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Originally published In Press as doi:10.1074/jbc.M411005200 on January 6, 2005

J. Biol. Chem., Vol. 280, Issue 15, 14829-14835, April 15, 2005
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Differential Contribution of Two Peroxisomal Protein Receptors to the Maintenance of Peroxisomal Functions in Arabidopsis*

Makoto Hayashi{ddagger}§, Mina Yagi{ddagger}, Kazumasa Nito{ddagger}, Tomoe Kamada{ddagger}, and Mikio Nishimura{ddagger}§

From the {ddagger}Department of Cell Biology, National Institute for Basic Biology, and the §Department of Molecular Biomechanics, School of Life Science, Graduate University of Advanced Studies, Okazaki 444-8585, Japan

Received for publication, September 24, 2004 , and in revised form, January 5, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Peroxisomes in higher plant cells are known to differentiate in function depending on the cell type. Because of the functional differentiation, plant peroxisomes are subdivided into several classes, such as glyoxysomes and leaf peroxisomes. These peroxisomal functions are maintained by import of newly synthesized proteins containing one of two peroxisomal targeting signals known as PTS1 and PTS2. These targeting signals are known to be recognized by the cytosolic receptors, Pex5p and Pex7p, respectively. To demonstrate the contribution of Pex5p and Pex7p to the maintenance of peroxisomal functions in plants, double-stranded RNA constructs were introduced into the genome of Arabidopsis thaliana. Expression of the PEX5 and PEX7 genes was efficiently reduced by the double-stranded RNA-mediated interference in the transgenic Arabidopsis. The Pex5p-deficient Arabidopsis showed reduced activities for both glyoxysomal and leaf peroxisomal functions. An identical phenotype was observed in a transgenic Arabidopsis overexpressing functionally defective Pex5p. In contrast, the Pex7p-deficient Arabidopsis showed reduced activity for glyoxysomal function but not for leaf peroxisomal function. Analyses of peroxisomal protein import in the transgenic Arabidopsis revealed that Pex5p was involved in import of both PTS1-containing proteins and PTS2-containing proteins, whereas Pex7p contributed to the import of only PTS2-containing proteins. Overall, the results indicated that Pex5p and Pex7p play different roles in the maintenance of glyoxysomal and leaf peroxisomal functions in plants.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Peroxisomal enzymes are synthesized in the cytosol and function after their post-translational transport into peroxisomes. Most of the plant peroxisomal enzymes have been shown to contain one of two peroxisome targeting signals within their amino acid sequences (1, 2). One type of targeting signal, called PTS1 1 (peroxisome targeting signal), is a unique tripeptide sequence found in the carboxyl terminus of the proteins (3, 4). In higher plant cells, the permissible combinations of tripeptide sequence for PTS1 are (C/A/S/P)(K/R)(I/L/M) (5). Another type of targeting signal, PTS2, is involved in a cleavable amino-terminal presequence (6). The amino-terminal presequences contain a consensus sequence (A/L/Q/I)X5(H)(L/I/F) (where X indicates any amino acid) (7, 8). PTS2 is processed to form the mature protein after its transport into peroxisomes.

Peroxisomes in higher plant cells have been shown to differentiate into at least three different classes, namely glyoxysomes, leaf peroxisomes, and unspecialized peroxisomes (9). Each organelle contains a unique set of enzymes that provides special functions in various organs in higher plants. Glyoxysomes are present in cells of storage organs, such as endosperms and cotyledons during post-germinative growth of oilseed plants, as well as in senescent organs (10). They contain enzymes for fatty acid {beta}-oxidation and the glyoxylate cycle and play a pivotal role in the conversion of seed-reserved lipid into sucrose. It has been suggested that fatty acids produced from the lipid are exclusively degraded in glyoxysomes (i.e. not in mitochondria) during germination and post-germinative growth in plants (11). By contrast, leaf peroxisomes are widely found in cells of photosynthetic organs. It has been shown that some enzymes responsible for photorespiration are localized in leaf peroxisomes even though the entire photorespiratory process involves a combination of enzymatic reactions that occur in chloroplasts, leaf peroxisomes, and mitochondria (12). We recently identified 256 candidates of peroxisomal genes encoding either PTS1-containing proteins or PTS2-containing proteins within the entire Arabidopsis genome (13). Of these, the functions of only 29 gene products have been identified. By using a custom-made microarray, we extensively examined the expression of all these genes in various organs. Statistical analyses of the expression profiles revealed that peroxisomal genes could be classified into five groups. From the results, we suggested that plant peroxisomes can be subdivided into at least five different classes, namely glyoxysomes, cotyledonary peroxisomes, leaf peroxisomes, root peroxisomes, and unspecialized peroxisomes. However, the mechanism to maintain the functional differentiation of plant peroxisomes is still unknown.

