Free Access Two small protein families, DYNAMIN-RELATED PROTEIN3 and FISSION1, are required for peroxisome fission in Arabidopsis Xinchun Zhang, MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, MI 48824, USASearch for more papers by this authorJianping Hu, Corresponding Author MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, MI 48824, USA Plant Biology Department, Michigan State University, East Lansing, MI 48824, USA*For correspondence (fax +15173539168; e-mail huji@msu.edu).Search for more papers by this author Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URLShare a linkShare onEmailFacebookTwitterLinked InRedditWechat Summary Peroxisomes are multi-functional organelles that differ in size and abundance depending on the species, cell type, developmental stage, and metabolic and environmental conditions. The PEROXIN11 protein family and the DYNAMIN-RELATED PROTEIN3A (DRP3A) protein have been shown previously to play key roles in peroxisome division in Arabidopsis. To establish a mechanistic model of peroxisome division in plants, we employed forward and reverse genetic approaches to identify more proteins involved in this process. In this study, we identified three new components of the Arabidopsis peroxisome division apparatus: DRP3B, a homolog of DRP3A, and FISSION1A and 1B (FIS1A and 1B), two homologs of the yeast and mammalian FIS1 proteins that mediate the fission of peroxisomes and mitochondria by tethering the DRP proteins to the membrane. DRP3B is partially targeted to peroxisomes and causes defects in peroxisome fission when the gene function is disrupted. drp3A drp3B double mutants display stronger deficiencies than each single mutant parent with respect to peroxisome abundance, seedling establishment and plant growth, suggesting partial functional redundancy between DRP3A and DRP3B. In addition, FIS1A and FIS1B are each dual-targeted to peroxisomes and mitochondria; their mutants show growth inhibition and contain peroxisomes and mitochondria with incomplete fission, enlarged size and reduced number. Our results demonstrate that both DRP3 and FIS1 protein families contribute to peroxisome fission in Arabidopsis, and support the view that DRP and FIS1 orthologs are common components of the peroxisomal and mitochondrial division machineries in diverse eukaryotic species. Introduction Plant peroxisomes contain a wide array of metabolic activities, such as fatty acid β-oxidation, the glyoxylate cycle, photo-respiration, jasmonate biosynthesis, H2O2 detoxification, and metabolism of nitrogen and indole-butyric acid (Hayashi and Nishimura, 2003; Nyathi and Baker, 2006; Olsen and Harada, 1995; Reumann and Weber, 2006; Zolman etal., 2000). Peroxisomes can form de novo from the endoplasmic reticulum (ER) or arise from division/proliferation of pre-existing peroxisomes via multiple steps involving organelle elongation/enlargement, membrane constriction and peroxisome fission (Fagarasanu etal., 2007; Hoepfner etal., 2005; Motley and Hettema, 2007; Titorenko and Mullen, 2006). Peroxisome division (from one to at least two peroxisomes) takes place constitutively or under induced conditions; induced division (or peroxisome proliferation) is often considered to be the increase in peroxisome abundance/volume in response to environmental and metabolic stimuli (Yan etal., 2005). Several major components of the peroxisome division machineries are conserved in eukaryotes. For example, orthologs of the peroxisomal membrane protein PEROXIN11 (PEX11) in fungi, animals, trypanosomes and plants promote the first step of peroxisome division, namely peroxisome elongation/tubulation (Fagarasanu etal., 2007). Arabidopsis contains five PEX11 isoforms, PEX11a–e, all of which are targeted to peroxisome membranes and able to induce peroxisome elongation and population increase with various degrees of functional specificity and redundancy (Lingard and Trelease, 2006; Nito etal., 2007; Orth etal., 2007). Decreasing the expression of individual PEX11 genes or a subfamily of PEX11 genes led to a reduction in the total number of peroxisomes (Orth etal., 2007) or slightly enlarged peroxisomes (Nito etal., 2007). Arabidopsis plants overexpressing individual PEX11 genes displayed significant peroxisome tubulation and an increase in peroxisome abundance (Orth etal., 2007). Understanding of the precise mode of action for PEX11 proteins remains elusive; membrane modification through phospholipid binding, metabolite transport, and recruitment of downstream proteins are some of the proposed functions (Fagarasanu etal., 2007; Thoms and Erdmann, 2005). The second class of conserved constituents of the peroxisome division apparatus consists of dynamin-related proteins (DRPs), which mediate peroxisome fission after membrane constriction has occurred (Fagarasanu etal., 2007). Dynamin and dynamin-related proteins are large self-assembling GTPases that are involved in the fission and fusion of membranes by forming spiral-like structures around the membranes and acting as mechanochemical enzymes or signaling GTPases (Hoppins etal., 2007; Koch etal., 2004; Osteryoung and Nunnari, 2003; Praefcke and McMahon, 2004). DRPs share with the conventional dynamins an N-terminal GTPase domain, a middle domain (MD), and a C-terminal GTPase effector domain (GED), but lack the pleckstrin homology domain (PH) for binding to membrane lipids and the C-terminal proline- and arginine-rich domain (PRD) that mediates interactions with SH3 motif-containing proteins (Thoms and Erdmann, 2005). The Saccharomyces cerevisiae DRPs Vps1p and Dnm1p and the mammalian DLP1/Drp1 proteins are required for peroxisome division in addition to their roles in mitochondrial division (Dnm1p and DLP1/Drp1) and Golgi (DLP1/Drp1) and vacuole (Vps1p) morphogenesis (Hoepfner etal., 2001; Koch etal., 2003, 2004; Kuravi etal., 2006; Li and Gould, 2003; Schrader, 2006; Wilsbach and Payne, 1993). Of the 16-protein superfamily of dynamins and DRPs in Arabidopsis (Hong etal., 2003), family 3 consists of DRP3A and DRP3B, which share 77% amino acid sequence identity (Figure1a). Both proteins are involved in mitochondrial division, and DRP3A also controls the division of peroxisomes (Arimura and Tsutsumi, 2002; Arimura etal., 2004; Logan etal., 2004; Mano etal., 2004). Whether or not DRP3B functions in peroxisome fission is unclear. Sequence alignments of DRP3 (YLL001W) and FIS1 proteins (YIL065C).