Plant and Cell Physiology Advance Access originally published online on May 3, 2007
Plant and Cell Physiology 2007 48(6):763-774; doi:10.1093/pcp/pcm053
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Functional Classification of Arabidopsis Peroxisome Biogenesis Factors Proposed from Analyses of Knockdown Mutants
1Department of Cell Biology, National Institute for Basic Biology, Okazaki, 444-8585 Japan
2Department of Molecular Biomechanics, School of Life Science, The Graduate University for Advanced Studies, Okazaki, 444-8585 Japan
*Corresponding author: E-mail, mikosome{at}nibb.ac.jp; Fax, +81-564-55-7505.
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Abstract |
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In higher plants, peroxisomes accomplish a variety of physiological functions such as lipid catabolism, photorespiration and hormone biosynthesis. Recently, many factors regulating peroxisomal biogenesis, so-called PEX genes, have been identified not only in plants but also in yeasts and mammals. In the Arabidopsis genome, the presence of at least 22 PEX genes has been proposed. Here, we clarify the physiological functions of 18 PEX genes for peroxisomal biogenesis by analyzing transgenic Arabidopsis plants that suppressed the PEX gene expression using RNA interference. The results indicated that the function of these PEX genes could be divided into two groups. One group involves PEX1, PEX2, PEX4, PEX6, PEX10, PEX12 and PEX13 together with previously characterized PEX5, PEX7 and PEX14. Defects in these genes caused loss of peroxisomal function due to misdistribution of peroxisomal matrix proteins in the cytosol. Of these, the pex10 mutant showed pleiotropic phenotypes that were not observed in any other pex mutants. In contrast, reduced peroxisomal function of the second group, including PEX3, PEX11, PEX16 and PEX19, was induced by morphological changes of the peroxisomes. Cells of the pex16 mutant in particular possessed reduced numbers of large peroxisome(s) that contained unknown vesicles. These results provide experimental evidence indicating that all of these PEX genes play pivotal roles in regulating peroxisomal biogenesis. We conclude that PEX genes belonging to the former group are involved in regulating peroxisomal protein import, whereas those of the latter group are important in maintaining the structure of peroxisome.
Keywords: Double-Stranded RNA interference - Glyoxysome - Peroxin - Peroxisome biogenesis - PEX - Protein targeting
Abbreviations: 2,4-DB, 2,4-dichlorophenoxybutyric acid; dsRNAi, double-stranded RNA interference; GFP, green fluorescent protein; PEX, peroxisome biogenesis factor, PTS, peroxisomal targeting signal.
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Introduction |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsAcknowledgmentsReferences |
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In higher plants, peroxisomes, an organelle about 1 µm in diameter, accomplish a variety of physiological functions such as lipid catabolism, photorespiration and hormone biosynthesis. For example, peroxisomes found in the cells of storage organs, such as cotyledons and endosperms, possess enzymes for fatty acid ß-oxidation and the glyoxylate cycle, and play a pivotal role in the conversion of seed reserve lipids into sucrose during post-germinative growth of oilseed plants (Beevers 1979
). After greening, plants produce sucrose from photosynthesis. During the greening process, peroxisomes lose enzymes necessary for lipid catabolism, and acquire those for photorespiratory glycolate metabolism (Titus and Becker 1985, Nishimura et al. 1986, Sautter 1986).
Peroxisomes do not have their own DNA. Therefore, peroxisomal functions are completely regulated by the nuclear genome. Peroxisomal matrix proteins encoded by the genome are translated on free polysomes in the cytosol and then imported into peroxisomes (Lazarow and Fujiki 1985). Two types of peroxisome targeting signals, i.e. PTS1 and PTS2, have been elucidated (Hayashi 2000). PTS1 is a tripeptide sequence found in the C-terminus of proteins. In higher plant cells, the permissible combinations of tripeptide sequence for PTS1 are [C/A/S/P]-[K/R]-[I/L/M]. On the other hand, PTS2 exists in a cleavable N-terminal pre-sequence. The N-terminal pre-sequences contain a consensus sequence, [R]-[A/L/Q/I]-X5-[H]-[L/I/F] (X stands for any amino acid). Recent bioinformatic analysis reveals that the Arabidopsis genome contains almost 300 genes, which encode proteins with either PTS1 or PTS2 (Kamada et al. 2003, Reumann 2004).