Recent analyses of peroxisome-deficient mutants allowed the identification of over 25 PEX genes and their products, peroxins (14), from various organisms. Fifteen PEX gene orthologues exist in the Arabidopsis genome (15), and Arabidopsis mutants with defective PEX2, PEX5, PEX6, PEX10, PEX14, and PEX16 were reported (16–22). Of these, Pex14p is a peroxisomal membrane protein encoded by PED2 (Arabidopsis orthologue of PEX14) and is believed to contribute to both PTS1- and PTS2-dependent protein targeting pathways, because the Arabidopsis ped2 mutant had defects in intracellular transport of both PTS1- and PTS2-containing proteins (16). In contrast, Pex5p (PEX5 product) and Pex7p (PEX7 product) function as cytosolic receptors for PTS1- and PTS2-containing proteins, respectively (23).

Here we report on transgenic plants that had defects in Pex5p and Pex7p. Based upon the phenotypes of the transgenic plants, we show evidence that Pex5p and Pex7p contributed differently to the maintenance of the peroxisomal functions in plants.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Plant Materials and Growth Conditions—Arabidopsis thaliana ecotype Columbia was used as the wild-type plant. Construction of transgenic plants expressing GFP-PTS1 and PTS2-GFP and characterization of the ped2 mutant were described previously (16, 24). All seeds were surface-sterilized and grown on growth medium (2.3 mg/ml Murashige and Skoog salts (Wako, Osaka, Japan), 1% sucrose, 100 µg/ml myoinositol, 1 µg/ml thiamine-HCl, 0.5 µg/ml pyridoxine, 0.5 µg/ml nicotinic acid, 0.5 mg/ml MES-KOH, pH 5.7, and 0.8% agar) with or without antibiotic(s). In some experiments, 0.2 µg/ml of 2,4-dichlorophenoxybutyric acid was added, or sucrose was removed from the growth medium. Seedlings were then transferred and grown on a 1:1 mixture of perlite and vermiculite under a 16-h-light (100 µE m–2 s–1)/8-h-dark light cycle at 22 °C in a normal atmosphere or in an atmosphere containing 1000 Pa CO2.

Construction of Artificial Genes—Isolation of PEX5 and PEX7 cDNAs was reported previously (23). For making the pex5i and pex7i constructs, the gene-specific sequences in the sense orientation were amplified by PCR to introduce SpeI and SacI sites at each end, whereas BamHI and XhoI sites were introduced into the same sequence of the antisense orientation. The sense and antisense DNA fragments were inserted between SpeI-SacI and XhoI-BamHI sites of pBluescript KS– (Stratagene, La Jolla, CA), respectively. A BamHI-BglII fragment encoding a bar gene from the pARK22 plasmid was also inserted at a BamHI site of the same plasmid as a linker. The XhoI-SacI DNA fragments were removed from the plasmids and ligated into the XhoI-SacI site of pBI121-Hm linker. The pBI121-Hm linker was constructed from a Ti vector, pBI121, by replacing the GUS gene with a linker sequence containing XbaI, XhoI, SmaI, SpeI, KpnI, and SacI sites. It also contained a hygromycin-resistant gene as a selectable marker in plants. To make the PEX5{Delta}7 construct, a DNA fragment encoding the Met1–Tyr657 region of Pex5p was amplified by PCR. It was inserted into a pDONR221 plasmid (Invitrogen) and then transferred into a Ti vector, pGWB2 (kindly gifted from Dr. Nakagawa), using BP and LR clonases according to the manufacturer's specifications (Invitrogen).

Agrobacterium-mediated Transformation—Ti vectors carrying pex5i, pex7i, and PEX5{Delta}7 were independently introduced into kanamycin-resistant parental transgenic Arabidopsis plants expressing GFP-PTS1 and PTS2-GFP (24) by vacuum infiltration (25) using Agrobacterium tumefaciens (strain C58C1RifR). Transformed Arabidopsis lines were selected on growth medium containing 50 µg/ml kanamycin and 25 µg/ml hygromycin.

Immunoblotting—Sample preparation and immunoblot analyses using antibodies against AtPex5p and AtPex7p were performed as previously reported (23). Crude extract was prepared from leaves of the 3-week-old T1 plant. Ten µg of the total protein was used for immunoblotting.

Measurements of Maximum Quantum Yield of Photosystem II (Fv/Fm)—To reduce the effect of photoinhibition, plants were grown for 4 weeks in an atmosphere containing 1000 Pa CO2 under low light (50 µE m–2 s–1). These plants were then illuminated with strong light (450 µE m–2 s–1) for 0 and 4 h in a normal atmosphere (36 Pa CO2). At the end of each illumination period, plants were kept for 30 min in the dark. The ratio of variable fluorescence to maximum fluorescence (Fv/Fm) was automatically calculated from the results of the modulated chlorophyll fluorescence emission from the upper surface of dark-adapted leaves that was measured using a pulse amplitude modulation fluorometer; Mini-PAM (H. Walz, Effeltrich, Germany).

