The apem1 (previously known as apm1) mutant exhibits elongated and a reduced number of peroxisomes in a variety of cells throughout the plant (Figure 2). Mitochondria are also elongated, but other organelles such as chloroplasts, nuclei, the Golgi apparatus, and the endoplasmic reticulum (ER) are not. The APEM1 gene encodes DYNAMIN-RELATED PROTEIN 3A (DRP3A), a member of the dynamin superfamily that has a pivotal role in vesicle division and organelle fission and fusion (Mano et al., 2004; Praefcke and McMahon, 2004). In addition to DRP3A, its closest homolog, DRP3B, is also involved in peroxisome and mitochondria fission, and plant- and alga-specific DRP5B affects peroxisome, chloroplast, and mitochondria fission (Fujimoto et al., 2009; Aung and Hu, 2012). Interestingly, forward genetic screening isolated a number of independent lines possessing mutations at the DRP3A locus (Mano et al., 2004; Praefcke and McMahon, 2004), but not the DRP3B or DRP5B locus (Aung and Hu, 2012). Various experimental data indicate that DRP3A is the primary protein responsible for peroxisome fission (Fujimoto et al., 2009; Zhang and Hu, 2009; Aung and Hu, 2012).

In the apem2 (previously known as apm2) mutant, GFP-PTS1 protein is located in the cytosol as well as in peroxisomes (Figure 2). The APEM2 gene encodes the integral peroxisomal membrane protein PEX13 (Mano et al., 2006). Peroxisomal matrix proteins are transported to peroxisomes by their receptor PEX5 or PEX7, and translocate into the peroxisomal matrix through the pore formed by the receptor and the docking complex consisting of PEX14 and PEX13 (and PEX17 in fungi) on the peroxisomal membrane. The apem2 mutation introduces a stop codon at position 263 instead of glutamine in the C-terminal region. The C-terminus of PEX13 interacts with PEX14 and PEX5 via the Src homology 3 (SH3) domain in fungi (Bottger et al., 2000; Douangamath et al., 2002). However, A. thaliana PEX13 lacks an obvious SH3 domain, and a yeast two-hybrid assay showed that PEX13 interacts with PEX7, but not PEX14 or PEX5 (Mano et al., 2006; Boisson-Dernier et al., 2008). Another group reported a different pex13 mutation in which Glu is substituted by Lys only 20 amino acids upstream of the apem2 mutation (Woodward et al., 2014), supporting the importance of the C-terminal region of plant PEX13. Boisson-Dernier et al. (2008) isolated the A. thaliana abstinence by mutual consent (amc) mutant, which disrupts male-female gametophyte recognition (Boisson-Dernier et al., 2008). amc is a PEX13 loss-of-function mutant, showing that peroxisomes play a role in the reproductive process (Boisson-Dernier et al., 2008; Goto-Yamada et al., 2014b). These results indicate that PEX13 and other peroxisomal biogenesis factors evolved differently in plants in comparison with other organisms.

The apem3 mutant exhibits enlarged peroxisomes, and the diameter of some can reach more than 10 µm (Figure 2; Mano et al., 2011). Introduction of the apem1/drp3a mutation, which impairs peroxisome division, into apem3 does not affect the enlarged peroxisome phenotype, and DRP3A protein is detected on apem3 enlarged peroxisomes. In addition, division-arrested apem1/drp3a peroxisomes do not show the same level of enlargement as those in the apem3 mutant (Mano et al., 2011). These results suggest that the enlarged peroxisomes observed in apem3 do not arise due to perturbation of peroxisome division. The APEM3 gene encodes PEROXISOMAL MEMBRANE PROTEIN 38 (PMP38), which is also known as PEROXISOMAL NAD⁺ CARRIER (PXN) because it can transport NAD⁺ into the peroxisomal matrix (Fukao et al., 2001; Eubel et al., 2008; Bernhardt et al., 2012). Blockade of NAD⁺ supply reduces the efficiency of lipid metabolism in peroxisomes and induces the accumulation of long-chain fatty acids (Bernhardt et al., 2012). Other mutants defective in fatty acid β-oxidation also contain enlarged peroxisomes (Hayashi et al., 2001). In addition, the enlargement of peroxisomes in the pmp38/pxn mutant is suppressed by disruption of PEROXISOMAL ABC TRANSPORTER1 (PXA1), which transports fatty acids into peroxisomes (Rinaldi et al., 2016). These results demonstrate that the enlargement of peroxisomes in apem3/pmp38/pxn mutants is due to the accumulation of fatty acids, which may produce hydrogen peroxide and damage peroxisomes (Rinaldi et al., 2016).