A recent advance in peroxisomal research is the identification of peroxisomal biogenesis factors (Hayashi and Nishimura 2003, Lazarow 2003, Baker and Sparkes 2005, Hayashi and Nishimura 2006). Since the 1990s, peroxisome-defective mutants have been isolated in diverse organisms, such as yeasts, mammals and plants, and many genes responsible for these mutations have been identified. Analyses of these genes allowed characterization of many factors regulating peroxisomal biogenesis not only in plants but also in yeasts and mammals. These genes and their gene products have been given numbers with the acronym ‘PEX’ and ‘peroxin’, respectively (Distel et al. 1996).
Of these, Arabidopsis genes AtPEX5, AtPEX7 and AtPEX14 have been demonstrated to be involved in peroxisomal protein targeting (Hayashi et al. 2000, Nito et al. 2002, Hayashi et al. 2005). AtPEX5 and AtPEX7 gene products, AtPex5p and AtPex7p, have been shown to function as specific receptors recognizing PTS1 and PTS2, respectively. They form a PTS1–PTS2 receptor complex by binding between the N-terminal domain of AtPex5p and the C-terminal domain of AtPex7p. Nascent polypeptides containing PTS1 bind to tetratricopeptide repeats (TPRs) in AtPex5p, whereas nascent polypeptides containing PTS2 bind to the WD40 repeat in AtPex7p. After the formation of the receptor–cargo complex, the two N-terminal domains (58I–65L and 78R–97R) of AtPex14p, a peroxisomal membrane protein, capture the receptor–cargo complex by binding to a WXXXF/Y repeat that exists in the middle of AtPex5p. These results indicate that AtPex5p and AtPex14p mediate import of both PTS1- and PTS2-containing proteins, while AtPex7p carries out the role of transferring only PTS2-containing proteins to the import pathway. This conclusion was proven recently in vivo by analyzing transgenic plants with reduced PEX5 and PEX7 gene expression using RNA interference (RNAi) (Hayashi et al. 2005).
Many other PEX genes were experimentally characterized using a variety of methods. For instance, AtPEX4, AtPEX5, AtPEX6 and AtPEX14 were identified from studies of Arabidopsis mutants with defects in peroxisomal fatty acid ß-oxidation (Hayashi et al. 2000, Zolman et al. 2000, Zolman and Bartel 2004, Zolman et al. 2005). Analysis of AtPEX4 also allowed identification of its binding protein, AtPex22p. PEX12 and PEX13 were identified from studies of apm mutants whose peroxisomes showed aberrant morphology (Mano et al. 2006). In contrast, two PEX genes, i.e. AtPEX2 and AtPEX16, were found from studies not focused on peroxisomes. AtPEX2 was identified by studying the TED3 mutant that was screened as a suppresser of the de-etiolated 1 mutation (Hu et al. 2002), while AtPEX16 was identified by studying the sse1 mutant that showed a shrunken seed phenotype (Lin et al. 1999).
The completion of sequencing of the Arabidopsis genome has enabled bioinformatic prediction of Arabidopsis PEX orthologs (Mullen et al. 2001, Charlton and Lopez-Huertas 2002). Of these, T-DNA insertion mutants in the PEX10 and PEX12 genes have been identified by a reverse genetic approach and characterized (Schumann et al. 2003, Sparkes et al. 2003, Fan et al. 2005). The prediction was also used to demonstrate the subcellular localization of the PEX3 product, the effect of PEX11 overexpression and the biochemical properties of PEX19 products (Hunt and Trelease 2004, Hadden et al. 2006, Lingard and Trelease 2006). However, the functional contribution to peroxisomal biogenesis at the molecular level of these PEX genes, with the exception of PEX5, PEX7 and PEX14, is still obscure (Baker and Sparkes 2005).
Here we classified Arabidopsis PEX genes by evaluating their physiological contribution in peroxisome biogenesis using the same technique, i.e. analysis of knockdown mutants established by double-stranded RNA interference (dsRNAi). The data provide experimental evidence that PEX genes can be subdivided into two groups, i.e. genes involved in peroxisomal protein import and those involved in regulating peroxisomal morphology.