Detection of Fluorescence Derived from GFP-PTS1 and PTS2-GFP— For analyzing root cells, T1 plants, grown 10 days on growth medium, were mounted under a coverslip. For analyzing leaf cells, leaves removed from the 3-week-old plants were mounted under a coverslip. Fluorescent images of the specimens were captured using an LSM 510 laser-scanning confocal microscope with an argon laser and a fluorescein isothiocyanate filter set (emission 500–550 nm) (Carl Zeiss, Oberkochen, Germany).

Yeast Two-hybrid Analyses—Yeast two-hybrid analyses of PEX5{Delta}7 were performed according to protocols reported previously (23). A DNA fragment encoding PEX5{Delta}7 in a pDONR plasmid was transferred into a pGAD-GW plasmid. The yeast two-hybrid vector, pGAD-GW, was constructed from pGAD-C1 (26) by replacing the multicloning site with a gateway conversion cassette (Invitrogen). Construction of other vectors used in this study have been described elsewhere (23). Transformants were tested for growth on synthetic medium containing 50 mM 3-aminotriazole without histidine and were assayed for quantitative {beta}-galactosidase activity.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Generation of Transgenic Plants That Have a Defect in Pex5p or Pex7p—Double-stranded RNA interference was used to induce post-transcriptional gene silencing of PEX5 and PEX7.To induce double-stranded RNA interference in Arabidopsis, we constructed two artificial genes, pex5i and pex7i, that encode RNAs capable of double strand formation at gene-specific sequences. Transformation and selection were performed by a method reported previously (27). As shown in Fig. 1A, both constructs contain two identical sequences encoding a part of the corresponding protein in antisense and sense orientations and were linked together by a bar gene. In parallel, we constructed PEX5{Delta}7 overexpressing the Met1–Tyr657 region of Pex5p lacking 71 amino acid residues at the carboxyl terminus (Fig. 1A). This transgenic plant was constructed because we expected that the defective Pex5p protein would induce a dominant negative phenotype. These DNA fragments were inserted under the control of a constitutive 35S promoter from cauliflower mosaic virus in a Ti vector containing a hygromycin-resistant gene as a selectable marker in plants, and then integrated into the genomes of two kanamycin-resistant transgenic plants, AtGFP-PTS1 and AtPTS2-GFP, by Agrobacterium-mediated transformation. Construction of these parental lines has been described previously elsewhere (24). Peroxisomes of the AtGFP-PTS1 and AtPTS2-GFP had punctated green fluorescence because of the peroxisomal targeting of GFP-PTS1 (a GFP fusion protein containing 10 carboxyl-terminal amino acid residues of hydoxypyruvate reductase) and PTS2-GFP (a GFP fusion protein containing 49 amino-terminal amino acid residues of citrate synthase at its amino terminus) (see Fig. 4, bottom panels). The retransformation of pex5i, pex7i, and PEX5{Delta}7 into the AtGFP-PTS1 and AtPTS2-GFP plants allowed us to determine the effects of these DNA constructs on import of the GFP-PTS1 and PTS2-GFP in vivo. The primary transformants were designated as T0 plants. T1 progenies showing both kanamycin and hygromycin resistance were designated as pex5i/GFP-PTS1, pex5i/PTS2-GFP, pex7i/GFP-PTS1, pex7i/PTS2-GFP, PEX5{Delta}7/GFP-PTS1, and PEX5{Delta}7/PTS2-GFP.



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  		FIG. 1.
Generation of pex5i, pex7i, and PEX5{Delta}7 transgenic plants expressing GFP-PTS1 and PTS2-GFP. A, gene constructs for pex5i, pex7i, and PEX5{Delta}7. For pex5i, DNA fragments encoding the Leu28–Gln207 region of AtPex5p (solid boxes with arrow indicating the orientation) in the antisense and sense orientations were linked with a 550-bp fragment of the bar gene (open box marked with bar). For pex7i, DNA fragments encoding the Gly50–Val239 region of AtPex7p (hatched box) were used. PEX5{Delta}7 encodes the Met1–Tyr657 region of Pex5p (solid box). These genes were inserted downstream of the cauliflower mosaic virus 35S promoter (open arrow marked with 35SP), followed by nopaline synthase or 35S terminator (open box marked with nosT or 35ST). B, immunodetection of AtPex5p and AtPex7p in transgenic plants. Extracts were prepared from leaves of transgenic plants (pex5i, pex7i, and PEX5{Delta}7) expressing GFP-PTS1 (lane 1) or PTS2-GFP (lane 2), and wild-type plants (cont). For each sample, 10 µg of the total protein was subjected to SDS-PAGE analysis. Immunoblot analysis was performed using the antibodies raised against AtPex5p (Pex5p) and AtPex7p (Pex7p). Arrows indicate positions of the corresponding proteins, respectively, and the arrowhead represents the position of PEX5{Delta}7.

 



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  		FIG. 4.
Subcellular localization of GFP-PTS1 and PTS2-GFP fusion proteins in pex5i transgenic plants. Fluorescent images of pex5i/GFP-PTS1 (PTS1) and pex5i/PTS2-GFP (PTS2) leaf epidermal cells (leaf) and root cells (root) were taken by confocal laser microscope. As controls, fluorescent images of the parental transgenic plant expressing GFP-PTS1 in leaf epidermal cells and the parental transgenic plant expressing PTS2-GFP in root cells were also shown (cont). Magnifications of these panels are the same. Bar = 30 µm.