(a) Alignment of Arabidopsis DRP3A and DRP3B and the yeast Saccharomyces cerevisiae Dnm1p protein. The arrowhead shows the mutation in pdd1. Shaded residues are identical amino acid residues.(b) Alignment of Arabidopsis FIS1A and FIS1B and the S. cerevisiae Fis1p protein. Positions of the tetratricopeptide (TPR)-like domain in the three proteins are: Fis1p, 6–129; FIS1A, 36–142; FIS1B, 92–125. The putative transmembrane domain (TMD) is underlined. Shaded residues are identical amino acid residues. The third group of proteins with a conserved function in peroxisome division, at least in yeasts and mammals, is composed of the FISSION1 (FIS1) proteins. FIS1 orthologs are integral membrane proteins targeted to both peroxisomes and mitochondria, acting as adaptors for the mammalian DLP1 and yeast Dnm1 proteins by recruiting these DRPs to the organelles to perform membrane fission (Kobayashi etal., 2007; Koch etal., 2003, 2005; Kuravi etal., 2006). Structural features shared by FIS1 orthologs include a highly conserved C-terminal transmembrane domain (TMD) and a tetratricopeptide repeat (TPR)-like binding domain that spans over two-thirds of the protein from the N-terminus and mediates protein–protein interaction (Figure1b). Arabidopsis contains two homologs of FIS1 (FIS1A and FIS1B) that share 58% protein sequence identity (Figure1b). The Arabidopsis mutant bigyin (FIS1A) shows a reduced number of mitochondria and an increase in mitochondrial size (Scott etal., 2006). It remains to be determined whether these two Arabidopsis FIS1 proteins are involved in controlling the number and size of peroxisomes, and whether FIS1B is also required for mitochondrial division. We are interested in elucidating molecular pathways underlying the environmental and metabolic control of the abundance of plant peroxisomes, which will ultimately lead to answers to the question of how peroxisomal dynamics correlate with plant physiology and development. Transmission electron microscopic studies have demonstrated that plants increase their peroxisome numbers by mostly unknown mechanisms in response to environmental stresses such as ozone, the herbicide isoproturon, the hypolipidemic drug clofibrate, and high light conditions (de Felipe etal., 1988; Ferreira etal., 1989; Oksanen etal., 2003; Palma etal., 1991). We recently provided evidence that light induces the proliferation of peroxisomes in Arabidopsis seedlings through the far-red light receptor phytochrome A (phyA) and the bZIP transcription factor HY5 HOMOLOG (HYH), which coordinately activate expression of the PEX11b gene (Desai and Hu, 2008). To further unravel the molecular pathways governing the environmental control of plant peroxisome abundance, we need first to establish a precise mechanistic model for peroxisome division in plants. All players in the division machinery need to be identified, as some of them could be targets for regulation by plant peroxisome proliferators. To this end, we screened for mutants deficient in peroxisome division and performed reverse genetic studies to characterize plant orthologs of the yeast and mammalian proteins involved in peroxisome division. In this paper, we describe the role of DRP3B and the two FIS1 proteins in peroxisome division in Arabidopsis. The role of the FIS1 proteins in mitochondrial division is also described. To identify more players in the peroxisome division and proliferation pathways in Arabidopsis, we performed ethyl methane sulfonate (EMS) mutagenesis on seeds from the peroxisome marker plant YFP–PTS1, which expresses the yellow fluorescent protein with the Peroxisome-Targeting Signal Type1 (Ser-Lys-Leu) sequence attached to the C-terminus (Desai and Hu, 2008; Fan etal., 2005; Orth etal., 2007). Screening of the M2 population for peroxisome division/proliferation deficient (pdd) mutants enabled us to identify several classes of mutants showing changes in peroxisome size, shape or number from the wild-type plants (Figure2a). The pdd1 mutant exhibited highly aggregated/inseparable (Figure2b) and massively elongated (Figure S1) peroxisomes, phenotypes that are reminiscent of those of the aberrant peroxisome morphology 1 (apm1) mutants, which contain mutations in the DRP3A gene (Mano etal., 2004). Sequencing of the DRP3A gene in the pdd1 mutant revealed a nonsense mutation at Gln319 (Figures1a and 2i). Peroxisome phenotypes of the drp3 mutants.(a–h) Confocal micrographs of leaf mesophyll cells from wild-type and drp3 mutant plants expressing the YFP–PTS1 peroxisomal marker gene and grown for 4 weeks. Green signals indicate YFP–PTS1-labelled peroxisomes; red signals indicate chloroplasts. Scale bars = 10 μm. Insets show RT-PCR analyses of RNA from corresponding mutants. In each inset, the left lane is wild-type and the right lane is the mutant; the top panel represents transcripts of the individual DRP3 gene and bottom panel shows the UBIQUITIN10 transcript.