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Results |
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Establishment of knockdown mutants
We have previously demonstrated that dsRNAi has a great advantage for producing specific and heritable gene silencing and for analyzing the functions of PEX5 and PEX7 in planta (Hayashi et al. 2005
). Therefore, we applied this technique for establishing knockdown mutants of the Arabidopsis PEX genes listed in Table 1, involving two paralogous genes for PEX3, two for PEX19 and five for PEX11. To reduce gene expression by dsRNAi, a 300–530 bp cDNA fragment derived from each PEX gene transcript was amplified by reverse transcripion–PCR (RT–PCR) (see Table 1, region used for RNAi). We carefully designed the DNA fragments of approximately 450 bp with no significant similarity to any other open reading frames (ORFs) in the Arabidopsis genome. In the case of PEX genes having paralogous genes (PEX3, PEX11 and PEX19), we particularly paid more attention to the choice of a region specific for each single gene. They were designed not to have a perfect match of more than 22 bp with their counterpart(s), since a 22 bp perfect match has been proved to induce RNA silencing in related gene expression in plants (Ishihara et al. 2005).
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Duplicate cDNA fragments in sense and antisense orientations were simultaneously recombined into two positions within the T-DNA region of pHB-F, a Ti plasmid containing a phosphinothricin-resistant gene as a selectable marker in the plants. The dsRNAi construct was designed to express RNA capable of forming a double strand at the two identical PEX gene-specific sequences under the control of the cauliflower mosaic virus 35S promoter, and independently integrated into the genomes of AtGFP-PTS1 and AtPTS2-GFP, kanamycin-resistant transgenic plants expressing green fluorescent protein (GFP)–PTS1 and PTS2–GFP, respectively, by Agrobacterium-mediated transformation. Construction of these transgenic plants has previously been described elsewhere (Mano et al. 2002
). Peroxisomes in the cells of AtGFP-PTS1 and AtPTS2-GFP had punctated green fluorescence due to the peroxisomal targeting of GFP–PTS1 and PTS2–GFP. Retransformation of the dsRNAi construct into AtGFP-PTS1 and AtPTS2-GFP allowed us to visualize the function of the corresponding PEX gene in vital plant cells.
Table 2 summarizes the process of establishing knockdown mutants produced by transformation of dsRNAi into AtGFP-PTS1. We designated the knockdown mutant as pexNi where pexN referred to a PEX gene or a combination of PEX genes silenced by the dsRNAi. We selected certain numbers of T1 progeny showing both kanamycin and phosphinothricin resistance in each construct. Intracellular localization of GFP–PTS1 in the cells of selected T1 progeny was examined using fluorescent microscopy. We found that all or most kanamycin/phosphinothricin-resistant T1 progeny of pex1i, pex2i pex4i, pex6i, pex10i, pex12i, pex13i, pex16i, pex19-1i and pex19-2i showed fluorescent patterns different from that of their parent, AtGFP-PTS1 (Table 2, see below in detail). In contrast, single knockdown mutants that silenced any of the PEX3 and PEX11 paralogs failed to show mutant phenotypes (data not shown). Therefore, we decided to make double/triple knockdown mutants. For the construction of the double and triple knockdown mutant, DNA fragments of the corresponding genes shown in Table 1 were ligated to make a single DNA fragment by a three-step amplification of PCR following the method reported by Higuchi (1990). The single DNA fragment was used to suppress expression of the multiple genes. Multiple knockdown mutants, pex3-1/3-2i, pex11a/11bi and pex11c/11d/11ei, showed altered fluorescent patterns (Table 2, see below in detail).
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Since these T1 plants were heterozygous in dsRNAi, the observed phenotypes appeared to be dominant (Table 2, T1). We selected one of the T1 progeny showing the most severe phenotype in order to obtain T2 progeny. We used the selected T2 progeny for further analyses. All, or a majority of the kanamycin/phosphinothricin-resistant T2 progeny showed the same fluorescent pattern as the T1 progeny, suggesting that the phenotype described in this study was heritable and dominant (Table 2, T2). The effect of dsRNAi in the selected T1 progeny was determined by quantitative RT–PCR. The result indicated that expression of the target gene in these knockdown mutants was decreased to 14.7–58.3% of target gene expression in the wild type (Table 2, percentage expression of the gene). In the case of pex17i, however, none of 16 pex17i plants showing kanamycin/phosphinothricin resistance had any mutant phenotype (Table 2, pex17i) in spite of the existence of a single PEX17 gene in the Arabidopsis genome. We decided to select one of the pex17i T1 progeny to obtain T2 progeny. The effect of dsRNAi in pex17i was determined using the T2 progeny.