 
The effects of the transgenes in the T1 progenies were examined by immunoblot analysis. In both pex5i/GFP-PTS1 and pex5i/PTS2-GFP the amounts of Pex5p were greatly reduced, whereas no effect was observed for the amounts of Pex7p (Fig. 1B, pex5i (lane 1) and pex5i (lane 2)). Inversely, the amounts of Pex7p were greatly reduced, whereas no effect was observed for the amounts of Pex5p in both pex7i/GFP-PTS1 and pex7i/PTS2-GFP (Fig. 1B, pex7i (lane 1) and pex7i (lane 2)). PEX5{Delta}7/GFP-PTS1 and PEX5{Delta}7/PTS2-GFP contained an additional form of Pex5p whose molecular mass was well matched with the polypeptide encoded by PEX5{Delta}7, whereas no effect was observed for Pex7p (Fig. 1B, PEX5{Delta}7 (lane 1) and PEX5{Delta}7 (lane 2)). It is noteworthy that all of these samples contained similar amounts of ribulose-bisphosphate carboxylase/oxygenase (data not shown). All phenotypes described in this study were dominant.

Effects of 2,4-Dichlorophenoxybutyric Acid and Sucrose on Growth of Pex5i, Pex7i, and PEX5{Delta}7—We reported previously (28) that plants lacking glyoxysomal fatty acid {beta}-oxidation become resistant to 2,4-dichlorophenoxybytyric acid (2,4-DB) and require sucrose during postgerminative growth. To analyze the contribution of Pex5p and Pex7p to glyoxysomal function, the effects of 2,4-DB and sucrose were examined using T2 progenies of the transgenic lines. When these T2 populations were grown on medium containing 2,4-DB, ~75% of the T2 seedlings had elongated roots, indicating resistance to 2,4-DB. The 2,4-DB-resistant phenotypes are shown in Fig. 2, upper panel. Because of genetic segregation of the transgene in the T2 population, the rest of the seedlings showed strong growth inhibition identical with their parental plants. In addition, ~75% of T2 seedlings required sucrose for post-germinative growth. These seedlings could not grow normally in the absence of sucrose (Fig. 2, middle panel). All seedlings that showed 2,4-DB resistance or required sucrose were resistant to both hygromycin and kanamycin (data not shown). These results indicated that introduction of the pex5i, PEX5{Delta}7, and pex7i constructs into the transgenic plants caused the defect in glyoxysomal function.



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  		FIG. 2.
Effects of 2,4-DB and sucrose on the growth of pex5i, PEX5{Delta}7, and pex7i transgenic plants. T2 progenies of the transgenic plants (pex5i, PEX5{Delta}7, and pex7i) and parental plants (cont) expressing GFP-PTS1 (lane 1) or PTS2-GFP (lane 2) were grown for 10 days on growth medium containing 0.2 µg/ml of 2,4-DB, growth medium without sucrose (–sucrose), and normal growth medium (GM) under constant illumination. Seedlings were removed from the media and arranged on agar plates. Bar = 1 cm.

 
Activity of Photorespiration in pex5i, PEX5{Delta}7, and Pex7i—T2 progenies of pex5i/GFP-PTS1, PEX5{Delta}7/GFP-PTS1, and pex7i/PTS2-GFP, showing 2,4-DB resistance and cytosolic GFP fluorescence (see following description), were further cultivated in order to examine photorespiration in the leaf peroxisomes. When pex5i/GFP-PTS1 and PEX5{Delta}7/GFP-PTS1 were grown in a normal atmosphere (36 Pa CO2), they had yellow-green leaves and showed a dwarf phenotype compared with wild-type plants (Fig. 3A, pex5i(air) and PEX5{Delta}7(air)). This phenotype was recovered when the plants were grown under high CO2 conditions (1000 Pa CO2) (Fig. 3A, pex5i(CO2) and PEX5{Delta}7(CO2)). A similar phenomenon was observed in the ped2 mutant that has a defective PEX14 gene conferring reduced activity for photorespiration (16). In contrast, pex7i/PTS2-GFP grew normally even in the normal atmosphere (Fig. 3A, pex7i(air)). Based on these results, we assume that loss of Pex5p function reduces the activity of photorespiration in leaf peroxisomes, whereas loss of Pex7p does not.