In the apem4 (previously known as apm4) mutant, GFP-PTS1 protein is located in the cytosol as well as in peroxisomes (Figure 2). The APEM4 gene encodes PEX12 (Mano et al., 2006), which is one of the RING-finger domain-containing peroxins (PEX2, PEX10, and PEX12) involved in peroxisomal protein transport. In fungi, mono-ubiquitination of the peroxisomal protein receptor Pex5 is mediated by Pex4 and Pex12, which are E2 and E3 ligases, respectively (Platta et al., 2009), and this is required for recycling of Pex5 from the peroxisomal membrane to the cytosol. Three A. thaliana RING peroxins, PEX2, PEX10, and PEX12, exhibit E3 ubiquitin ligase activity in vitro (Kaur et al., 2013). Indeed, the apem4/pex12 mutant displayed accumulation of PEX5 protein in the peroxisomal membrane fraction, while the pex12-1 mutant exhibited elevated PEX5 and PEX7 levels (Mano et al., 2006; Kao et al., 2016). The apem4 mutant, as well as another pex12 mutant, exhibits suppression of not only PTS1- but also PTS2-directed protein transport. These results indicate that a PEX12 defect impairs PEX5 and PEX7 recycling. A. thaliana PEX12 can bind to PEX7 (Singh et al., 2009). Therefore, a defect of PEX12 also can lead to a decrease in the efficiency of PEX7-dependent PTS2 transport. Interestingly, the apem4 mutation, which replaces Arg170 with Lys, is adjacent to the pex12-1 mutation, which replaces Glu171 with Lys (Mano et al., 2006; Kao et al., 2016). However, these amino acid residues are not conserved among organisms, and the function of the region containing these two amino acid residues has not been clarified. The findings that mutations at two positions affect peroxisome transport imply the specific function of this region of PEX12.

Like apem2 and apem4, the apem9 mutant was isolated on the basis of a phenotype in which peroxisomal proteins accumulate in the cytosol and exhibits defects in both PTS1- and PTS2-dependent transport (Figure 2; Goto et al., 2011). APEM9 encodes a protein of unknown function, homologs of which are found only in plant genomes. Hydropathy profile analysis of APEM9 suggested that it is similar to yeast PEX15, which can recruit the PEX1/PEX6 complex from the cytosol to peroxisomal membranes (Goto et al., 2011). PEX15 is a tail-anchored peroxisomal membrane protein that is involved in recycling of PEX5 in fungi (Birschmann et al., 2003), and PEX26 was reported to be a PEX15 ortholog in mammalian cells (Matsumoto et al., 2003). The secondary structure of APEM9 appears to be more similar to that of PEX26 than PEX15, although sequence similarity is quite low (Goto et al., 2011). The AAA+ ATPases PEX1 and PEX6 form a heterooligomer and can function as an unfoldase to extract PEX5 from the membrane (Ciniawsky et al., 2015; Gardner et al., 2018; Pedrosa et al., 2018). The apem9 mutation substitutes Gly278 with Glu in the transmembrane domain, which affects the peroxisomal localization of APEM9 and the PEX1/PEX6 complex (Goto et al., 2011). DAYU (a synonym of APEM9) binds to PEX13 and PEX16 (Li et al., 2014). As described above, PEX13 is a component of the PEX5 docking complex, and bridging the docking complex closer to the recycling machinery may make export of PEX5 efficient. Unlike apem2/pex13, mutants defective in APEM9/PEX15/PEX26 or PEX6 displayed a decreased amount of PEX5, and the PEX5 level was increased by treatment with the proteasome inhibitor MG132, suggesting that PEX5 undergoes proteasomal degradation when recycling machinery does not extract it properly (Gonzalez et al., 2017).