Effects of 2,4-dichlorophenoxybutyric acid and sucrose on post-germinative growth
Fig. 1 shows post-germinative growth of the knockdown mutants. Of all the knockdown mutants we analyzed, only pex10i showed defects in root elongation (Fig 1, GM) and a dwarf phenotype. The growth of other knockdown mutants was similar to that of their parental plant, AtGFP-PTS1. We previously reported that pex5i and pex7i have defects in peroxisomal fatty acid ß-oxidation, became resistant to 2,4-dichlorophenoxybytyric acid (2,4-DB) and required sucrose for post-germinative growth (Fig. 1, pex5i and pex7i) (Hayashi et al. 2005). To analyze the contribution of PEX genes to peroxisomal fatty acid ß-oxidation, we examined the effects of 2,4-DB and sucrose on post-germinative growth of the knockdown mutants. As shown in Fig. 1, roots of pex1i, pex2i, pex4i, pex6i, pex10i, pex12i and pex13i elongated to similar lengths regardless of the presence or absence of 2,4-DB in the medium, and could not germinate properly on medium without sucrose. Resistance to the toxic level of 2,4-DB and requirement of sucrose for post-germinative growth clearly demonstrated that these mutants had defects in peroxisomal fatty acid ß-oxidation.
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In contrast, pex3-1/3-2i, pex11a/11bi, pex11c/11d/11ei, pex16i, pex17i, pex19-1i and pex19-2i did not require sucrose for post-germinative growth. All of these mutants except pex16i were sensitive to 2,4-DB (Fig. 1). This result indicated that these knockdown mutants had complete peroxisomal fatty acid ß-oxidation activity. pex16i, however, showed resistance to 2,4-DB. We have previously demonstrated that peroxisome-defective (ped) mutants were dominant against 2,4-DB resistance, while being recessive against the requirement for sucrose (Hayashi et al. 1998
). This suggested that pex16i had reduced peroxisomal fatty acid ß-oxidation activity.
Knockdown mutants showed mis-sorting of peroxisomal proteins
Of all the knockdown mutants analyzed in this study, the pleiotropic phenotype of pex10i was the most severe. It had shortened roots due to the reduced size of cells in the longitudinal axis (Fig. 1, GM), variegated leaves (Fig. 2A) and small siliques. Although it produced a small number of viable seeds, most of the embryos in the siliques were white, immature and lethal phenotypes (Fig. 2C) that were different from the control plant (Fig. 2B, D).
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We also examined the subcellular localization of GFP fusion proteins, GFP–PTS1 and PTS2–GFP, in the cells of pex10i using a confocal laser-scanning microscope. Both root and leaf cells of pex10i expressing GFP–PTS1 showed green fluorescence only in the periphery of the cells (Fig. 2E, G) that coincided with the cytosol surrounding the central vacuoles. Cytosolic fluorescence was also obtained in leaf and root cells of pex10i expressing PTS2–GFP (Fig. 2F, H). In contrast, cells of parental transgenic plants, AtGFP-PTS1 and AtPTS2-GFP, showed punctated green fluorescence that coincided with peroxisomes (Fig. 2I, J).
In addition to pex10i, GFP–PTS1 in cells of pex1i, pex2i, pex4i, pex6i, pex12i and pex13i was also predominantly detected in the cytosol of both root cells (Fig. 3A–F) and leaf cells (data not shown). Similar fluorescent images were also observed in these knockdown mutants expressing PTS2–GFP (data not shown). These data indicate that pex1i, pex2i, pex4i, pex6i, pex10i, pex12i and pex13i had defects in the import of both PTS1-containing and PTS2-containing proteins into peroxisomes, although they did not show the visible phenotypes that were detected in pex10i.