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  		FIG. 3.
Photorespiratory glycolate metabolism in pex5i, PEX5{Delta}7, and pex7i transgenic plants. A, effect of CO2 on the growth of pex5i, PEX5{Delta}7, and pex7i transgenic plants. The parental plants expressing AtGFP-PTS1 (cont), pex5i/GFP-PTS1 (pex5i), PEX5{Delta}7/GFP-PTS1 (PEX5{Delta}7), and pex7i/PTS2-GFP (pex7i) were grown for 8 weeks in an atmosphere containing 1000 Pa CO2 (CO2) or in a normal atmosphere (air) under constant illumination. Arrows indicated top of an inflorescence apex. B, effect of strong irradiation on maximal quantum yield of photosystem II (Fv/Fm). AtGFP-PTS1 (GFP-PTS1), pex5i/GFP-PTS1 (pex5i/PTS1), AtPTS2-GFP (PTS2-GFP), pex7i/PTS2-GFP (pex7i/PTS2), and the ped2 mutant (ped2) were grown for 4 weeks in an atmosphere containing 1000 Pa CO2 under low light (50 µE m–2 s–1). These plants were illuminated with strong light (450 µE m–2 s–1) for 4 h in a normal atmosphere. Maximum quantum yields of photosystem II (Fv/Fm) of the leaves at a 0-h (0h) and a 4-h illumination (4h) were measured after these plants were kept for 30 min in darkness. Each point represents the average Fv/Fm measured in six leaves of independent plants with standard error.

 
We tested this hypothesis by measuring the maximum quantum yield of photosystem II, which can be estimated from the ratio of the variable fluorescence of dark-adapted chlorophyll a to the maximum fluorescence (Fv/Fm) (29–31). We compared Fv/Fm of dark-adapted leaves of the parental plants with pex5i/GFP-PTS1 plants and pex7i/PTS2-GFP plants (Fig. 3B). They were initially grown for 3 weeks in an atmosphere containing high CO2 (1000 Pa) under low light (50 µE m–2 s–1) to reduce the effects of photoinhibition. All plants grown in this condition showed normal growth and had similar Fv/Fm values (Fig. 3B, 0h). These plants were then transferred to a normal atmosphere (36 Pa CO2), where they were illuminated with a strong light (450 µE m–2 s–1). Under these conditions, ribulose-bisphosphate carboxylase/oxygenase acts as an oxygenase in addition to its carboxylase activity, which is necessary for CO2 fixation in photosynthesis. Phosphoglycolate, a byproduct of the oxygenase reaction, is metabolized by photorespiration enzymes and finally returned to the Calvin-Benson cycle. Therefore, the Fv/Fm of the wild-type plants would not be expected to change after the strong illumination, and this is what was observed (Fig. 3B, GFP-PTS1(4 h) and PTS2-GFP(4 h)). In contrast, significant reduction of Fv/Fm was observed in the pex5i/GFP-PTS1 as well as the ped2 mutant, which has a defective PEX14 gene (Fig. 3B, pex5i/PTS1(4h) and ped2(4h)). Only a weak reduction of Fv/Fm was observed in pex7i/PTS2-GFP (Fig. 3B, pex7i/PTS2(4 h)). These results indicate that pex5i/GFP-PTS1 plants were unable to maintain sufficient activity of the Calvin-Benson cycle under low CO2/strong light conditions because of the defect in photorespiration. The imbalance between the light reaction and the Calvin-Benson cycle might have been responsible for the observed reduction of the Fv/Fm. These results indicated that Pex5p, but not Pex7p, is necessary for the maintenance of the activity of photorespiration in leaf peroxisomes.

Intracellular Transport of Peroxisomal Proteins in the Absence of Pex5p—Subcellular localization of GFP fusion proteins, GFP-PTS1 and PTS2-GFP, in the cells of pex5i/GFP-PTS1 and pex5i/PTS2-GFP was determined using a confocal laser-scanning microscope. Both leaf and root cells of pex5i/GFP-PTS1 showed green fluorescence only in the periphery of the cells (Fig. 4, upper panels) that coincided with the cytosol surrounding the central vacuoles. Identical fluorescent images were obtained in 19 independently isolated transformants for pex5i/GFP-PTS1 out of 20 T1 progenies showing hygromycin/kanamycin resistance. Cytosolic fluorescence was also obtained in leaf and root cells of 35 independent transformants for pex5i/PTS2-GFP out of the 42 T1 progenies (Fig. 4, middle panels). In contrast, cells of parental transgenic plants, AtGFP-PTS1 and AtPTS2-GFP, showed punctated green fluorescence that coincided with peroxisomes (Fig. 4, lower panels). These data indicate that Pex5p mediates intracellular transport of both PTS1-containing and PTS2-containing proteins.

