion in the fifth intron of DEG15 (deg15-1; Helm et al., 2007
To determine whether the protease mutations impaired peroxisomal fatty acid β-oxidation, we assessed seedling hypocotyl elongation in the dark and root elongation in the light with and without supplemental Suc. Peroxisome biogenesis mutants often display defects in peroxisomal β-oxidation of fatty acids stored in seeds and consequently require Suc during early seedling development (Hayashi et al., 1998
As previously reported (Schuhmann et al., 2008 ), we found that the deg15-1 mutant, like the wild type, had long hypocotyls and roots regardless of presence or absence of Suc (Fig. 2). Similarly, pxm16-1 and pxm16-2 displayed Suc-independent hypocotyl and root elongation (Fig. 2). By contrast, although the lon2 mutant hypocotyls were not significantly Suc dependent in the dark, lon2-1 and lon2-2 displayed root elongation defects in the light that were partially restored by Suc (Fig. 2).
Many pex mutants and other β-oxidation mutants display apparent defects in converting the protoauxin IBA to the active auxin indole-3-acetic acid and therefore are resistant to both the inhibitory effects of IBA on root elongation and to the stimulatory effects of IBA on lateral root production (Zolman et al., 2000
In contrast to the mild IBA resistance exhibited by lon2 mutants when assayed for root elongation inhibition (Fig. 3, A and B), lon2-1 and lon2-2 seedlings were both dramatically resistant to the stimulatory effects of IBA on lateral root formation, similar to ped1-96 and pex6-1 seedlings (Fig. 3C). In contrast, deg15 and pxm16 mutants resembled the wild type in the lateral root assay, efficiently producing lateral roots in response to IBA (Fig. 3C). All lines produced lateral roots when grown on the synthetic auxin 1-naphthaleneacetic acid, suggesting that the lateral root deficiency in the lon2 mutants stemmed from defective peroxisomal IBA metabolism, rather than a general inability to produce lateral roots or respond to auxin. The striking lon2 resistance to IBA-stimulated lateral root formation, accompanied by only mild resistance to the inhibitory effects of IBA on primary root elongation, suggested that LON2 may be more important for peroxisomal IBA metabolism as root cells mature and differentiate. In addition to the defects displayed in specific assays of peroxisomal function described above, lon2-1 and lon2-2 mutants were smaller than the wild type both as seedlings (Fig. 2D) and as adult plants (Fig. 4 ), whereas deg15-1 and pxm16 seedlings and adult plants resembled the wild type.
We examined mutants defective in two other putative peroxisomal proteases (At2g18080 and At2g35615; Table I) but did not find any differences from the wild type in IBA resistance or Suc dependence assays (data not shown). The only putative peroxisomal protease with a major peroxisome-targeting signal for which we did not identify a T-DNA insertion was the ovarian tumor-like Cys protease (At3g57810; Table I). At3g57810 is most highly expressed in flowers (Winter et al., 2007 ), which might explain the absence of this protein from peroxisomal proteome studies that have not sampled flowers (Fukao et al., 2002; Reumann et al., 2007; Arai et al., 2008; Eubel et al., 2008; Reumann et al., 2009).
To assess genetic interactions among the peroxisomal proteases, we isolated lon2 deg15 and lon2 pxm16 double mutants. The deg15 mutant enhanced several lon2 defects. lon2-1 deg15-1 and lon2-2 deg15-1 double mutant seedlings (Figs. 2D and 3B) and adult plants (Fig. 4) were small and exhibited significantly enhanced Suc dependence both in the dark and in the light (Fig. 2). Similarly, the lon2-1 deg15-1 and lon2-2 deg15-1 double mutant seedlings were more resistant to the inhibitory effect of IBA on root elongation than the single mutants (Fig. 3A). The lon2 mutants were completely insensitive to the tested IBA concentration in the lateral root promotion assay; lon2 deg15 double mutants resembled lon2 single mutants in this assay (Fig. 3C). In contrast, the pxm16 mutants did not appear to enhance lon2 mutant defects; lon2 pxm16 double mutants resembled lon2 single mutants in Suc dependence (Fig. 2), IBA resistance (Fig. 3), and adult size (Fig. 4).