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Knockdown mutants containing aberrant peroxisomes
In contrast to the knockdown mutants described above, pex3-i/3-2i, pex11a/11bi, pex11c/11d/11ei, pex16i, pex19-1i and pex19-2i were competent in importing both GFP–PTS1 and PTS2–GFP into peroxisomes. These knockdown mutants showed no obvious, visible phenotype. However, fluorescent microscopic observation of GFP–PTS1 (and PTS2–GFP, not shown) visualized aberrant peroxisomes in cells of these mutants. Of these, peroxisomes in pex16i showed the most drastic changes in their number and morphology. As shown in Fig. 4A, peroxisomes in the mutant became fewer and larger than those in AtGFP-PTS1 (Fig. 4A). We often found a cell containing just one large peroxisome. The large peroxisomes were still competent in importing both GFP–PTS1 and PTS2–RFP (red fluorescent protein), and often included vesicles which had no fluorescence (Fig. 4B). We compared the diameters of peroxisomes in the cells of pex16i with those of control plants. As shown in Fig. 4C, the average diameter of peroxisomes in pex16i was 4.1 µm, and the largest peroxisomes were >10.0 µm, whereas the diameter of peroxisomes in control plants was approximately 1.0 µm. An immunogold labeling experiment revealed that the large peroxisomes in pex16i, as well as peroxisomes in AtGFP-PTS1, were double stained with antibodies against GFP and catalase, a marker enzyme for peroxisomes (Fig. 4D), demonstrating that the large organelles with GFP fluorescence in pex16i are peroxisomes.
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There are two PEX19 paralogs, PEX19-1 and PEX19-2, in the Arabidopsis genome (Table 1). By using dsRNAi, we succeeded in specifically reducing expression of each paralog in pex19-1i and pex19-2i, respectively (Table 2). As shown in Fig. 5A, GFP–PTS1 was imported into large peroxisomes in cells of both pex19-1i and pex19-2i. The average diameter of peroxisomes in pex19-1i and pex19-2i was 2.7 µm, and the largest peroxisome was >6 µm (Fig. 5B). It is noteworthy that GFP–PTS1 was also detected in the cytosol of both pex19-1i and pex19-2i (Fig. 5A), suggesting that they had reduced activity for import of peroxisomal proteins.
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PEX3 and PEX11 also belong to multigene families. However, our efforts to produce single knockdown mutants failed to show any obvious phenotypes. Arabidopsis has five paralogs for PEX11 (Table 1), which were subdivided into two groups. The first group included PEX11a and PEX11b, and the other PEX11c, PEX11d and PEX11e (Fig. 6A). Therefore, we decided to establish the double and triple knockdown mutants, pex11a/11bi and pex11c/11d/11ei. Simultaneous suppression of these combinations led to the formation of large peroxisomes in the cells of roots and leaves (Fig. 6B). The average size of peroxisomes in pex11a/11bi and pex11c/11d/11ei is 1.55 and 2.36 µm, respectively (Fig. 6C). As shown in Fig. 7A and B, the double knockdown mutant pex3-1/3-2i had elongated, tubular peroxisomes. The length of the longest peroxisome was >20 µm and these peroxisomes had globular heads and long filamentous tails (Fig. 7B, C).
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Overall, the results indicated that peroxins analyzed in this study can be functionally subdivided into two groups, one for peroxins regulating peroxisomal protein import and another for peroxins maintaining the structure of the peroxisome (Table 2).
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Discussion |
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It is now widely recognized that many PEX genes are involved in regulating biogenesis of peroxisomes in different species (Heiland and Erdmann 2005
). This is also true in the case of plant peroxisomes (Hayashi and Nishimura 2006). A variety of techniques, such as forward genetic screening, reverse genetic screening and bioinformatics, have allowed the identification of 22 PEX genes in the Arabidopsis genome (Table 1). However, the different approaches used for analyzing each PEX gene have made it difficult to compare their functional contribution to peroxisomal biogenesis in plants.
We previously reported the establishment of pex5 and pex7 mutants using a dsRNAi technique (Hayashi et al. 2005). Here we succeeded in establishing knockdown mutants with reduced gene expression of 18 PEX genes by the same dsRNAi technique. This technique, together with the use of transgenic plants expressing GFP–PTS1, allowed us to compare the functional contributions of these PEX genes to peroxisomal biogenesis from versatile view points, i.e. peroxisomal function, peroxisomal protein import and peroxisome morphology, in planta.
Of these knockdown mutants, pex1i, pex2i, pex4i, pex6i, pex10i, pex12i and pex13i had defects in peroxisomal function, i.e. fatty acid ß-oxidation. All of these mutants accumulated GFP–PTS1 in the cytosol, indicating that peroxisomal protein import is disordered in these mutants. The loss of peroxisomal protein caused peroxisomal remnants that do not have proper function. The result clearly demonstrated that these PEX genes directly or indirectly contribute to peroxisomal protein targeting together with the PTS1–PTS2 receptor complex (Pex5p and Pex7p) and the docking protein, Pex14p (Hayashi et al. 2000, Nito et al. 2002, Hayashi et al. 2005).