Protein-Protein Interaction of PEX5{Delta}7 Gene Product—By employing a yeast two-hybrid system, co-immunoprecipitation assays, and pull-down assays, we demonstrated previously (23) that Arabidopsis Pex5p has the ability to bind not only to PTS1-containing proteins but also Pex7p and Pex14p in vitro (also see Fig. 5). Pex5p possesses a tetratricopeptide repeat motif at the carboxyl terminus (32, 33). The tetratricopeptide repeat motif consists of seven tandem repetitive consensus amino acid sequences that are necessary to interact with PTS1 (34, 35). The PEX5{Delta}7 gene product, Pex5{Delta}7p, is missing the last repeat in the tetratricopeptide repeat motif. Binding activities of Pex5{Delta}7p with PTS1 proteins Pex7p and Pex14p were examined by using yeast two-hybrid analysis according to a method reported previously (23). Each protein was fused to Gal4-AD or Gal4-BD and expressed in a yeast tester strain PJ69-4A (26). Protein-protein interactions were monitored by the Gal4-dependent transcriptional activation of HIS3 and {beta}-galactosidase reporter genes (Fig. 5). No significant transcriptional activation of HIS3 and {beta}-galactosidase reporter genes indicated that Pex5{Delta}7p could not bind with PTS1-containing protein. Binding activity between Pex5{Delta}7p and Pex7p, however, was identical to that between Pex5p and Pex7p, whereas Pex14p showed weaker binding activity to Pex5{Delta}7p than Pex5p.



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  		FIG. 5.
Binding activities of Pex5{Delta}7p with AtPex14p, AtPex7p, and PTS1 analyzed by yeast two-hybrid system. Pex5{Delta}7p, AtPex5p, AtPex14p, amino-terminal domain (Met1–Arg180) of AtPex7p and PTS1 were fused to Gal4-BD (BD) or Gal4-AD (AD) as indicated. Transformants expressing each set of proteins were tested for growth on synthetic medium without histidine (–His), and assayed for quantitative {beta}-galactosidase activity ({beta}-gal activity).

 
Effect of PEX5{Delta}7 Gene Product on Intracellular Transport of Peroxisomal Proteins—Fig. 6 shows subcellular localization of GFP-PTS1 and PTS2-GFP in leaf and root cells of PEX5{Delta}7/GFP-PTS1 and PEX5{Delta}7/PTS2-GFP transformants. Leaf and root cells of both PEX5{Delta}7/GFP-PTS1 and PEX5{Delta}7/PTS2-GFP had green fluorescence only in the cytosol. Identical fluorescent images were obtained in 5 transformants for PEX5{Delta}7/GFP-PTS1 and 6 for PEX5{Delta}7/PTS2-GFP out of 10 and 8 T1 progenies, respectively. These data indicate that the ability for intracellular transport of both PTS1-containing and PTS2-containing proteins was reduced when the PEX5{Delta}7 gene product was overexpressed in the cells.



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  		FIG. 6.
Subcellular localization of GFP-PTS1 and PTS2-GFP fusion proteins in PEX5{Delta}7 transgenic plants. Fluorescent images of the PEX5{Delta}7/GFP-PTS1 (PTS1) and PEX5{Delta}7/PTS2-GFP (PTS2) in leaf epidermal cells (leaf) and root cells (root) were taken by a confocal laser microscope. Bar = 30 µm.

 
Intracellular Transport of Peroxisomal Proteins in the Absence of Pex7p—Fig. 7 shows subcellular localization of GFP-PTS1 and PTS2-GFP in leaf and root cells of the pex7i/GFP-PTS1 and pex7i/PTS2-GFP. All 55 independent transformants for pex7i/GFP-PTS1 showed punctated fluorescence that was identical to their parental plants (Fig. 7, upper panels), although we confirmed that these plants contained reduced amounts of Pex7p (Fig. 1B, pex7i (lane 1)). In contrast, cells of 32 independent transformants for pex7i/PTS2-GFP out of the 34 T1 progenies had green fluorescence only in the cytosol (Fig. 7, lower panels). These data indicate that loss of Pex7p disrupted intracellular transport of only PTS2-containing proteins, but not PTS1-containing proteins.



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  		FIG. 7.
Subcellular localization of GFP-PTS1 and PTS2-GFP fusion proteins in pex7i transgenic plants. Fluorescent images of pex7i/GFP-PTS1 (PTS1) and pex7i/PTS2-GFP (PTS2) leaf epidermal cells (leaf) and root cells (root) were taken by confocal laser microscope. Bar = 30 µm.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Double-stranded RNA interference has been shown to be an effective trigger of gene silencing in a number of organisms, including higher plants (36). This silencing is induced by sequence-specific RNA degradation of double-stranded RNA. To analyze the functions of Pex5p and Pex7p in vivo, we induced gene silencing of endogenous PEX5 and PEX7 by making transgenic Arabidopsis plants transformed with artificial genes encoding a hairpin RNA consisting of an inverted repeat of a fragment for the corresponding gene sequence separated by a spacer. It had been reported that this method could efficiently induce specific and heritable gene silencing in a number of plant genes (27, 37, 38). Here we showed that this method also efficiently reduced the amount of plant peroxins. This technique allowed us to present the experimental data analyzing the relationship between the functional differentiation of peroxisomes and the two targeting receptors, Pex5p and Pex7p, in relation to their protein-protein interaction in planta (Figs. 2 and 3).

