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First published online August 19, 2005; 10.1104/pp.105.066811 Plant Physiology 139:231-239 (2005) © 2005 American Society of Plant Biologists The Arabidopsis PEX12 Gene Is Required for Peroxisome Biogenesis and Is Essential for Development1,[w]Department of Energy Plant Research Laboratory (J.F., S.Q., T.O., C.A., J.H.), and Department of Plant Biology (J.H.), Michigan State University, East Lansing, Michigan 48824; and Howard Hughes Medical Institute, Plant Biology Laboratory, The Salk Institute, La Jolla, California 92037 (J.C.)
Peroxisomes perform diverse and vital functions in eukaryotes, and abnormalities in peroxisomal function lead to severe developmental disorders in humans. Peroxisomes are also involved in a wide array of physiological and metabolic functions unique to plants, yet many aspects of this important organelle are poorly understood. In yeast and mammals, various steps in peroxisome biogenesis require the function of peroxin (PEX) proteins, among which PEX12 is a RING finger peroxisomal membrane protein involved in the import of matrix proteins. To investigate the role of PEX12 in plants, we identified a T-DNA knockout allele of PEX12 and generated partial loss-of-function pex12 mutants using RNA interference. We show that pex12 null mutants are developmentally arrested during early embryogenesis, and that the embryo-lethal phenotype can be rescued by overexpression of the PEX12-cyan fluorescent protein fusion protein, which targets to the peroxisome. Using virus-induced gene-silencing techniques, we demonstrate that peroxisomal number and fluorescence of the yellow fluorescent protein-peroxisome targeting signal type 1 protein are greatly reduced when PEX12 is silenced. RNA interference plants with partial reduction of the PEX12 transcript exhibit impaired peroxisome biogenesis and function, inhibition of plant growth, and reduced fertility. Our work provides evidence that the Arabidopsis (Arabidopsis thaliana) PEX12 protein is required for peroxisome biogenesis and plays an essential role throughout plant development.
Peroxisomes perform diverse and crucial functions in eukaryotes, and peroxisomal deficiencies cause developmental disorders in humans and disrupt many physiological and developmental processes in fungi (Powers and Moser, 1998  ; Gould and Valle, 2000; Parsons et al., 2001; Titorenko and Rachubinski, 2004). Plant peroxisomes, which include glyoxysomes, leaf-type peroxisomes, and unspecialized peroxisomes, contain different sets of enzymes depending on the cell type and developmental stage. They are involved in a wide range of functions, including lipid metabolism, photorespiration, nitrogen metabolism, stress response, and synthesis of some plant hormones (Beevers, 1979; Olsen and Harada, 1995; Hayashi and Nishimura, 2003).
More than 30 yeast genes have been identified that encode components for various aspects of peroxisome biogenesis, including peroxisome assembly, matrix protein import, and peroxisome proliferation (Purdue and Lazarow, 2001
The Arabidopsis (Arabidopsis thaliana) genome is predicted to encode about 15 proteins homologous to the yeast peroxins (Mullen et al., 2001
Mutations in PEX12 lead to failure of matrix protein import in yeast and mammals and result in the Zellweger syndrome, a lethal neurological disorder in humans (Gould and Valle, 2000
Null Mutants of PEX12 Cease to Develop during Early Embryogenesis
AtPEX12 (At3g04460) is a single-copy gene encoding a putative protein of 44 kD. It shares approximately 27% protein sequence identity with its yeast and mammalian orthologs and contains a C5-type RING finger motif with five conserved Cys (Fig. 1), which is different from the C3HC4-type RING found in AtPEX2 and AtPEX10. To study the function of PEX12 in plant development, we first identified from the Salk Arabidopsis T-DNA knockout collection (Alonso et al., 2003
To confirm that the abnormal embryos indeed were homozygous for the T-DNA insertion allele, we dissected both abnormal and normal embryos (torpedo stage) from the seeds and pooled approximately 10 embryos of each type for DNA extraction. PCR analysis demonstrated that normal embryos contained both the insertion and the wild-type alleles (Fig. 2D, lanes 1 and 2), suggesting that they were composed of both wild-type and heterozygous embryos. However, only the T-DNA insertion-specific band was amplified from the abnormal embryos (Fig. 2D, lanes 3 and 4), indicating that these embryos were homozygous knockouts. To determine which step of embryo development was disrupted by the pex12 null mutation, we analyzed developing seeds in heterozygous plants using Nomarski optics. Knockout embryos of pex12 showed retarded growth and in most cases were able to develop to the heart stage, but failed to grow further into torpedo and mature embryos (Fig. 3, C and D). Occasionally, the mutant embryos ceased to grow at globular or early torpedo stage (data not shown). Our data suggest that homozygous pex12 null embryos were delayed in growth and eventually stopped developing during early stages of embryogenesis.