(i) Genomic structures of DRP3A and DRP3B. Boxes represent exons, with the coding region in black. Positions of the mutant alleles are also indicated.(j) Quantification of total YFP fluorescence and peroxisome number per 2500 μm2 of the mesophyll cells in drp3 mutants (n   8, P   0.05 ). Phylogenetic analyses of DRP sequences from various species suggested that DRP3A and DRP3B are more closely related to yeast Vps1p and Dnm1p and human Drp1 than to members of the other Arabidopsis DRP families (Arimura and Tsutsumi, 2002; Konopka and Bednarek, 2008). Searches of public Arabidopsis microarray databases (http://www.genevestigator.ethz.ch/; Zimmermann etal., 2004) showed that DRP3A and DRP3B are both fairly ubiquitously expressed (Figure S2). Given the roles of DRP3A, Vps1p, Dnm1p and Drp1 in peroxisome division, it is highly likely that DRP3B is also involved in the same process in Arabidopsis. To test this hypothesis, we obtained two T-DNA insertion mutants of DRP3B, drp3B-1 (SALK_045316) and drp3B-2 (SALK_112233), as well as two additional mutant alleles of DRP3A, drp3A-1 (SALK_008706) and drp3A-2 (SALK_147485) (Figure2i). Semi-quantitative RT-PCR of RNA from the mutants provided evidence that expression of DRP3A or DRP3B was strongly reduced in the respective mutants (insets in Figure2c–f). The YFP–PTS1 peroxisomal marker gene was introduced into these mutants via Agrobacterium-mediated transformation (Clough and Bent, 1998); transgenic plants were analyzed for peroxisome phenotypes. Both drp3B mutant alleles expressing YFP–PTS1 contained many peroxisome aggregates that were constricted but for which fission failed (Figure2e,f). These phenotypes were comparable to some extent with those observed in pdd1, drp3A-1 and drp3A-2 (Figure2b–d). However, the long and extended tails associated with peroxisomes that were frequently seen in the drp3A alleles (Figure2b–d) and named by Scott etal. (2007) as ‘peroxules’ were not observed in the drp3B mutants (Figure2e,f). Most of the individual peroxisomes in the drp3A and drp3B mutants were larger than those in the wild-type (Figure2b–f). Thus, DRP3B, like its homolog DRP3A, is required for peroxisome division. However, DRP3B’s role may be weaker than that of DRP3A. Single mutants of DRP3A and DRP3B each showed deficiency in peroxisome division, suggesting that the functions of DRP3A and DRP3B may not be completely overlapping. To further address this question, we generated drp3A drp3B double mutants. For convenience in genotyping, the two T-DNA insertion lines of DRP3A (instead of the EMS mutant pdd1) were used to perform crosses. Two double mutants were obtained: drp3A-1 drp3B-1 and drp3A-2 drp3B-2. Similar to the single mutants, the double mutants (F2) also contained many clumped peroxisomes that were each slightly larger than those of the wild-type (Figure2g,h). However, these morphological defects of peroxisomes in the double mutants were not stronger than those of the single mutants. To determine whether the total number of peroxisomes was changed in these mutants, we used ImageJ software to quantify peroxisome abundance. We measured the planar area of YFP fluorescence and the number of peroxisomes in a given field in leaf mesophyll cells using information extrapolated from at least eight confocal images from each genotype. Whereas the total area of fluorescence per 2500 μm2 remained virtually unchanged between wild-type and drp3 single mutants, it was decreased in the two drp3A drp3B double mutants (Figure2j). Compared with the wild-type, the number of peroxisomes per 2500 μm2 was noticeably decreased in the single mutants; the double mutants contained the lowest number of peroxisomes (Figure2j). Of the two double mutants, drp3A-1 drp3B-1 has a slightly weaker phenotype, probably due to the fact that a small amount of DRP3A mRNA was detected in drp3A-1 (Figure2c). At the seedling stage, pdd1, drp3A-1 and drp3A-2 grew more slowly than the wild-type YFP–PTS1 control plants, with pdd1 exhibiting the strongest growth inhibition (Figure3a). It seems that, despite our inability to detect DRP3A transcripts in drp3A-2, it is not a null mutant. The drp3B mutant alleles showed comparable phenotypes to those of drp3A. The two double mutants displayed stronger defects in plant size than the single mutants (Figure3a) and were slightly pale green at the seedling stage (data not shown). Adult plants of the wild-type and single mutants were largely undistinguishable in appearance, but double mutants showed reduced plant size (Figure3b). The pale-green phenotype reflects defects in photo-respiration, a pathway in which peroxisomes and mitochondria play essential roles. Reduced division of both peroxisomes and mitochondria in the drp3 mutants obviously reduced plant growth. Growth and germination phenotypes of the drp3 mutants.(a, b) Wild-type and drp3 mutants grown for 3 (a) and 7 weeks (b).