The physiological defects of the lon2 mutants suggested that LON2 was required for peroxisomal function. Peroxisomal proteases might function to degrade damaged or obsolete peroxisomal proteins, such as the glyoxylate cycle enzymes malate synthase (MLS) and isocitrate lyase (ICL), which are synthesized early in Arabidopsis postgerminative growth and then degraded after the onset of photoautotrophic growth (Cornah et al., 2004
LON2 Is Necessary for Thiolase Accumulation and PTS2 Protein Processing
DEG15 is a PTS2-processing protease (Helm et al., 2007
To determine whether the reduced thiolase accumulation in lon2 required PTS2 removal, we examined thiolase levels in lon2 deg15 double mutants. We found that, as in deg15-1, thiolase was wholly unprocessed in lon2-1 deg15-1 and lon2-2 deg15-1 and that the unprocessed thiolase appeared to be destabilized, as in lon2 (Fig. 6, A and B). Thus, the apparent thiolase instability in lon2 did not require PTS2 removal by DEG15. In the lon2 pxm16 double mutants, thiolase displayed similar accumulation and processing as in the lon2 single mutants (Fig. 6, C and D), consistent with the lack of enhancement of lon2 physiological phenotypes by the pxm16 mutants (Figs. 2–4
We also examined processing and accumulation of a second PTS2-containing protein, peroxisomal malate dehydrogenase (PMDH), in the protease mutants. PMDH accumulated in a pattern reciprocal to thiolase during seedling development, with relatively low levels immediately following germination that increased as seedlings matured (Fig. 5). As previously reported (Helm et al., 2007
Because blocking PTS2 processing did not restore thiolase accumulation in the lon2 mutants, we tested whether the apparent thiolase instability and PMDH processing defects might reflect peroxisomal import defects. We examined a 35S-PTS2-GFP reporter (Woodward and Bartel, 2005
To determine the subcellular localization of PTS2-GFP and explore the apparent correlation between PTS2-GFP import defects and cell maturity, we analyzed PTS2-GFP via confocal fluorescence microscopy in wild-type, lon2-2, and pex5-10 root cells. In wild-type roots, PTS2-GFP displayed punctate fluorescence typical of peroxisomal localization in both root tips (Fig. 7C) and in maturing cells several millimeters above the tip (Fig. 7B), whereas in pex5-10, PTS2-GFP appeared to be partially cytosolic and peroxisomal in both root tip and maturation zone cells (Fig. 7, F and G). In lon2-2 root tip cells, PTS2-GFP appeared to be predominantly peroxisomal (Fig. 7E). However, PTS2-GFP displayed only partial peroxisomal localization in the lon2-2 vasculature, and PTS2-GFP appeared to be predominantly cytosolic in epidermis and cortex cells of the root maturation zone (Fig. 7D).