We performed electron microscopic analysis with the knockout embryos to determine the effect of PEX12 deficiency at the ultrastructural level. Embryos subjected to examination were from heterozygous plants 6 d after fertilization, when wild-type embryos are usually at the torpedo stage of embryogenesis. Wild-type embryos contained well-developed peroxisomes (also called glyoxysomes in seeds), lipid bodies, and plastids (Fig. 3E). The pex12 null embryos, however, lacked peroxisomes, contained small and underdeveloped plastids, and were missing characteristic lipid body structures (Fig. 3F). Based on these observations, we conclude that PEX12 is required at least for the formation of the peroxisome. The impairment of other subcellular structures, such as lipid bodies and plastids, could be an indirect consequence of the loss of PEX12. For example, lipid bodies are physically associated with peroxisomes during seed germination, providing them with fatty acid substrates. It is thus conceivable that the function and presence of lipid bodies can be affected by a feedback mechanism when no functional peroxisomes are present to carry out lipid metabolism; however, a direct role of PEX12 in lipid body formation cannot be completely ruled out.
To determine the subcellular localization of AtPEX12, we analyzed wild-type Arabidopsis plants coexpressing yellow fluorescent protein (YFP)-peroxisome targeting signal type 1 (PTS1) and PEX12-cyan fluorescent protein (CFP) by fluorescence microscopy. PTS1, which is composed of Ser, Lys, and Leu, is widely used as a tag to localize proteins to the peroxisome in diverse systems. It is the targeting sequence for the majority of known peroxisomal matrix proteins and is recognized by the PTS1 receptor PEX5 (Subramani et al., 2000
We also transformed plants heterozygous for the knockout allele with the PEX12-CFP fusion construct driven by the 35S constitutive promoter. T2 plants were screened for homozygosity for the pex12 knockout allele and overexpression of the PEX12-CFP protein. The embryo-lethal phenotype of the knockout plants was rescued by the transgene (Fig. 4D), providing further evidence that the lack of a functional PEX12 gene was responsible for the lethal phenotype and suggesting that the PEX12-CFP fusion protein functions properly.
The embryo-lethal phenotype of the pex12 knockout plants prevented us from further elucidating the potential roles of PEX12 in peroxisome biogenesis and in later stages of development; thus, mutants with reduced levels of PEX12 were needed. To this end, two RNAi strategies were employed to knock down the expression of PEX12: (1) infecting plants with viruses containing a fragment of the PEX12 coding sequence and (2) making transgenic plants stably expressing a PEX12 double-stranded RNAi (dsRNAi) construct.
A gene-silencing system based on the bipartite geminivirus cabbage leaf curl virus (CbLCV) was recently developed that can efficiently induce diffusible, homology-based systemic silencing of endogenous genes in Arabidopsis (Turnage et al., 2002 Plants infected by both the sense and antisense PEX12-silencing constructs exhibited a strong reduction in the number of peroxisomes as well as peroxisomal fluorescence of the YFP-PTS1 protein in new leaves (Fig. 5, C and E) compared to old leaves (Fig. 5, D and F), whereas plants infected by the empty vector control did not show a significant difference between old and new leaf tissue (Fig. 5, A and B). Reverse transcriptase (RT)-PCR analysis was subsequently performed to determine the expression level of PEX12 in these tissues. Figure 5G shows that, in plants bombarded with the PEX12-silencing constructs, the transcript level of PEX12 in the new tissue was significantly lower than in the old tissue, suggesting that PEX12 is required for peroxisome biogenesis in leaves. Despite the fact that the CbLCV virus used in this work was attenuated by removal of the AR1 gene, plants still displayed mild viral symptoms after infection, such as wrinkled leaves, stunted growth, and lack of inflorescence. As such, the mutant phenotypes caused by PEX12 silencing in adult plants could not be unambiguously determined by this approach.