(c) Sucrose-dependence assay of drp3 mutants. Hypocotyl lengths of dark-grown seedlings grown for 5 days on MS plates with or without 1% sucrose were measured. Error bars indicate standard deviations (n  P   0.05 ). To determine whether disruption of the DRP3 genes led to impaired seedling establishment, we measured the hypocotyl lengths of dark-grown seedlings germinated in the presence or absence of sucrose. The pex14 null mutant, which is defective in a peroxisome biogenesis factor involved in peroxisomal matrix protein import and has a sugar-dependent phenotype (Fan etal., 2005; Orth etal., 2007), was used as a control (Figure3c). On sucrose-free medium, hypocotyl elongation was more inhibited in the drp3A drp3B seedlings than in the single mutants and the wild-type; this deficiency was largely rescued by exogenous sucrose (Figure3c). It is likely that the drp3 mutants are defective in lipid metabolism as a result of reduced division of peroxisomes and mitochondria, two key locations of this physiological process. Hence, insufficient energy in the form of carbohydrates was available for the seedlings to become established. Collectively, results from the sugar-dependence assays and the peroxisome and plant growth phenotype analyses of the drp3 mutants provide evidence that DRP3A and DRP3B mediate peroxisome division in a partially redundant manner. Given that DRP3B clearly plays a role in peroxisome division, we next sought to determine whether this protein is indeed sorted to peroxisomes. Full-length cDNA encoding DRP3B (At2g14120) was inserted to the C-terminus of YFP in a plant expression vector driven by the CaMV 35 S promoter. This construct was co-expressed with CFP–PTS1 (cyan fluorescent protein linked to PTS1) in Arabidopsis. Transgenic plants expressing both transgenes exhibited many YFP signals that were tightly associated with small circular structures labeled with CFP–PTS1 (Figure4a). Likewise, many YFP–DRP3B proteins were also associated with mitochondria, which were stained using MitoTracker (Figure4b). Subcellular localization of DRP3B.Confocal images were taken from leaf epidermal cells of 4-week-old plants co-expressing CFP–PTS1 and YFP–DRP3B. Scale bars = 10 μm.(a) Association of YFP–DRP3B (green signals) with some CFP–PTS1-labelled peroxisomes (red signals).(b) Association of YFP–DRP3B (green signals) with some MitoTracker-stained mitochondria (magenta signals). Rather than being distributed throughout these organelles, the YFP–DRP3B protein was targeted to spots on peroxisomes and mitochondria or showed juxtaposition to these compartments (Figure4). A similar localization pattern was previously shown for DRP3A and DRP3B on mitochondria (Arimura and Tsutsumi, 2002; Arimura etal., 2004) and for DRP3A on peroxisomes (Mano etal., 2004); the locations of these spots were suggested to be possible sites for membrane constriction (Arimura and Tsutsumi, 2002; Arimura etal., 2004). Taken together, our data demonstrate that the DRP3B protein is partially localized to peroxisomes in addition to targeting to mitochondria. In yeast and mammals, DRP proteins are tethered to the membrane of peroxisomes and mitochondria by the small membrane-anchored protein FIS1 before participatation in division by pinching off small organelles from tubules that are already constricted (Kobayashi etal., 2007; Koch etal., 2003, 2005; Kuravi etal., 2006). Data collected from online microarray databases (http://www.genevestigator.ethz.ch/; Zimmermann etal., 2004) revealed that both FIS1A and FIS1B from Arabidopsis are constitutively expressed. The expression level of FIS1A is higher than that of FIS1B in most tissues, but FIS1B shows very high expression in pollen (Figure S2). FIS1A (BIGYIN) was previously shown to control the size and number of mitochondria (Scott etal., 2006). However, whether FIS1B plays a role in mitochondrial division and whether these two FIS1 proteins are targeted to mitochondria have not been clearly demonstrated. Here, we characterized Arabidopsis FIS1A and FIS1B to determine whether they play a role in the division of both peroxisomes and mitochondria. The subcellular localization of these two proteins was determined. We transformed 35 S promoter-driven constructs containing YFP–FIS1 or FIS1–YFP into Arabidopsis plants that were already expressing the peroxisomal marker protein CFP–PTS1. Transgenic plants expressing YFP–FIS1A or YFP–FIS1B fusions displayed partial co-localization of the YFP signals with CFP–PTS1 (Figure5a,b). Unlike DRP3B, which was concentrated at spots on the peroxisome (Figure4), the FIS1 proteins were evenly distributed along peroxisomes (Figure5a,b). In contrast, when fused to the N-terminus of YFP, FIS1A and FIS1B were mostly diffused in the cytosol and nucleus (Figure5c,d), indicating that the C-terminus of FIS1, which contains the transmembrane domain, is important for targeting FIS1 to the peroxisome. These data suggest that Arabidopsis FIS1A and FIS1B are partially targeted to peroxisomes, and that the C-terminus of the proteins is required for this targeting. We also used MitoTracker dye to stain leaf epidermal cells from transgenic plants expressing YFP–FIS1A or YFP–FIS1B. Confocal microscopy showed that some of the YFP–FIS1A and YFP–FIS1B fusion proteins clearly co-localized with MitoTracker (Figure6a,b), thus validating the partial mitochondrial localization of these two proteins. Peroxisome targeting of the FIS1 proteins.Confocal images were taken from leaf epidermal cells of 4-week old plants expressing CFP–PTS1 combined with YFP–FIS1 or FIS1-YFP, as indicated at the top. Each inset is an immunoblot analysis of proteins extracted from wild-type plants expressing CFP–PTS1 only (left lane) and plants co-expressing CFP–PTS1 and the indicated FIS1 construct (right lane). The α-GFP antiserum detected CFP–PTS1 (bottom band) and the FIS1–YFP (or YFP–FIS1) fusion proteins (top band). Scale bars = 10 μm.Co-localization of YFP–FIS1 fusion proteins with mitochondria.Images were captured from epidermal cells of 6-week-old leaves from the YFP–FIS1 transgenic plants stained by MitoTracker. Scale bars = 10 μm. We obtained loss-of-function mutants to further examine the role of FIS1 proteins in the division of peroxisomes and mitochondria. The fis1A mutant (SALK_086794) has a T-DNA insertion in the last exon (Figure7a) and is the same allele (bigyin1-2) used by Scott