The defects in PMDH and PTS2-GFP processing and PTS2-GFP localization that we observed in older lon2 seedling extracts (Figs. 5 and 7A) and differentiated root cells (Fig. 7D) suggested that LON2 becomes necessary for peroxisomal import of matrix proteins as seedlings (or cells) mature. To determine whether LON2 also was necessary for import of native peroxisomal proteins, we immunolabeled cotyledon cells with antibodies to peroxisomal catalase (CAT), the PTS1 proteins MLS and hydroxypyruvate reductase (HPR), the PTS2 proteins thiolase and PMDH, and the membrane peroxin PEX14 (Fig. 8A
). Cotyledons from 4- and 8-d-old seedlings were selected for labeling because Arabidopsis cotyledon cells do not divide during this period (Mansfield and Briarty, 1996
In cotyledons from both 4- and 8-d-old wild-type seedlings, we found the peroxisomal matrix and membrane proteins in punctate structures typical of peroxisomes, whereas all but the membrane protein PEX14 appeared to be largely cytosolic in the pex5-10 receptor mutant (Fig. 8A). In 4-d-old lon2-1 and lon2-2 seedlings, CAT, MLS, and thiolase appeared to be peroxisomal, whereas PMDH showed both peroxisomal and cytosolic labeling. The peroxisomal localization of thiolase and the dual localization of PMDH detected by immunolocalization at day 4 (Fig. 8A) were consistent with the presence of only processed thiolase but both processed and unprocessed PMDH detected by immunoblotting at day 4 (Fig. 8B). In 8-d-old seedlings, all of the monitored peroxisome matrix proteins (CAT2, HPR, and PMDH) appeared to be largely cytosolic in the lon2 mutants, although we observed occasional punctate labeling (Fig. 8A), consistent with the possibility that some peroxisomal import was still occurring and suggested by the detection of not only unprocessed but also processed PMDH in lon2 seedling extracts (Fig. 8B). Immunolabeling with anti-PEX14 antibodies revealed the presence of peroxisomes in the wild type, lon2-1, lon2-2, and pex5-10 (Fig. 8A), indicating that the mutant defects likely resulted from impaired import of peroxisomal matrix proteins, rather than impaired peroxisome formation. We concluded that the lon2 mutant import defect extends beyond PTS2 proteins, such as PMDH and PTS2-GFP, and includes a variety of matrix proteins, including at least one PTS1 protein (HPR) as well as catalase, which has an atypical PTS (Kamigaki et al., 2003 ).
Proteases function to degrade entire proteins and to process proproteins and can remove damaged or obsolete proteins and regulate metabolic and signaling pathways (Schaller, 2004 ). In this work, we demonstrated that the peroxisomal LON2 protease is needed for importing PTS1 and PTS2 proteins into the peroxisome matrix of maturing cotyledon and root cells of Arabidopsis seedlings.
One peroxisome-associated proteolytic process during Arabidopsis development is the degradation of the glyoxylate cycle enzymes ICL and MLS a few days after germination (Lingard et al., 2009
Our mutant analysis confirmed previous findings that DEG15 is required for PTS2 processing (Helm et al., 2007
The pxm16 mutants showed no aberrant phenotypes and did not enhance lon2 defects under any of the examined conditions (Figs. 2–4
In contrast to deg15 and pxm16, the Arabidopsis lon2 mutants displayed severe defects in certain facets of peroxisome function and were smaller than the wild type following growth in soil (Fig. 4). Although the lon2 alleles displayed only minor defects in seedling growth without Suc supplementation (Fig. 2) and primary root elongation inhibition by IBA (Fig. 3, A and B), lon2 mutants were severely impaired in IBA-stimulated lateral root formation (Fig. 3C). In this regard, the lon2 mutant phenotype was unique compared to other peroxisome-defective mutants. Many mutants, like pex6-1 and pex5-10, show severe defects in both IBA-responsive root elongation inhibition and lateral root promotion (Zolman et al., 2000 The lateral root assay, in which 4-d-old seedlings are transferred to IBA-containing medium for four additional days, may be particularly sensitive to developmentally delayed defects in peroxisome metabolism. The lon2 mutants displayed matrix protein import defects that appeared to correlate with cell age; differentiated root cells imported PTS2-GFP into peroxisomes much less efficiently than recently divided cells near the root tip (Fig. 7, D and E). Similarly, several PTS1 and PTS2 proteins were predominantly peroxisomal in 4-d-old lon2 cotyledon cells but largely cytosolic in 8-d-old cotyledon cells (Fig. 8A). These localization defects were accompanied by accumulation of both processed and unprocessed PMDH and PTS2-GFP in older lon2-2 seedlings (Figs. 5 and 7A). Although the T-DNA insertions that we identified in LON2 are near the C terminus and may not completely disrupt LON2 function, the lon2 protein products would lack the C-terminal PTS1 (Fig. 1A) and would be predicted to remain cytosolic. If present, it seems unlikely that cytosolic lon2 protein is responsible for the accelerated thiolase degradation we observed in lon2-1 and lon2-2, as thiolase stability in pex5-10 resembles the wild type (Fig. 7A) even though thiolase (and presumably LON2) is largely cytosolic in pex5-10 (Fig. 8A). Moreover, we cannot eliminate the possibility that some lon2 protein enters the peroxisome, either through a cryptic PTS1 provided by the T-DNA border or by association with an interacting peroxisome-bound protein. The future characterization of LON2 antibodies and demonstrated lon2 null alleles would aid in resolving these ambiguities.