The apem10 mutant exhibits a decreased level of punctate peroxisomal GFP signals and accumulation of GFP fluorescence in the cytosol (Figure 2). The apem10 mutation replaces Gln144 with a stop codon in the peroxisomal LON PROTEASE 2 (LON2) protein. Immunostaining of the peroxisomal membrane protein ASCORBATE PEROXIDASE (APX) showed that the number of peroxisomes is reduced in the apem10 mutant (Goto-Yamada et al., 2014a). This indicates that peroxisomes are degraded and that matrix proteins, such as GFP-PTS1, accumulate in the cytosol (Lingard and Bartel, 2009; Goto-Yamada et al., 2014a). The apem10/lon2 phenotype is accentuated with age. Accumulation of GFP-PTS1 in the cytosol is not observed in newly emerging young leaves, but is prominent in well-developed mature leaves in which peroxisomes are enlarged occasionally (Farmer et al., 2013; Goto-Yamada et al., 2014a). In addition, the phenotype of enlarged peroxisomes in apem10 mutants was suppressed under high CO₂ conditions, in which the photorespiratory pathway was not required, indicating that the apem10 phenotype correlates with peroxisomal activity (Goto-Yamada et al., 2014a). Induction of autophagy deficiency rescued the apem10/lon2 phenotype (Farmer et al., 2013; Goto-Yamada et al., 2014a). Peroxisomal metabolic systems contain a variety of oxidases and produce hydrogen peroxide, which is a threat to peroxisomal proteins and membranes (Nishimura et al., 1983; Corpas et al., 2020). Studies of plant LON2 and autophagy revealed the existence of two independent peroxisome maintenance processes: 1) LON2 degrades abnormal and/or obsolete matrix proteins inside peroxisomes and 2) when peroxisomes are not sufficiently restored by LON2, autophagy degrades abnormal peroxisomes (Farmer et al., 2013; Shibata et al., 2013; Goto-Yamada et al., 2014a). A lack of LON2 induces peroxisome degradation via autophagy (called pexophagy). Plant peroxisomes alter their metabolic systems in response to their environment and developmental changes. The molecular mechanisms to replace enzymes responsible for each type of metabolism have long been discussed. The quality control system of peroxisomes, which involves the two aforementioned coordinated degradation processes, explains the mechanism underlying peroxisomal functional transition and a new model was proposed (Goto-Yamada et al., 2015). Interestingly, the protease activity of the C-terminal serine peptidase domain seems to contribute to degradation of peroxisomal proteins, but not to inhibition of pexophagy, which is dependent on the N-terminal chaperone domain (Goto-Yamada et al., 2014a). The mechanisms underlying inhibition and induction of pexophagy remain to be investigated.