etal. (2006) for mitochondrial phenotype analysis. Using RT-PCR analysis, we were unable to detect FIS1A transcripts in this mutant (Figure7b), which was later crossed into the YFP–PTS1 background. As T-DNA insertion lines for FIS1B were not available, we used RNAi to reduce the expression of this gene. The full-length cDNA of FIS1b (513 bp) was cloned into the pFGC5941 dsRNAi vector as inverted repeats; the 35 S-driven construct was later transformed into plants expressing YFP–PTS1. We generated a total of 59 T1 transformants that contained both sense and antisense repeats of FIS1B, seven of which were randomly chosen for RT-PCR analysis. Two RNAi lines, R15 and R47, which showed silencing of FIS1B but wild-type levels of FIS1A mRNA (Figure7c), were selected for further analysis in T3. The fis1A T-DNA insertion mutant and the FIS1B RNAi lines both displayed growth inhibition compared with the wild-type plant (Figure7d). Growth phenotype of the fis1 mutants.(a) FIS1A gene. Boxes indicate exons; the coding region is in black. The arrowhead indicates the position of the T-DNA insertion in the fis1A mutant.(b, c) RT-PCR analysis of FIS1A, FIS1B and UBIQUITIN10 transcripts in wild-type, fis1A and the two FIS1B RNAi lines.(d) Growth comparison of 6-week-old fis1 mutants and wild-type plants. Two plants were grown in each pot. Confocal microscopic analysis of peroxisomes in the mesophyll cells of rosette leaves demonstrated that these mutants contained many enlarged peroxisomes, some of which were clustered together and showed failed fission, in contrast to the mostly spherical and separated peroxisomes in the wild-type plants (Figure8a–d). Quantification of YFP fluorescence area and peroxisome abundance (per 2500 μm2) from over eight images from each genotype revealed that, whereas the total volume of peroxisomes (measured by YFP fluorescence area in the given field) remained largely constant, the number of peroxisomes was significantly reduced in the fis1 mutants (Figure8i). The fis1A mutant (bigyin-2, SALK_086794) used in this study was shown previously to contain enlarged mitochondria as well as having a reduced mitochondrial number per cell (Scott etal., 2006). When stained with MitoTracker, the two FIS1B RNAi lines (R15 and R47) also showed many mitochondria that were enlarged in size and decreased in number, similar to the fis1A mutant (Figure8e–h). The MitoTracker fluorescence area per 2500 μm2 remained constant between wild-type and the single fis1 mutants, but the total number of mitochondria strongly decreased in the mutant lines (Figure8i). Sugar-dependence assays were also performed on the fis1 mutants. Both fis1A and the two FIS1B RNAi lines showed partial growth inhibition on sucrose-free medium (Figure8j), indicating weak deficiencies in lipid metabolism during germination. Taken together, our results show that FIS1A and FIS1B are targeted to both peroxisomes and mitochondria and are required for the division of both types of organelle in Arabidopsis. Peroxisomal and mitochondrial phenotypes of the fis1 mutants.(a–h) Confocal micrographs of leaf mesophyll (a–d) or leaf epidermal (e–h) cells from 6-week-old wild-type and fis1 mutant plants, all of which contained the YFP–PTS1 peroxisomal marker gene. Green signals, YFP–PTS1-tagged peroxisomes; red signals, autofluorescent chloroplasts; magenta signals, MitoTracker-stained mitochondria. Scale bars = 10 μm.(i) Quantification of total peroxisome (YFP) and mitochondrial (MitoTracker) fluorescence and the number of these two types of organelles within 2500 μm2 of leaf cells in wild-type and fis1 mutants (n   8, P   0.05). Error bars indicate standard deviations.(j) Sucrose-dependence assays of the fis1 mutants. Hypocotyl lengths of 5-day-old etiolated seedlings grown on MS plates with or without 1% sucrose were measured (n  P   0.05 ). Error bars indicate standard deviations. In Arabidopsis, the PEX11 protein family and the DRP3A protein have been shown to be involved in peroxisome division (Lingard and Trelease, 2006; Mano etal., 2004; Nito etal., 2007; Orth etal., 2007). In this study, we identified three additional components of the Arabidopsis peroxisome division apparatus – DRP3B, FIS1A and FIS1B. Whereas PEX11 proteins are primarily responsible for the elongation/tubulation of peroxisomes, DRP3A/3B and FIS1A/1B proteins mediate the fission of peroxisomes. We have provided genetic evidence that DRP3A and DRP3B play partially redundant roles in peroxisome division, seedling establishment and plant growth. First, some of the YFP–DRP3B proteins were found to be associated with spots on peroxisomes, similar to what was discovered for DRP3A (Mano etal., 2004). Second, single and double mutants of DRP3A and DRP3B were impaired in peroxisome division, and the dry3A drp3B double mutants showed stronger phenotypes than either single mutant parent with respect to peroxisome number, sugar dependence, and plant stature and pigmentation. The degree of dwarfness in the DRP3A null allele pdd1 shown in this study was weaker than that of apm1-6, the strongest mutant allele of DRP3A identified from a previous study, which contained a Gly71→Asp substitution (Mano etal., 2004). This phenotypic difference implies that the truncated DRP3A protein encoded by pdd1 may still be partially functional, whereas mutation of the N-terminal GTPase domain in apm1-6 may have completely abolished the function of this protein. Alternatively, the mutant protein encoded by the apm1-6 allele may have a dominant negative effect by disrupting the function of both endogenous DRP3 proteins and possibly other DRPs that play a role in the division of peroxisomes and