Silencing of PEX12 by dsRNAi in Transgenic Plants To elucidate more clearly the impact of PEX12 on plant development, a second RNAi approach was performed simultaneously. Given the lethal phenotype of the null mutants, we aimed to generate weaker and partial loss-of-function mutants of PEX12 by using a smaller fragment of the gene, rather than the entire coding region, in the RNAi construct. The same 247-bp cDNA fragment of PEX12 was cloned as inverted repeats into the dsRNAi vector pFGC5941, obtained from the Arabidopsis Biological Resource Center (ABRC). Plants containing YFP-PTS1 were transformed with the pFGC5941-derived PEX12-silencing construct under the control of the 35S promoter. Approximately 20% of the 50 T1 transgenic plants showed fairly strong reduction of the PEX12 mRNA. The level of PEX12 suppression correlated with the severity of the mutant phenotypes in both T1 plants and their progeny. Compared with wild-type plants and RNAi plants with a subtle reduction of PEX12 expression (such as RNAi no. 3), RNAi plants with stronger reduction of PEX12 gene expression (such as RNAi nos. 1 and 2) were smaller and paler green, and developed more slowly (Fig. 6, A and B). The mutants were also less fertile with smaller gynoecia and shorter stamens in many mutant flowers (Fig. 6, C and D), and contained reduced numbers of peroxisomes and significantly weakened YFP-PTS1 fluorescence in peroxisomes (Fig. 6, E–H).
We also tested sugar dependence and indole-3-butyric acid (IBA) response of the RNAi plants. Through  -oxidation and the glyoxylate cycle, oilseed peroxisomes participate in lipid mobilization, which provides seedlings with energy to grow. As such, peroxisomal mutant seedlings of oilseed species such as Arabidopsis usually grow poorly in the absence of exogenous sugar. Mutants defective in -oxidation are also resistant to the inhibitory effect of IBA on root elongation because mutant peroxisomes cannot efficiently convert IBA into IAA inside this organelle (Zolman et al., 2000). T3 seeds were plated on medium with or without 1% Suc and on medium containing 20 µM IBA combined with 1% Suc; root length was measured after 7 d of growth in the light. RNAi plants numbers 1 and 2 exhibited defects in root elongation without supplemental sugar, a phenotype that was mostly rescued by Suc (Fig. 6I). In addition, these mutants were also less responsive to IBA (Fig. 6I), which is indicative of a deficiency in -oxidation. Taken together, our data support the role of PEX12 as a critical player in peroxisome formation and function, and in plant vegetative and reproductive growth.
The essential role of PEX12 throughout Arabidopsis development led us to examine its expression pattern in the plant. An RT-PCR analysis of the PEX12 transcript suggested that this gene was ubiquitously expressed in young seedlings, leaves, roots, and flowers (data not shown). To assess its expression more completely, we used GENEVESTIGATOR, an Arabidopsis microarray gene expression database and analysis toolbox (Zimmermann et al., 2004
We have presented evidence that PEX12 is a peroxisomal protein in Arabidopsis and is essential for peroxisome biogenesis and plant development. First, null pex12 embryos (SALK_13612) were slow growing and eventually aborted during early embryogenesis. Second, PEX12-CFP was localized to the peroxisome and complemented the lethal phenotype of the pex12 knockout mutants. In addition, YFP-PTS1 plants infected by the CbLCV virus carrying part of the PEX12 coding sequence displayed strong reduction of the number of peroxisomes and import of PTS1-containing matrix proteins. Furthermore, transgenic plants, in which PEX12 gene expression was partially reduced by a dsRNAi construct, showed partial deficiency in peroxisome biogenesis and function, a smaller stature, and reduced fertility. Finally, a search of the GENEVESTIGATOR microarray database revealed similar expression patterns of the three RING peroxins in some tissues, supporting the essential roles of these PEX genes in seed development, germination, and flower formation.