We should emphasize that this is the first analysis of a Pex7p-deficient plant (pex7i), and we have shown the differential contribution of two peroxisomal protein receptors, Pex7p and Pex5p, to the maintenance of peroxisomal functions in planta. Specifically, phenotypes of this mutant indicated that Pex7p is necessary for the maintenance of glyoxysomal function but not for the maintenance of leaf peroxisomal function. These data indicated that Pex7p plays an important role in regulating functional differentiation of peroxisomes and defines temporally and spatially limited differentiation of peroxisomes, i.e. glyoxysomes found only in etiolated cotyledons during the early stage of postgerminative growth. It is noteworthy that the amount of Pex7p reduced in parallel with the functional differentiation of peroxisomes from glyoxysomes to leaf peroxisomes induced by illumination of the cotyledons (23). It has been shown that Rhizomelic chondrodysplasia punctata, a human autosomal recessive disorder, is caused by mutations in the PEX7 gene. In a recent study (39) of Pex7–/– mice, the model animals for this disorder indicated that symptoms of this disorder, such as cataracts, rhizomelia, and epiphyseal calcifications, may be induced by abnormalities of peroxisomal functions, i.e. the biosynthesis of plasmalogens, the {alpha}-oxidation of phytanic acid, and {beta}-oxidation of very long chain fatty acid. These results indicated that Pex7p plays an important role in defining the distinct characteristics of peroxisomal function(s) in different multicellular organisms, such as higher plants and mammals.

Phenotypes of the Pex5p-deficient plant (pex5i) clearly indicated that Pex5p is necessary for the maintenance of both glyoxysomal and leaf peroxisomal functions. This conclusion was also supported by the dominant negative phenotype observed in PEX5{Delta}7, a transgenic Arabidopsis overexpressing functionally defective Pex5p, that had defective glyoxysomes and leaf peroxisomes. An Arabidopsis pex5 mutant has been identified previously (21) from progenies of ethyl methanesulfonate-mutagenized seeds. Although this mutant also had defective glyoxysomes, defects in leaf peroxisomal function were not observed. This may be because the missense mutation (Ser318 to Leu) may cause a leaky phenotype in the mutant.

Despite the advantage of RNA interference, significant numbers of the later generations lost their cytosolic fluorescence and regained fluorescence in their peroxisomes during growth and development (data not shown). One of the possible explanations for this is that the severe phenotypes might tend to release the gene silencing effects by an unknown influence of defective peroxisomes. It is noteworthy that Arabidopsis mutants that had homozygous T-DNA insertion in PEX2, PEX10, and PEX16 were known to be embryonic lethal (17–20).

We previously reported protein-protein interaction among Pex5p, Pex7p, and Pex14p (23). In this study, we showed that the Met1–Val230 region of Pex5p and the Tyr266–Ser317 region of Pex7p could bind to each other. Binding of nine WXXX(F/Y) repeats in Pex5p with two domains (Ile58–Leu65 and Arg78–Arg97) of Pex14p was also seen, whereas no binding was observed between Pex7p and Pex14p. From these in vitro analyses, we suggested that the mechanism of peroxisomal protein targeting in higher plant cells is as follows. In the cytosol, Pex5p and Pex7p bind to each other to form a Pex5p-Pex7p receptor complex that catches both PTS1- and PTS2-containing proteins. The receptor-cargo complex captures peroxisomes by binding with Pex14p, a peroxisomal membrane protein, and with Pex5p, and the cargo is then transferred into the peroxisomal matrix.

This hypothesis is well matched to our present study showing peroxisomal protein import in the Pex5p- and Pex7-defecient plants. When Pex5p is missing, import of not only PTS1-containing proteins but also PTS2-containing proteins is abolished because of the loss of the Pex5p-Pex7p receptor complex (Fig. 4). Dependence of PTS2-containing protein import on Pex5p was also examined by overexpression of Pex5{Delta}7p in which binding activity with the cargo, PTS1-containing proteins, is completely lost (Fig. 6). In this case, excess amounts of Pex5{Delta}7p bind with Pex7p to form a disordered complex. Because of the weak binding activity of Pex5{Delta}7p to Pex14p and the loss of cargo, the disordered complex could not be identified by Pex14p, resulting in a disturbance of the import of not only PTS1-but also PTS2-containing proteins (Fig. 5). When Pex7p is missing, however, transport of only PTS2-containing proteins is abolished. Pex7p-deficient Arabidopsis showed normal activity for import of PTS1-containing proteins, because Pex5p might act as a receptor for PTS1-containing proteins and bind to Pex14p, without forming receptor complex with Pex7p (Fig. 7).

The contribution of Pex5p on the import of PTS2-containing proteins is species-dependent. Arabidopsis has only one PEX5 orthologue. Our extensive search for this transcript suggests that Arabidopsis may only produce a single form of Pex5p (15, 23). Therefore, all Pex5p expressed in Arabidopsis cells could mediate not only PTS1-but also PTS2-dependent protein targeting pathways. In mammalian cells, however, two isoforms of Pex5p (Pex5pL and Pex5pS) are produced by alternative splicing (40). Pex5pL, containing an additional 37 amino acids, mediates a PTS2-dependent protein targeting pathway, whereas Pex5pS does not (41, 42). In yeast, PTS1- and PTS2-dependent protein targeting pathways are independently mediated by Pex5p and Pex7p and combine into one pathway when each peroxin binds to Pex14p (43). In contrast, Caenorhabitis elegans is known to have no PTS2-dependent protein targeting pathway (44).