The H. polymorpha Lon isoform Pln appears to degrade certain peroxisome-targeted, non-native proteins (Aksam et al., 2007
Plant peroxisomal LON isoforms are more similar to chordate peroxisomal LON isoforms than either are to their yeast relatives (Fig. 1C). Moreover, phylogenetic analysis suggests that a subset of yeasts that includes H. polymorpha may have acquired a peroxisomal LON isoform in an evolutionary event distinct from the event in which the peroxisomal LON now found in plants and animals was obtained (Fig. 1C), consistent with the possibility that peroxisomal LON may function differently in these lineages. Arabidopsis lon2 mutants displayed striking matrix protein import defects (Figs. 7 and 8). Similarly, a mammalian cell line expressing a dominant negative pLon variant mis-sorts catalase to the cytosol (Omi et al., 2008
Plant Material and Growth Conditions
The wild type and mutants were in the Arabidopsis (Arabidopsis thaliana) Columbia-0 (Col-0) accession. Seeds were surface sterilized, stratified in 0.1% [w/v] agar at 4°C for the indicated times, and germinated on nutrient media supplemented with Suc and hormones as previously described (Zolman and Bartel, 2004
The wild type (35S-PTS2-GFP; Woodward and Bartel, 2005
Cotyledons (Lingard et al., 2009
Samples for immunofluorescence microscopy were prepared as described previously (Lingard et al., 2009 A Zeiss Axioplan 2 fluorescence compound microscope equipped with narrow-band GFP filters (Chroma) and a 40x lens was used for imaging immunolabeled tissues. Images were acquired with a CoolSNAP HQ camera (Photometrics) using MetaMorph 7 imaging software (Molecular Devices). Confocal images of GFP-expressing tissues were acquired using a Zeiss LSM 510 confocal microscope (excitation, 488 nm; emission, 500–550 nm) equipped with a 20x lens. Images were cropped using NIH ImageJ and adjusted for brightness in Adobe Photoshop CS. Sequence data from this article can be found in the GenBank/EMBL data libraries under the accession numbers listed in Table I and Supplemental Table S2.
The following materials are available in the online version of this article.
We thank John Harada, Masayoshi Maeshima, Douglas Randall, Steven Smith, and Richard Trelease for the MLS, ICL, HPR, PMDH, and CAT antibodies, respectively. We thank Randall Baldasarre for assistance in isolating double mutants, Andrew Woodward for assistance with figures, Steven Smith for pmdh1 pmdh2 seeds, the Arabidopsis Biological Resource Center at Ohio State University for seeds from Salk Institute insertion lines, and Naxhiely Martinez, Sarah Ratzel, Lucia Strader, Andrew Woodward, and Bethany Zolman for critical comments on the manuscript. Received June 5, 2009; accepted September 7, 2009; published September 11, 2009.
1 This research was supported by the National Science Foundation (MCB–0745122), the National Institutes of Health (R01GM079177), the Robert A. Welch Foundation (C–1309), and a postdoctoral fellowship to M.J.L. (USDA 2008–20659). 
2 Present address: 700 Chesterfield Parkway, Chesterfield, MO 63017. 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: Bonnie Bartel (bartel{at}rice.edu).
[C] Some figures in this article are displayed in color online but in black and white in the print edition.
[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.109.142505 * Corresponding author; e-mail bartel{at}rice.edu.
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12), numbers above light-gray bars indicate the mean percentages of elongation on media without Suc, and asterisks denote no Suc values significantly different than Suc values (Student's t test, P < 0.001). B and D show three seedlings representing the range of observed phenotypes. [See online article for color version of this figure.]
-MLS (Olsen et al., 1993