It has been reported that mutants having nucleotide substitutions in PEX4, PEX6, PEX12, PEX13 and PEX14 genes are defective in peroxisomal function (Hayashi et al. 2000, Zolman et al. 2000, Zolman and Bartel 2004, Zolman et al. 2005, Mano et al. 2006). However, all of these mutants showed partial defects in peroxisomal protein import. Our results suggest that the point mutation in these genes caused partial inhibition of peroxisomal protein import due to the leaky phenotype of the mutants. In the case of PEX2, PEX10 and PEX12 genes, T-DNA-inserted homozygous knockout mutants showed an embryo-lethal phenotype (Hayashi et al. 2000, Hu et al. 2002, Sparkes et al. 2003, Flynn et al. 2005). This result suggests the importance of these PEX genes; however, the lethal phenotype prevented analysis of their function in planta. Overall, we concluded that reduction of PEX gene expression by dsRNAi can induce a more severe but not lethal phenotype, and is technically superior to both point mutation and T-DNA insertion.
It is noteworthy that the phenotypes of pex1 and pex10 mutants were described here for the first time. In particular, pex10i could produce a small number of viable seeds that allowed analysis of the function of the PEX10 gene in planta, although most of the embryos were lethal, as are the embryos of T-DNA-inserted pex10 knockout mutants (Sparkes et al. 2003, Flynn et al. 2005). We succeeded in showing the pleiotropic phenotypes of pex10i, such as reduced cell size in roots and variegated leaves, which were not found in other pex mutants. These phenotypes cannot be explained by defects in known peroxisomal functions in plants, such as lipid catabolism, photorespiration, amino acid catabolism and hormone biosynthesis. pex10i may shed light on currently unknown functions of plant peroxisomes. Indeed, only a few dozen of the PEX genes have been functionally characterized, while our bioinfomatic analysis revealed that the Arabidopsis genome contains at least 286 peroxisomal genes (Kamada et al. 2003). Further analyses of these uncharacterized genes in conjunction with pex10i phenotypes will be needed.
The results of the present study categorized the function of PEX3, PEX11, PEX16 and PEX19 into the same group. Knockdown mutants of these genes did not require exogenously supplied sucrose for germination, and had the ability to transport GFP–PTS1 into peroxisomes. In addition, peroxisomes in the cells of these knockdown mutants were aberrant in size, number or shape. The result suggested that PEX3, PEX11, PEX16 and PEX19 contribute to morphology determination of peroxisomes, but not to import of peroxisomal matrix proteins. From these results, phenotypes of mutant plants defective in PEX3, PEX11 and PEX19 expression are described for the first time. These genes have been known to exist as a multigene family in the Arabidopsis genome. It would seem that the existence of multiple genes might prevent forward genetic isolation of the mutants. Indeed, we failed to detect phenotypes of knockdown mutants that suppressed either PEX3 or PEX11 gene expression by dsRNAi. However, the dsRNAi technique allowed us to establish multiple knockdown mutants, i.e. pex3-1/3-2i, pex11a/11bi and pex11c/11d/11ei, with defects in peroxisomal morphology in planta.
Pex3p, Pex16p and Pex19p have been suggested as essential factors for assembly of peroxisomal membrane proteins in yeast and mammals (Fujiki et al. 2006). However, this hypothesis is still a matter of debate as data are limited, particularly in plants (Lin et al. 1999, Hunt and Trelease 2004, Lin et al. 2004, Karnik and Trelease 2005, Hadden et al. 2006). In the present study, we showed evidence that double mutation of PEX3-1 and PEX3-2 caused elongated and tubular peroxisomes. The morphology of peroxisomes in the double mutant was similar to that found in the apm1 mutant. The APM1 gene encodes DRP3A, dynamin-related protein 3A, that regulates division of peroxisomes (Mano et al. 2004). It is possible that Pex3p recruits not only peroxisomal membrane proteins but also a protein necessary for peroxisomal division. Pex3p is believed to be a docking factor for Pex19p. Pex19p is a predominantly cytoplasmic protein, and is believed to play a role as a chaperon by binding to peroxisomal membrane proteins (Fujiki et al. 2006). Previous data have shown distinct expression profiles for Arabidopsis PEX19-1 and PEX19-2 (Hadden et al. 2006), which coincides well with our present data showing that a single mutation in these genes causes aberrant peroxisomal morphology. These results suggest that PEX19-1 and PEX19-2 are not functionally redundant.