Leaf peroxisomes function in many metabolic pathways, some of which also involve other organelles such as mitochondria and chloroplasts (Mano and Nishimura, 2005; Hayashi and Nishimura, 2006; Nyathi and Baker, 2006; Hu et al., 2012; Kao et al., 2018; Oikawa et al., 2019). Therefore, it is thought that the close localization of peroxisomes, mitochondria, and chloroplasts contributes to efficient metabolite flow. In fact, electron micrographs showed these three organelles in close contact with each other (Frederick and Newcomb, 1969; Tolbert, 1982; Nishimura et al., 1986; Oikawa et al., 2019; Baillie et al., 2020). As described above, visualization of peroxisomes using fluorescent proteins enables analysis of their positioning in living cells (Jedd and Chua, 2002; Mano et al., 2002; Mathur et al., 2002). Peroxisomes actively move on actin filaments using myosin motors and interact with other organelles such as chloroplasts and mitochondria (Jedd and Chua, 2002; Mano et al., 2002; Mathur et al., 2002; Goto-Yamada et al., 2015; Oikawa et al., 2015; Oikawa et al., 2019; Baillie et al., 2020; Mathur, 2021). Peroxisomes in dark-adapted cells change their shape from spherical to elliptical in order to strengthen their interactions with chloroplasts under the photosynthetic condition (Oikawa et al., 2015; Oikawa et al., 2019). The strength of interactions between peroxisomes and chloroplasts in the dark and light was measured using a femtosecond laser to evaluate adhesion strength directly in living leaf cells. This revealed that light has a strong positive effect on adhesion (Oikawa et al., 2015; Hosokawa et al., 2016). An optical tweezer was used to measure the interaction strength between a peroxisome and a chloroplast in vitro (Gao et al., 2016). These studies revealed the existence of a physical interaction between peroxisomes and chloroplasts, and suggest that this interaction has physiological significance for plant cellular function.

The tethering factor(s) that connects a peroxisome and a chloroplast remains unclear, but PEX10, a C3HC4 zing RING-finger peroxisomal membrane protein, is one candidate (Schumann et al., 2007). Expression of dominant-negative PEX10 disturbed the interaction of peroxisomes with chloroplasts and photorespiration. Further studies are required to clarify whether PEX10 functions as a tethering factor between a peroxisome and a chloroplast directly and whether other PEXs are involved in this interaction.

It was recently reported that a large complex of glycolysis enzymes, a phosphoglycerate mutase-enolase metabolon, plays a role in the interaction between mitochondria and chloroplasts (Zhang et al., 2020). A direct interaction between mitochondria and chloroplasts has been clearly shown by analyzing mitochondrial movement (Oikawa et al., 2021). It is interesting to investigate whether enzymes in the metabolite pathway participate in the interaction between peroxisomes and chloroplasts similar to the interaction between mitochondria and chloroplasts. Determination of the mechanism underlying the peroxisome-chloroplast interaction will help to elucidate the role of organelle interactions in plants.

Glyoxysomes, one of the peroxisomes, engage in the degradation of reserve oil stored in the oil body via β-oxidation and the glyoxylate cycle. A. thaliana peroxisome defective 1 (ped1) was defective in fatty acid β-oxidation (Hayashi et al., 1998). Detailed electron microscopic analysis revealed that the glyoxysomes in etiolated cotyledons of the ped1 mutant appeared abnormal, having tubular structures that are derived from invagination of the glyoxysomal membrane. (Hayashi et al., 2001). These invagination sites were always in contact with oil bodies, proposing that direct interaction between glyoxysomes and lipid bodies is involved in the process of fatty acid metabolism (Hayashi et al., 2001). A. thaliana sugar dependent 1 (sdp1) mutant was identified from the pool of ethyl methanesulfonate (EMS)-mutagenized A. thaliana, which expressed the fusion gene encoding OLEOSIN, one of oil body membrane proteins, with GFP, as having larger and more oil body aggregates compared with the wild-type plant (Cui et al., 2016). SDP1 is a triacylglycerol (TAG) lipase that resides on the oil body membrane, and hydrolyzes TAG to produce fatty acids. From the analyses using the sdp1 mutant, Cui et al. (2016) showed that sucrose is a key factor for peroxisome-oil body interaction dependent on actin filaments, and that PEROXISOME DEFFECTIVE 3 (PED3), a peroxisomal ATP binding cassette transporter, is the potential anchor protein to the membranes of these organelles (Cui et al., 2016).