mitochondria. It will be necessary to obtain a mutant in which both DRP3 proteins are completely non-functional in order to determine the full capacity of this subfamily of DRPs in peroxisome division and plant development. Furthermore, given that the drp3A and drp3B single mutants each displayed apparent morphological deficiencies in peroxisomes, each gene must contain some unique functions in peroxisome division. The slightly different peroxisomal phenotypes of the drp3A and drp3B mutants shown in this study provide support for this prediction. Dynamins and dynamin-related proteins are engaged in endocytosis, cell division and expansion, intracellular vesicle trafficking, and division of organelles such as plastids, mitochondria, peroxisomes and Golgi vesicles (Osteryoung and Nunnari, 2003; Praefcke and McMahon, 2004). The complete functional spectra of many of the Arabidopsis DRPs have not been characterized. Members from different DRP subfamilies may be involved in the same function. For example, in addition to DRP3A and DRP3B (Arimura and Tsutsumi, 2002b; Arimura etal., 2004; Logan etal., 2004; Mano etal., 2004), two members of family 1 were also shown to participate in mitochondrial division. Mutants of DRP1C (ADL1C) and DRP1E (ADL1E) exhibited abnormal mitochondrial elongation; the two proteins also partially co-localized with a mitochondrial marker (Jin etal., 2003). Thus, it is likely that other Arabidopsis DRP subfamilies are also involved in peroxisome division. In addition, the same DRP may participate in the fission of multiple types of membranes. Such examples include yeast Vps1p protein (vacuolar protein sorting protein 1), which plays a role in the division of peroxisomes and biogenesis of vacuoles, and mammalian DLP1 protein, which participates in the fission of peroxisomes, mitochondria and Golgi bodies (Hoepfner etal., 2001; Koch etal., 2003; Li and Gould, 2003). Therefore, despite the finding that DRP3A is involved in the division of only peroxisomes and mitochondria (Mano etal., 2004), we cannot completely exclude the possibility that DRP3B is also targeted to other subcellular compartments and contributes to the division or morphogenesis of other organelles. Peroxisomes and mitochondria both move fast. Thus, we were unable to clearly address the question of whether or not YFP–DRP3B targets to spherical structures other than peroxisomes and mitochondria by visualizing YFP–DRP3B, peroxisomes and mitochondria simultaneously in a single image. We also show in this study that the two Arabidopsis FIS1 homologs, FIS1A and FIS1B, are targeted to both peroxisomes and mitochondria and play significant roles in the division of these organelles. FIS1 is one of very few proteins known to target to the membrane of both peroxisomes and mitochondria. The C-terminus appears to be critical for targeting of FIS1A and FIS1B to peroxisomes (Figure5) in Arabidopsis, consistent with the finding that the C-terminal region of human FIS1 (including the transmembrane domain) is both necessary and sufficient for targeting to both peroxisomes and mitochondria in human cells (Koch etal., 2005). An open question remains as to how targeting signals are specified within the C-terminus of the FIS1 protein, and which organelle-specific proteins mediate these targeting events. Among the three essential components of the mitochondrial division machinery in yeast, namely Dnm1p, Fis1p and Mdv1p (or its homolog Cav4p), Mdv1p/Cav4p (molecular adaptor) appears to be species-specific and does not have apparent structural orthologs in higher eukaryotes (Hoppins etal., 2007). It is possible that some unidentified proteins exclusively localized to peroxisomes mediate the specific targeting of FIS1 to peroxisomes in Arabidopsis and in other eukaryotes as well. Our study shows that, despite deficiencies in peroxisome fission, the peroxisomal volume (indicated by fluorescent areas) in the drp3 and fis1 single mutants is largely unchanged from that of the wild-type. This compensation for the reduced number of peroxisomes by enlarged individual peroxisomes may be a mechanism utilized by the cell to maintain a sufficient volume of organelles in order to carry out their normal function. However, when both members of the gene family are dysfunctional, as in the case for the drp3 double mutants, this balance was lost. We expect to see a similar trend in fis1 double mutants. In addition to partial redundancy of function, we also expect to see a unique function for each FIS1. For example, it is possible that FIS1A and FIS1B each have a specific DRP target. Whereas ectopic expression of Arabidopsis DRP3 genes (this study and that by Mano etal., 2004) or the human DLP1 gene (Li and Gould, 2003) did not cause any apparent peroxisome phenotypes, overexpressing YFP–FIS1 fusion proteins seems to lead to some degree of increased proliferation and clustering of peroxisomes (Figure5a,b versus c,d). Over-producing Myc–hFIS1 in mammalian cells led to more numerous peroxisomes and segmented mitochondria, suggesting that FIS1 is the limiting factor for peroxisomal and mitochondrial fission (Koch etal., 2005). To address this question in Arabidopsis, we will need to express untagged FIS1 or FIS fused to small tags to avoid possible dominant negative effects caused by attaching the 27 kDa YFP protein to wild-type FIS1. A very recent study using Arabidopsis suspension cell cultures failed to show co-localization of Myc –FIS1A or Myc–FIS1B with peroxisomes that were immunolabeled with α-catalase antibodies; however, an increase in the number of peroxisomes was observed in cells expressing Myc–FIS1B (Lingard etal., 2008). Their