Analysis of the pex16 knockdown mutant (pex16i) revealed that reduction of PEX16 function produced cells with fewer and enlarged peroxisomes containing unknown vesicles that may have been produced by invagination of the peroxisomal membrane. Although sse1, a T-DNA-inserted pex16 null mutant, has been characterized, the morphology of peroxisomes in the homozygous mutant remains unknown due to its embryonic lethal phenotype (Lin et al. 1999, Lin et al. 2004). Our present results suggest the contribution of plant PEX16 to peroxisomal morphology. Pex16p was demonstrated to be synthesized in the endoplasmic reticulum and transported to peroxisomes (Karnik and Trelease 2005, Mullen and Trelease 2006). It is now believed that Pex16p is involved in the early steps of peroxisomal biogenesis. However, the function of Pex16p at the molecular level remains largely unknown. In this context, we are interested in the reported morphology of the peroxisomal multivesicular bodies induced by infection of tobacco BY-2 cells with tomato bushy stunt virus (Mccarthy et al. 2005). Peroxisomal multivesicular bodies became fewer and larger, and contained unknown vesicles that may have been produced by invagination of the peroxisomal membrane, somewhat similar to the peroxisomes found in cells of pex16i. These data may reflect an unknown process required for peroxisomal membrane biogenesis. Further analysis of pex16i may give us insight into the early steps of peroxisomal biogenesis in plants.
Lingard and Trelease (2006) analyzed the effects of transient expression of one of the five PEX11 genes on peroxisomal morphology, and found evidence that these PEX11 genes individually promote duplication (PEX11a and PEX11e), aggregation (PEX11b) or elongation without fission (PEX11c and PEX11d). Orth et al. (2007) recently reported on the overexpression of the individual PEX11 genes in transgenic Arabidopsis and found that overexpression of PEX11a and PEX11b resulted in peroxisome elongation, whereas overexpresson of PEX11c, PEX11d and PEX11e led to an increased number of peroxisomes. These data were, to some extent, inconsistent with each other and with our data. Indeed, we failed to detect any change in peroxisomal morphology in any single PEX11 gene knockdown mutant, although Orth et al. (2007) reported that reduced expression of each gene decreased peroxisome abundance. Our present study indicated that the five PEX11 genes could be divided into two functionally redundant groups, one containing PEX11a and PEX11b, and another containing PEX11c, PEX11d and PEX11e. Although PEX11 genes are believed to play a role in peroxisome proliferation, further analysis is needed to clarify the functions of individual PEX11 genes.
Bioinformatic analysis has suggested that At4g18200 encodes Arabidopsis PEX17 (Mullen et al. 2001, Charlton and Lopez-Huertas 2002). Recently, however, the At4g18200 gene was deleted and separated into three genes, At4g18195, At4g18197 and At4g18205 (http://www.arabidopsis.org/). Of these, At4g18197 contains a short domain of amino acid sequence showing weak similarity to PEX17 in yeast (Yarrowia lipolytica). In spite of the fact that our dsRNAi construct was designed to target RNA encoding this domain, no pex17i plants showed any phenotype related to peroxisomes. Expression of the target gene in these pex17i plants was low enough (minimum; 46.4%, average; 50.7%) to induce a visible phenotype in the other knockdown mutants (maximum percentage of gene expression; 58.3%) (Table 2). This result implies that At4g18197/At18200 may not be an Arabidopsis PEX17.
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Materials and Methods |
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Plant materials
Arabidopsis thaliana ecotype Columbia was used as the wild-type plant. Construction of transgenic plants expressing GFP–PTS1 and PTS2–GFP was described previously. All seeds were surface sterilized in 2% NaClO, 0.02% Triton X-100, and were grown on germination medium [2.3 mg ml–1 Murashige and Skoog salts (Wako, Osaka, Japan), 1% sucrose, 100 µg ml–1 myo-inositol, 1 µg ml–1 thiamine-HCl, 0.5 µg ml–1 pyridoxine, 0.5 µg ml–1 nicotinic acid, 0.5 mg ml–1 MES-KOH (pH 5.7), 0.2% INA agar (Ina shokuhin, Nagano, Japan)] in an atmosphere containing 1,000 Pa CO2 under a 16 h light (50 µE m–2 s–1)/8 h dark cycle at 22°C. Some 7-day-old seedlings on the medium were transferred to a 1 : 1 mixture of perlite and vermiculite and grown under a 16 h light (100 µE m–2 s–1)/8 h dark cycle at 22°C.