The analysis of mutants accumulating excess peroxisomes described below and several other reports indicate that autophagic peroxisomal degradation, or pexophagy, is one of the major peroxisomal quality control mechanisms, along with maintenance by the chaperone-proteinase LON2/APEM10 (Farmer et al., 2013; Kim et al., 2013; Shibata et al., 2013; Goto-Yamada et al., 2014a). Mutants with defective autophagy fail to form autophagosomes and subsequently degrade peroxisomes. In these mutants, the cisterna-like membrane structure associated with peroxisomes and the ATG8 protein, one of the autophagosome components, were detected on autophagosome membrane structures by immunoelectron microscopy (Yoshimoto et al., 2014). Reduction-oxidation sensitive green fluorescent protein (roGFP) analysis revealed that the peroxisomes of autophagy-deficient mutants are highly oxidized, and that mCherry-ATG8a proteins selectively assemble on the oxidized peroxisomes (Shibata et al., 2013). Peroxisomes are oxidized by hydrogen peroxide produced in the process of peroxisome function, and such damaged peroxisomes are selectively recognized and eliminated by autophagy.

It is crucial to study mutants in order to understand the biological significance of peroxisome movement and positioning (interactions with other organelles) for cellular function. A. thaliana peup mutants were isolated from the EMS-mutagenized seed pools that were used to obtain apem mutants by the following method (Table 1; Shibata et al., 2013; Goto-Yamada et al., 2019). Leaves of the mutant lines were put on an agar plate under 100 μmol m⁻² s⁻¹ light to distribute chloroplasts perpendicular in leaf mesophyll cells (Kagawa et al., 2001; Oikawa et al., 2008; Wada and Kong, 2018). In WT cells, peroxisomes reside in a similar location as chloroplasts at the cell periphery because they mostly interact with chloroplasts (Oikawa et al., 2015). It was expected that if the mutants were defective in the peroxisome motility system that regulates peroxisome localization or in tethering factors that connect a peroxisome with a chloroplast, peroxisomes would exhibit abnormal positioning or remain in the cytosol distant from the chloroplast. About 10,000 plants were screened under a fluorescence microscope, and more than 50 peup mutants, which displayed peroxisome aggregation and diffuse localization in the cytosol due to a defect in interactions with chloroplasts, were obtained. Of them, peup1, peup2, and peup4 exhibited remarkable peroxisome aggregation and an increased number of peroxisomes (Figure 3; Shibata et al., 2013). Furthermore, the mutants displayed earlier senescence than WT plants in normal air conditions (Shibata et al., 2013; Yoshimoto et al., 2014). PEUP1, PEUP2, and PUEP4 encode autophagy-related (ATG) 2, ATG18a, and ATG7 proteins, respectively (Table 1; Shibata et al., 2013). These mutants accumulated undegraded peroxisomes containing inactive catalase aggregates, which were observed as high-density regions in peroxisomes in electron micrographs (Shibata et al., 2013). The undegraded peroxisomes are defective in interactions with chloroplasts and movement in the cytosol (Yoshimoto et al., 2014).

Other PEUP genes, PEUP17 and PEUP22, were recently revealed to encode ATG5 and ATG7, respectively (Table 1). Analysis of peup17 and peup22 demonstrated that sucrose starvation induces a type of microautophagy in root tip cells and that ATG genes are involved in this process (Goto-Yamada et al., 2019). Peroxisomes in other peup mutants are spherical with reduced motility or form small aggregates with aberrant motility. These mutants are expected to have defects in gene products that regulate the interaction between a peroxisome and a chloroplast or peroxisome mobility, such as tethering factors or receptors of motor proteins. They will be useful materials to study peroxisome quality control via autophagy and organelle interactions.