study also demonstrated that FIS1B has a role in cell-cycle-associated peroxisome duplication and was targeted to peroxisomes only after co-expression with a PEX11 protein, whereas FIS1A does not seem to be involved in peroxisome duplication (Lingard etal., 2008). In contrast, our study clearly demonstrates that, on their own, both YFP–FIS1A and YFP–FIS1B are able to localize to peroxisomes and mitochondria, and that both proteins are involved in peroxisome fission in Arabidopsis plants. GFP and myc fusions of the mammalian FIS1 protein (hFis1) are also targeted to both mitochondria and peroxisomes when expressed by themselves (Koch etal., 2005). It is possible that the roles of FIS1A and FIS1B in cell-cycle-associated peroxisome division in cell cultures differ from their role in peroxisomal division in intact Arabidopsis plants. Despite their distinct evolutionary origins (endosymbiotic versus ER-derived) and different membrane structures (double membrane versus single membrane), mitochondria and peroxisomes share some of the same DRPs and anchor proteins in the division machinery across plant, fungal and animal kingdoms (this study; Schrader, 2006; Schrader and Yoon, 2007). However, given that peroxisomes and mitochondria are functionally linked with respect to many metabolic activities, such as lipid metabolism and photo-respiration (two of the major functions involving plant peroxisomes), it is not surprising that division of these two organelles is coordinated at some level. The peroxisome phenotypes of fis1a and the FIS1B RNAi mutants are similar to those of the drp3A and drp3B mutants, consistent with the notion that FIS1 and DRP3 proteins work closely in the same pathway. In mammalian cells, hFIS1 and DLP1 physically interact in vivo and in vitro (Yoon etal., 2003). However, our co-immunoprecipitation (co-IP) assays using HA–FIS1 and YFP–DRP3 proteins failed to show the co-existence of DRP3A/3B and FIS1A/1B in the same protein complex (data not shown). Using bimolecular fluorescence complementation (BiFC), Lingard etal. (2008) did not detect interaction between DRP3A and FIS1A/FIS1B in Arabidopsis cultured cells. Thus, it is possible that the interaction between FIS1 and DRP in Arabidopsis is rather transient; alternatively, other proteins may bring DRP3 to FIS1 at the organelle membrane, which would represent a unique feature of plant peroxisomal/mitochondrial fission. PEX11, DRP and FIS1 represent conserved members of the peroxisome division machineries. Recently, ternary complexes containing mammalian DLP1, FIS1 and PEX11β were identified through chemical linking methods; this result links the machineries controlling peroxisome elongation and fission together, suggesting that these three groups of proteins show coordinated functions in peroxisome multiplication (Kobayashi etal., 2007). Recently, a novel tail-anchored membrane protein, Mff, was identified from mammalian cells; this protein promotes the fission of both mitochondria and peroxisomes independently of the FIS1 protein and does not have an obvious homolog in yeast (Gandre-Babbe and van der Bliek, 2008). It is unclear whether a plant homolog of Mff exists. In addition, a number of yeast peroxisomal membrane proteins, such as Pex28p, Pex29p, Pex30p, Pex31p and Pex32p, which are known to be specifically involved in controlling peroxisome size and abundance by means of largely unknown mechanisms (Thoms and Erdmann, 2005), do not seem to have apparent orthologs in plants. Proteins that mediate peroxisomal membrane constriction are largely unidentified in any species. As such, further genetic and biochemical studies are required to reveal plant- and peroxisome-specific players in peroxisome division. All plants were in the Columbia-0 (Col-0) background and were germinated under 16 h light (60 μE m−2 sec−1)/8 h dark conditions on 0.6% w/v agar plates with half-strength Murashige and Skoog basal salt mixture (½MS) supplemented with 1% w/v sucrose. After 2 weeks, plants were transferred to soil and grown under a photosynthetic photon flux density of 70–80 μmol m−2 sec−1 at 21°C with a 14 h light/10 h dark period. Wild-type plants expressing the CFP–PTS1 or YFP–PTS1 transgene (Desai and Hu, 2008; Fan etal., 2005; Orth etal., 2007) were used to visualize peroxisomes in plants. DNA fragments used for cloning in this study were amplified by PCR using High-Phusion DNA polymerase according to the manufacturer’s instructions (New England Biolabs Inc., http://www.neb.com). A standard Gateway cloning system (Invitrogen, http://www.invitrogen.com/) was used to produce the constructs. The Gateway-compatible PCR products of DRP3B, FIS1A and FIS1B were cloned into binary vectors containing the attR1-Cmr-ccdB-attR2 integration region using the One-Tube Format Protocol as described by the manufacturer. Constructs and primers used for Gateway cloning are listed in Table S1. The resulting constructs were transformed into A. tumefaciens (C58C1) via electroporation. Agrobacteria containing the constructs were later transformed into CFP–PTS1 or YFP–PTS1 plants using the floral-dip method (Clough and Bent, 1998). Stable primary transformants were selected on ½MS medium containing kanamycin (50 μg ml−1) for DRP3B–YFP and FIS1–YFP, glufosinate ammonium (10 μg ml−1; Crescent Chemicals, http://www.crescentchemical.com) for YFP–FIS1, or gentamycin (60 μg ml−1) for CFP–PTS1, and then transferred to soil for further characterization. drp3A-1 (SALK_008706), drp3A-2 (SALK_147485), drp3B-1 (SALK_045316), drp3B-2 (SALK_112233) and fis1A (SALK_086794) seeds were obtained from the Arabidopsis Biological Resource Center (Ohio State