Plasmid constructions
We used the Ti vector, pHB-F, for constructions of Ti plasmids suitable for dsRNAi. This vector was constructed by introducing a phosphinothricin-resistant (bar) gene into the SacI site of pHELLSGATE8 (Wesley et al. 2001), a Gateway-based Ti plasmid suitable for RNA interference in plants. A partial cDNA fragment derived from each PEX gene (Table 2, region used for RNAi) was amplified by RT–PCR using the Superscript first-strand synthesis system (Invitrogen Corp., Carlsbad, CA, USA). It was subcloned into pDONR221, and then transferred to pHB-F by Gateway technology according to a protocol provided by the manufacturer (Invitrogen Corp.). The recombinant pHB-F contained two identical cDNA fragments in sense and antisense orientations within the T-DNA region.
Arabidopsis transformation
The recombinant pHB-F was independently introduced into kanamycin-resistant transgenic Arabidopsis plants expressing either AtGFP-PTS1, i.e. kanamycin-resistant transgenic plants expressing GFP–PTS1 (a GFP fusion protein containing the C-terminal 10 amino acid residues of hydoxypyruvate reductase at its C-terminus), or AtPTS2-GFP, i.e. kanamycin-resistant transgenic plants expressing PTS2–GFP (a GFP fusion protein containing the N-terminal 49 amino acid residues of citrate synthase at its N-terminus) (Mano et al. 1999) by vacuum infiltration using Agrobacterium tumefaciens (strain C58C1RifR) (Bechtold et al. 1993). In some experiments, the recombinant pHB-F was also transformed into kanamycin/hygromycin-resistant transgenic Arabidopsis plants expressing both GFP–PTS1 and PTS2–mRFP. The primary transformants were designated as T0 plants. Transformed Arabidopsis lines were selected on growth medium containing both 50 µg ml–1 kanamycin and 10 µg ml–1 phosphinothricin, and designated as T1 transformants. T2 progeny showing kanamycin/phosphinothricin resistance were used for further analyses.
Analyses of fluorescence in cells of the transgenic plants
Seven-day-old roots and 3-week-old leaves of the homozygous plants were mounted under coverslips. Fluorescent images of the specimens were captured using an LSM 510 confocal laser-scanning microscope (excitation; 488 nm, emission; 500–550 nm, Carl Zeiss, Göttingen, Germany). All images were scanned as 2.5 nm optical slice sections. The diameters of peroxisomes on the image were measured using Image Gauge, an image analysis software (Fujifilm, Tokyo, Japan).
Electron microscopy
The leaves of the homozygous plants were harvested. Ultrathin sectioning, immunogold labeling and electron microscopic observation were performed as described previously (Kamigaki et al. 2003).
Quantitative PCR
Total RNA was extracted from 3-week-old leaves using Concert Plant RNA Reagent (Invitrogen Corp.), and reverse transcribed using the Superscript first strand synthesis system (Invitrogen Corp.) according to protocols provided by the manufacturer. PCR amplification of the cDNA was performed by TaKaRa Ex Taq R-PCR (TAKARA SHUZO CO., LTD, Shiga, Japan) in the presence of SYBR Green I (BioWhittaker Molecular Applications, Rockland, ME, USA), and monitored by SmartCycler (Cepheid, Sunnyvale, CA, USA) according to the manufacturer's protocol. The relative quantity of the target mRNA was calculated using actin as a standard.
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The authors thank Dr. Peter M. Waterhouse of Australia for kindly providing pHELLSGATE8. We also thank Dr. Roger Y. Tsien from the USA for kindly providing monomeric red fluorecent protein, mRFP. This work was supported in part by a grant from the Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists, by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (15207005 to M.N. and 12640625 to M.H.) and by a grant from Core Research of Science and Technology (CREST) of the Japan Science and Technology to M.H.
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(Received February 27, 2007; Accepted April 27, 2007)
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