The screening strategy, e.g., the parental strain to be mutagenized and the criteria for isolation of mutants, depends on the experiments. The most reported imaging-based approach is mutagenesis and screening of strains with visualized peroxisomes as described in the above section. Zhang and Hu. (2009) reported the screening and identification of A. thaliana peroxisome division/proliferation deficient (pdd) mutants to identify factors involved in peroxisome division and proliferation pathways. pdd mutants were isolated from parental EMS-mutagenized A. thaliana, which expressed 35S promoter-driven YFP-PTS1. They identified pdd1 and pdd2 as DRP3A alleles (Aung and Hu, 2009; Zhang and Hu, 2009). Rinaldi et al. (2016) reported a massive number of mutants, with 34 novel alleles of 15 genes involved in oil body mobilization, fatty acid β-oxidation, the glyoxylate cycle, peroxisome fission, and pexophagy (Rinaldi et al., 2016). These mutants were isolated from EMS-mutagenized GFP-PTS1-expressing plants, and the mutations were determined by a combination of map-based cloning and direct or whole genome sequencing. Although it had been reported that deficiencies in enzymes involved in peroxisomal β-oxidation led to swollen peroxisomes, this had not been proven. One of the main achievements of this report is genetically proving that accumulation of fatty acids inside peroxisomes leads to enlargement of peroxisomes using a number of isolated mutants defective in peroxisomal metabolism and transporters (Rinaldi et al., 2016). The visualization of peroxisomes is also effective in mammalian cells to isolate peroxisome-deficient mutants. Ghaedi et al. (1999) generated Chinese Hamster Ovary cells stably transformed with GFP-PTS1 or PTS2-GFP, and mutagenized these cells with N-methyl-N’-nitro-N-nitrosoguanidine. The mutant phenotypes were investigated by observation under a fluorescence microscope (Ghaedi et al., 1999). Another group employed a similar technique, and these studies identified several genes related to peroxisome biogenesis (Ito et al., 2000; Ghaedi and Fujiki, 2008). Comprehensive, imaging-based, large-scale screening has been achieved in yeast. Yeast is a very useful tool for functional analysis of proteins because of the ease of genetic analyses and the large number of established analytical tools. A collection of gene deletions covering 96% of yeast open reading frames, called a modified synthetic genetic array (SGA), is available, and automated screening is also possible (Giaever et al., 2002). Wolinski et al. (2009) established an experimental platform that can be connected to a SGA, enabling qualitative, quantitative, and automated large-scale analysis of GFP-labeled peroxisomes in yeast cells (Wolinski et al., 2009). The technique identified two novel genes that had not been previously linked to peroxisome biogenesis as well as all known factors required for PTS1-dependent protein transport. Cohen et al. (2014) employed dual reporters to visualize peroxisomes. Cherry fused with PTS1 (Cherry-PTS1) and GFP-tagged peroxisomal membrane protein Ant1 (GFP-Ant1) allow assessment of the efficiency of not only matrix protein sorting but also membrane protein transport and peroxisome formation (Cohen et al., 2014). Use of a combination of the SGA approach and multiple reporters identified a P-type ATPase and ion transporter in the ER membrane (Spf1), which is required for delivery of peroxisomal membrane proteins to peroxisomes, and revealed that peroxisomes localize in close contact with mitochondria and ER (Cohen et al., 2014).

In addition to mutant screening using strains with visualized peroxisomes, mutant screenings focusing on specific peroxisomal factors or phenomena have also been reported. Burkhart et al. (2013) focused on degradation of peroxisomal enzymes in glyoxysomes, which are a type of peroxisomes in which the glyoxylate cycle occurs and are found in cotyledons during early germination of seedlings (Burkhart et al., 2013). ISOCITRATE LYASE (ICL), a glyoxysomal enzyme, is required for lipid conversion to sucrose during post-germinative growth and becomes unnecessary once photosynthesis starts in seedlings (Nishimura et al., 1982; Titus and Becker, 1985). To identify components required for degradation of unwanted peroxisomal matrix proteins, a transgenic plant expressing GFP-ICL was mutagenized with EMS. Mutants that retained GFP-ICL longer than the WT, named persistent GFP-ICL fluorescence (pfl) mutants, were isolated (Burkhart et al., 2013). In the screening, proteins involved in the docking complex (PEX14) and recycling complex (PEX2, PEX6, and PEX10) of the matrix protein transport machinery and a β-oxidation enzyme (PED1/KAT2) were identified from the pfl mutants (Burkhart et al., 2013; Burkhart et al., 2014). From these analyses, the authors concluded that efficient degradation of peroxisomal matrix proteins requires proteins to be sorted inside peroxisomes and also seems to require an appropriate metabolic level of peroxisomes and the entire peroxisomal protein transport system (Burkhart et al., 2013). A unique and large-scale screening was performed in yeast to study the priority of peroxisomal protein targeting. Peroxisomal proteins containing the PTS1 targeting signal are captured by the receptor PEX5 and transported to peroxisomes. If the level of cargo becomes high, the occupancy of PEX5 increases and only proteins with a high targeting priority will localize to peroxisomes. Rosenthal et al. (2020) generated yeast strains that express varying levels of PTS1 fused to mCherry (mCherry-SKL) by changing the copy number of mCherry-SKL in the construct (Rosenthal et al., 2020). Around 90 strains expressing peroxisomal proteins tagged with GFP were transformed with low or high levels of mCherry-SKL, and the localization of GFP to peroxisomes was measured in each strain using an automated microscopy platform.