In higher plant cells, all enzymes involved in fatty acid {beta}-oxidation and the glyoxylate cycle except cytosolic aconitase are predominantly localized in glyoxysomes (45, 46). Among these enzymes, short chain acyl-CoA oxidase, medium to long chain acyl-CoA oxidase, a multifunctional enzyme, malate synthase, and isocitrate lyase are PTS1-containing proteins, whereas medium chain acyl-CoA oxidase, long chain acyl-CoA oxidase, 3-ketoacyl-CoA thiolase, malate dehydrogenase, and citrate synthase are PTS2-containing proteins. The loss of Pex5p resulted in reduced amounts of all these enzymes in glyoxysomes. Therefore, plants had reduced glyoxysomal function, which was confirmed by their resistance to 2,4-DB and the requirement of sucrose for post-germinative growth (Fig. 2). In addition, the effects of CO2 and measurements of maximum quantum yield of photosystem II in adult plants indicated that loss of Pex5p also reduced leaf peroxisomal function (Fig. 3). It is known that leaf peroxisomes in photosynthetic organs contain enzymes responsible for photorespiration (12). All of these enzymes are PTS1-containing proteins, whose import into leaf peroxisomes is mediated by Pex5p. A similar pleiotropic phenotype was also observed in the Arabidopsis ped2 (PEX14 orthologue) mutant that is defective in the import of both PTS1- and PTS2-containing protein (16).

In contrast, the loss of Pex7p resulted in reduced glyoxysomal function, and no significant effect was observed in leaf peroxisomal function (Figs. 2 and 3). These results suggest that leaf peroxisomal function is predominantly maintained by Pex5p. This idea is supported by our recent report (13) showing a comprehensive survey for peroxisomal gene expression in Arabidopsis. We extensively examined the expression of all these genes by using a custom-made microarray. Statistical analyses of the expression profiles revealed that peroxisomal genes could be classified into five groups. One of the groups contains genes actively transcribed in photosynthetic organs containing leaf peroxisomes, i.e. green cotyledons and leaves. They encode glycolate oxidase, hydroxypyruvate reductase, serine-glyoxylate aminotransferase, and glutamate-glyoxylate aminotransferase and malate dehydrogenase. The former four enzymes, except malate dehydrogenase, are all PTS1-containing proteins and completely cover all of the reactions that are necessary for photorespiration to occur within leaf peroxisomes. This result indicates that leaf peroxisomal function is maintained predominantly by PTS1-containing proteins whose import is mediated by Pex5p. This might be a reason for why loss of Pex7p did not affect photorespiration. The only exception was malate dehydrogenase. This enzyme is a PTS2-containing protein that is believed to supply NADH that is necessary for the reaction catalyzed by hydroxypyruvate reductase. However, our present results suggest that peroxisomal malate dehydrogenase might not be necessary for photorespiration and that NADH could be supplied by an alternative pathway. Overall, the results indicated that functional differentiation of leaf peroxisomes was maintained by an appropriate use of the PTS1-containing proteins.

Our statistical analyses also allow us to propose more detailed differentiation of plant peroxisomes with unknown function(s) than has been classified previously (13). Indeed, Arabidopsis mutants that had defects in PEX2 and PEX16 suggested unidentified plant peroxisomal function involved in photomorphogenesis and development of storage organelles in seeds (17, 18). There are still a number of PEX orthologues in plants whose functions have not been analyzed (15, 47). Precise analyses of the plant PEX genes are necessary to determine functional differentiation of peroxisomes occurring during the entire life cycle.


   FOOTNOTES
 
* This work was supported in part by Grants-in-aid for Scientific Research 15207005 (to M. N.), 14340256, and 15657014 (to M. H.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan and by a grant from CREST of Japan Science and Technology (to M. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed: Dept. of Cell Biology, National Institute for Basic Biology, Okazaki 444-8585, Japan. Tel.: 81-564-55-7504; Fax: 81-564-55-7505; E-mail; makoto{at}nibb.ac.jp.

1 The abbreviations used are: PTS1, peroxisome targeting signal 1; PTS2, peroxisome targeting signal 2; 2,4-DB, 2,4-dichlorophenoxybutyric acid; GFP, green fluorescent protein; MES, 2-(N-morpholino)ethanesulfonic acid; Ti, tumor-inducing; Pa, pascal. Back


   ACKNOWLEDGMENTS
 
We thank Dr. P. James (University of Wisconsin) for kindly providing the host strain and cloning vectors for two-hybrid analysis, Dr. T. Nakagawa (Shimane University, Shimane, Japan) for the Ti vector pGWB2, and Dr. Hiroyuki Anzai (Meiji Seika Ltd., Yokohama, Japan) for the plasmid pARK22 containing the bar gene.



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TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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