The two classic and best-known functions for plant peroxisomes are (1) lipid mobilization through
Peroxisomal matrix proteins are mislocalized in the cytoplasm in yeast and animal cells with reduced function of PEX12, indicating that this protein is particularly required for protein import into peroxisomes (Chang et al., 1997
Electron microscopic analysis of an Arabidopsis pex10 null mutant demonstrated that, in addition to the lack of characteristic peroxisomes, the lethal embryos also contained small lipid bodies with a half-unit membrane studded with ribosomes and flat lipid body discs (Schumann et al., 2003
The mechanism for peroxisome biogenesis in various organisms shares a significant degree of conservation. All of the Arabidopsis PEX genes that have been cloned and characterized show some level of functional similarity with their yeast counterparts. For example, the PEX2, PEX10, PEX14, and PEX16 proteins all target to peroxisomes (Hayashi et al., 2000
Plant Material and Growth Conditions Arabidopsis (Arabidopsis thaliana) plants used in this study are of the Columbia background. Seeds were germinated on 1x Murashige and Skoog medium (Gibco), with or without 1% Suc, 20 µM IBA, and appropriate antibiotics, when necessary. Plants used for most experiments were grown with 16/8-h photoperiod under 80 to 100 µmol m–2 s–1 light conditions at 22°C. Plants used for bombardment in virus-induced gene silencing were grown in the same light intensity and temperature in short-day conditions with an 8/16-h photoperiod.
PCR was performed with genomic DNA using Taq DNA polymerase (Promega) and conditions suggested by the manufacturer. The primers were as follows: left border (LB) from the T-DNA vector, 5'-GCGTGGACCGCTTGCTGCAACT-3'; P12F from intron 5 and exon 6, 5'-ATGCCAAGATAGATGGATACATCCTCAAGG-3'; and P12R from exon 8, 5'-GGAGGGTACACTGTTGGAGCTGATAATCTC-3'. P12F and P12R amplify a 620-bp product from the wild-type allele. LB and P12R amplify an insertion-specific product of 730 bp. An approximately 110-bp piece of DNA of unknown origin was also found at the insertion site, between the T-DNA and the PEX12 genomic DNA.
A Leica MZ125 dissecting microscope (W. Nuhsbaum) was used to observe siliques and floral structures, and a Zeiss Axiophot microscope (Carl Zeiss) was used for Nomarski optics of seeds. For Nomarski optics, fresh siliques harvested 6 to 9 d after pollination were dissected. Developing seeds were treated with clearing solution containing chloral hydrate:water:glycerol (8:2:1, v/v/v) and cleared for 1 to 4 h at room temperature or overnight at 4°C. A Zeiss Axiophot microscope was used to visualize fluorescent proteins. For in vivo detection of YFP and CFP, leaf tissue was mounted in water and viewed with a YFP filter (excitation 500 ± 12.5 nm, emission 540 ± 20 nm) or a CFP filter (excitation 440 ± 10 nm, emission 480 ± 15 nm). Abnormal and normal seeds from the same silique (6–7 d after flowering) were fixed separately in 2.5% glutaraldehyde, 0.1 M phosphate buffer, pH 7.2, at room temperature for 2 h, followed by a secondary fixation in 1% (w/v) OsO4 in the same buffer. Samples were dehydrated in a graded series of acetone and embedded in Spurr's epoxy resin. Ultrathin sections (70–90 nm) were cut by a MT-X ultramicrotome, stained with 2% uranyl acetate and lead citrate, and observed under a JEM-100CX II transmission electron microscope (JEOL).