University). Homozygous mutants were identified by PCR analysis of genomic DNA using gene-specific forward (LP) and T-DNA left border primers (LBb1, 5′-GCGTGGACCGCTTGCTGCAACT-3′) and a gene-specific reverse primer (RP). PCR products were further sequenced to confirm insertion of the T-DNA into the gene. The primers for genotyping are shown in Table S1. YFP–PTS1 was expressed in the drp3A-1, drp3A-2, drp3B-1, drp3B-2 and fis1A mutants to visualize peroxisomes. The double mutants drp3A-1 drp3B-1 and drp3A-2 drp3B-2 were identified by genotyping of an F2 generation from crosses between the single mutants. Gene-specific primers (listed in Table S1) were used to amplify a 513 bp full-length cDNA fragment of FIS1B. The amplified fragment was cloned into pFGC5941 in sense and antisense orientations as described previously (Orth etal., 2007). The FIS1B RNAi construct was transformed into YFP–PTS1 plants, and T1 plants were screened on ½MS agar plates containing 50 μg ml−1 kanamycin and 10 μg ml−1 glufosinate ammonium. To make sure that both sense and antisense repeats of FIS1B are present, we genotyped T1 primary transformants using primers upstream (forward) and downstream (reverse) of the insertion sites (Table S1). Seeds of wild-type and mutants were plated on ½MS agar growth medium with or without 1% sucrose, following 4 days of cold treatment. All seeds were allowed to germinate and grow in the dark for 5 days. Five-day-old etiolated seedlings were scanned using an EPSON scanner (http://www.epson.com). Hypocotyl length was then measured using ImageJ (http://rsb.info.nih.gov/ij/). More than 50 seedlings of each genotype were used for hypocotyl length measurements (P   0.05). Total RNA was extracted using an RNeasy plant mini kit (Qiagen, http://www.qiagen.com/) according to the manufacturer’s protocol. First-strand cDNA synthesis was performed using Superscript II reverse transcriptase (Invitrogen) in a 20 μl standard reaction containing oligo(dT) primers. PCR amplification was performed using primers specific for DRP3A (At4g33650), DRP3B (At2g14120), FIS1A (At3g57090), FIS1B (At5g12390) and UBQ10 (At4g05320) genes (Table S1). PCR (Promega, http://www.promega.com) parameters were: 95°C for 2 min, followed by 26 cycles of 95°C for 30 sec, 54°C for 30 sec and 72°C for 1 min, and a final elongation step at 72°C for 10 min. Amplified DNA was run on a 0.8% agarose gel. Total protein was extracted from leaf discs of 4-week-old plants. Homogenized leaf tissue was placed in 1× SDS–PAGE sample buffer, boiled for 5 min, and centrifuged for 2 min at 13 000 g. The supernatant was run on SDS–PAGE gels and transferred to Immobilon-P membrane (Millipore Corp., http://www.millipore.com) for blotting. The primary antibody used to detect YFP and CFP proteins was a rabbit polyclonal GFP antibody (Santa Cruz Biotechnology, Inc., http://www.scbt.com). The secondary antibody used was goat anti-rabbit IgG (LI-COR Biosciences, http://www.licor.com). For in vivo detection of CFP and YFP, Arabidopsis tissue was mounted in water and viewed using a confocal laser scanning microscope (Zeiss Meta 510, http://www.zeiss.com/) to obtain confocal images of fluorescence proteins. To analyze subcellular localizations of FIS1A and FIS1B in mitochondria, leaves were treated with 500 nm MitoTracker Red CMXRos (Invitrogen) according to the method described by Arimura and Tsutsumi (2002). We used 458, 514, 543 and 633 nm lasers for excitation of CFP, YFP, MitoTracker and chlorophyll, respectively. For emission, we used 465–510 nm band-pass (CFP), 520–555 band-pass (YFP), 560–614 band-pass (Mitotracker) and 650 nm long-pass (chlorophyll) filters. All images were obtained from single optical sections of 0.14 μm in depth. We used ImageJ (http://rsb.info.nih.gov/ij/) to measure the fluorescence area and count organelle numbers in 50 μm × 50 μm confocal images. Color confocal images from single channels (YFP or MitoTracker) were converted to 8-bit grayscale. The scale for measurement was based on scale bars on the confocal images. We used manual settings of the threshold function to designate black pixels (peroxisomes or mitochondria) as objects to be measured or counted, and then the analyze particles function for fluorescence area measurements and organelle counting. Organelles that were clumped together without clear boundaries between, which probably indicates incomplete fission, were treated as a single organelle. The counting of the organelles was validated manually. Standard deviations and statistical significance for the data were calculated using the Excel program (Microsoft). At least eight images were used for all organelle counting and fluorescence measurements (P   0.05). We would like to thank the Arabidopsis Biological Resource Center (Ohio State University) for providing mutant seeds and the RNAi vector, Sarah Jacquart for assistance with mutant genotyping, Dr Melinda Frame for help with confocal microscopy, Marlene Cameron for graphic assistance, and Karen Bird for manuscript editing. This work was supported by the US Department of Energy, Michigan State University Intramural Research Grant Program (IRGP), and a grant from the National Science Foundation (MCB 0618335) to J.H.peroxisomes in the pdd1 mutant root cell. Green signals are YFP-PTS1-labelled peroxisomes. Scalebar=20μm. FigureS2.Expressionpatterns of the Arabidopsis DRP3 and FIS1genes. Expression is displayed as a signal expression value assigned byGENEVESTIGATOR (http://www.genevestigator.ethz.ch; (Zimmermannet al., 2004), where data used for the analysis weregathered. TableS3.Primers used inthis study. 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