In the correlative light and electron microscopy (CLEM) method, fluorescence and dyes in a sample are observed with an optical microscope, and then the same area is observed with an electron microscope (Razi and Tooze, 2009; Jahn et al., 2012). Although various CLEM methods have been developed and reported, they have mainly used cultured animal cells, and there are few reports on methods suitable for plant tissues and cells. Toyooka (2016) developed a new CLEM method to accurately capture the localization of fluorescently labeled biomolecules in plant tissues and cells at high resolution, and applied the method to A. thaliana with GFP-labeled peroxisomes (Toyooka, 2016). In yeast Hansenula polymorpha, the peroxisome-vacuole contact site was visualized using the CLEM method, and Pex3 is shown to be involved in the formation of peroxisome-vacuole contact sites (Wu et al., 2019). Bykov et al. (2019) developed a new methodology, MultiCLEM, to allow systematic, parallel, high-throughput screening for traits using the CLEM with computer image analysis (Bykov et al., 2019). By applying MultiCLEM to different yeast strains with GFP-labelled peroxisomes, they successfully identified peroxisomes in both fluorescence and electron microscopic images (Bykov et al., 2019). Since this methodology apparently can be scaled up to higher throughputs, not limited to yeast, it is expected to enable electron microscopy a powerful screening method.

Three-dimensional ultrastructural images with quantitative information can be reconstructed from image data obtained by transmission electron microscopy or focused ion beam scanning electron microscopy (FIB-SEM). Recently, Zechmann et al. (2021) reported that quantitative changes of the volumes of viral inclusion bodies, chloroplast fine structures, mitochondria, and peroxisomes using reconstituted 3D image data (Zechmann et al., 2021). They reconstituted 3D images during the process of Tobacco mosaic virus and Zucchini yellow mosaic virus infection in tobacco and pumpkin plants from serial sections obtained by transmission electron microscopy and extracted quantitative information on the size and number of peroxisomes and other organelles (Zechmann et al., 2021). In mouse liver hepatocytes, the wrappER, a curved wrapping type of rough ER accumulates fatty acid and fatty acid-binding proteins of the lipocalin family and regulates intracellular and systemic lipid flux by establishing extensive contact with almost all mitochondria. Ilacqua et al. (2022) showed that the wrappER contacts with peroxisomes in addition to mitochondria by analyzing a large portion of the cell volume of the hepatocytes by serial section electron tomography coupled to 3D reconstruction. Xu et al. (2017) reported an extended FIB-SEM system for high volume 3D imaging suitable for connectomics (Xu et al., 2017). Using this new system, the authors have successfully imaged large, complex samples of mammalian neural tissue, Drosophila brain, and Chlamydomonas reinhardtii in entirety with sufficient detail to allow high-quality reconstruction of connections. The introduction of these new imaging techniques is expected to make it possible to analyze peroxisome dynamics at higher resolution, more easily, and with a larger volume of data.