To make YFP-PTS1, a YFP-Ser-Lys-Leu fragment was amplified by PCR from the vector pEYFP-Peroxi (CLONTECH) and cloned into a vector derived from pPZP212 (Hajdukiewicz et al., 1994 To clone the PEX12 RNAi construct, the Arabidopsis vector pFGC5941 for dsRNA production was obtained from ABRC (stock no. CD3-447). A 247-bp fragment of PEX12 cDNA (position 364–611) was amplified by PCR using primers hpsipex12L (5'-GCTCTAGAGGCGCGCCGTTGTGTTACCGTATTTC-3') and hpsipex12R (5'-CGGGATCCATTTAAATTCACTTGATGCATGTATC-3'). The PCR product was digested with AscI and SwaI, and ligated into pFGC5941 to generate plasmid pFGC5941-PEX12a. The same PCR product was digested again with BamHI and XbaI, and ligated into pFGC5941-PEX12a to generate plasmid pFGC5941-PEX12. As a result, the pFGC5941-PEX12 plasmid has a copy of the 247-bp fragment inserted into AscI to SwaI sites, and an inverted repeat of the fragment inserted into BamHI to XbaI sites.
All PCR amplifications were carried out using Pfu DNA polymerase (Stratagene) and protocols suggested by the manufacturer. Agrobacterium-mediated transformation of Arabidopsis plants was performed using the floral dip method (Clough and Bent, 1998
A 247-bp fragment from PEX12 cDNA (position 364–611) was amplified by PCR using primers husi2L (5'-GCTCTAGAGTTGTGTTACCGTATTTC-3') and husi2R (5'-GAAGATCTTCACTTGATGCATGTATC-3'). The PCR product was digested with XbaI and BglII and cloned into the pCPCbLCV.007 vector (Turnage et al., 2002
A reverse copy of the 247-bp fragment was also generated by PCR using primers husi2aL (5'-GAAGATCTGTTGTGTTACCGTATTTC-3') and husi2aR (5'-GCTCTAGACTTCACTTGATGCATGTATC-3'). The PCR product was digested with XbaI and BglII and cloned into the pCPCbLCV.007 vector (Turnage et al., 2002
Arabidopsis plants in the YFP-PTS1 background were grown in individual pots in short-day conditions and bombarded with an equal amount of each silencing construct DNA (in CbLCV A) and the pCPCbLCV.008 DNA (CbLCV B) as described in a previous study (Turnage et al., 2002
Total RNA was extracted with TRIzol reagent (Invitrogen) and subjected to Reverse Transcription reaction (Gibco). The PEX12-specific primers PEX12F2 (5'-GCGAGATTGAGATTGAGGAAAGACAGTGCC-3') from exon 3 and PEX12R (5'-GGAGGGTACACTGTTGGAGCTGATAATCTC-3') from exon 8 amplify a 684-bp product from PEX12 cDNA. The ubiquitin-specific primers UBQ10-1 (5'-TCAATTCTCTCTACCGTGATCAAGATGCA-3') and UBQ10-2 (5'-GGTGTCAGAACTCTCCACCTCAAGAGTA-3') from the UBIQUITIN10 gene (At4g05320) amplify a cDNA product of approximately 320 bp. PCR conditions were as follows: 94°C for 3 min, followed by cycles (27 for Fig. 5 and 35 for Fig. 6) of 94°C for 45 s, 57°C for 45 s, 72°C for 1 min, and a final extension of 72°C for 7 min. Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers Q9M841, O00623, and Q01961.
We thank Drs. Andreas Weber and Gregg Howe for their comments on the manuscript, and Ms. Karen Bird for editing. We are also grateful to Renato Perez and Deb Lin for technical assistance, Dr. Dominique Robertson for the CbLCV vectors, Dr. Pablo Cerdan for the UBQ10 primers, and the Salk Institute and ABRC for the PEX12 T-DNA insertion lines and the dsRNA vector pFGC5941. Received June 7, 2005; returned for revision June 28, 2005; accepted July 13, 2005.
1 This work was supported by the U.S. Department of Energy and Michigan State University start-up funds to J.H., and by the National Science Foundation and the Howard Hughes Medical Institute (funding to J.C.). 
[w] The online version of this article contains Web-only data. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.066811. * Corresponding author; e-mail huji{at}msu.edu; fax 517–353–9168.
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