Vacuolar Degradation of Plant Organelles.

Plants continuously remodel and degrade their organelles due to damage from their metabolic activities and environmental stressors, as well as an integral part of their cell differentiation programs. Whereas certain organelles use local hydrolytic enzymes for limited remodeling, most of pathways that control the partial or complete dismantling of organelles rely on vacuolar degradation. Specifically, selective autophagic pathways play a crucial role in recognizing and sorting plant organelle cargo for vacuolar clearance, especially under cellular stress conditions induced by factors like heat, drought, and damaging light. In these short reviews, we discuss the mechanisms that control the vacuolar degradation of chloroplasts, mitochondria, endoplasmic reticulum, Golgi, and peroxisomes, with an emphasis on autophagy, recently discovered selective autophagy receptors for plant organelles, and crosstalk with other catabolic pathways.

Ubiquitin-conjugating activity by PEX4 is required for efficient protein transport to peroxisomes in Arabidopsis thaliana.

Protein transport to peroxisomes requires various proteins, such as receptors in the cytosol and components of the transport machinery on peroxisomal membranes. The Arabidopsis apem (aberrant peroxisome morphology) mutant apem7 shows decreased efficiency of peroxisome targeting signal 1-dependent protein transport to peroxisomes. In apem7 mutants, peroxisome targeting signal 2-dependent protein transport is also disturbed, and plant growth is repressed. The APEM7 gene encodes a protein homologous to peroxin 4 (PEX4), which belongs to the ubiquitin-conjugating (UBC) protein family; however, the UBC activity of Arabidopsis PEX4 remains to be investigated. Here, we show using electron microscopy and immunoblot analysis using specific PEX4 antibodies and in vitro transcription/translation assay that PEX4 localizes to peroxisomal membranes and possesses UBC activity. We found that the substitution of proline with leucine by apem7 mutation alters ubiquitination of PEX4. Furthermore, substitution of the active-site cysteine residue at position 90 in PEX4, which was predicted to be a ubiquitin-conjugation site, with alanine did not restore the apem7 phenotype. Taken together, these findings indicate that abnormal ubiquitination in the apem7 mutant alters ubiquitin signaling during the process of protein transport, suggesting that the UBC activity of PEX4 is indispensable for efficient protein transport to peroxisomes.

Autophagy controls reactive oxygen species homeostasis in guard cells that is essential for stomatal opening.

Reactive oxygen species (ROS) function as key signaling molecules to inhibit stomatal opening and promote stomatal closure in response to diverse environmental stresses. However, how guard cells maintain basal intracellular ROS levels is not yet known. This study aimed to determine the role of autophagy in the maintenance of basal ROS levels in guard cells. We isolated the Arabidopsis autophagy-related 2 (atg2) mutant, which is impaired in stomatal opening in response to light and low CO2 concentrations. Disruption of other autophagy genes, including ATG5, ATG7, ATG10, and ATG12, also caused similar stomatal defects. The atg mutants constitutively accumulated high levels of ROS in guard cells, and antioxidants such as ascorbate and glutathione rescued ROS accumulation and stomatal opening. Furthermore, the atg mutations increased the number and aggregation of peroxisomes in guard cells, and these peroxisomes exhibited reduced activity of the ROS scavenger catalase and elevated hydrogen peroxide (H2O2) as visualized using the peroxisome-targeted H2O2 sensor HyPer. Moreover, such ROS accumulation decreased by the application of 2-hydroxy-3-butynoate, an inhibitor of peroxisomal H2O2-producing glycolate oxidase. Our results showed that autophagy controls guard cell ROS homeostasis by eliminating oxidized peroxisomes, thereby allowing stomatal opening.

Regulation of Sugar and Storage Oil Metabolism by Phytochrome during De-etiolation.

Exposure of dark-grown (etiolated) seedlings to light induces the heterotrophic-to-photoautotrophic transition (de-etiolation) processes, including the formation of photosynthetic machinery in the chloroplast and cotyledon expansion. Phytochrome is a red (R)/far-red (FR) light photoreceptor that is involved in the various aspects of de-etiolation. However, how phytochrome regulates metabolic dynamics in response to light stimulus has remained largely unknown. In this study, to elucidate the involvement of phytochrome in the metabolic response during de-etiolation, we performed widely targeted metabolomics in Arabidopsis (Arabidopsis thaliana) wild-type and phytochrome A and B double mutant seedlings de-etiolated under R or FR light. The results revealed that phytochrome had strong impacts on the primary and secondary metabolism during the first 24 h of de-etiolation. Among those metabolites, sugar levels decreased during de-etiolation in a phytochrome-dependent manner. At the same time, phytochrome upregulated processes requiring sugars. Triacylglycerols are stored in the oil bodies as a source of sugars in Arabidopsis seedlings. Sugars are provided from triacylglycerols through fatty acid ß-oxidation and the glyoxylate cycle in glyoxysomes. We examined if and how phytochrome regulates sugar production from oil bodies. Irradiation of the etiolated seedlings with R and FR light dramatically accelerated oil body mobilization in a phytochrome-dependent manner. Glyoxylate cycle-deficient mutants not only failed to mobilize oil bodies but also failed to develop thylakoid membranes and expand cotyledon cells upon exposure to light. Hence, phytochrome plays a key role in the regulation of metabolism during de-etiolation.

NAI2 and TSA1 Drive Differentiation of Constitutive and Inducible ER Body Formation in Brassicaceae.

Brassicaceae and closely related species develop unique endoplasmic reticulum (ER)-derived structures called ER bodies, which accumulate ß-glucosidases/myrosinases that are involved in chemical defense. There are two different types of ER bodies: ER bodies constitutively present in seedlings (cER bodies) and ER bodies in rosette leaves induced by treatment with the wounding hormone jasmonate (JA) (iER bodies). Here, we show that At-α whole-genome duplication (WGD) generated the paralogous genes NAI2 and TSA1, which consequently drive differentiation of cER bodies and iER bodies in Brassicaceae plants. In Arabidopsis, NAI2 is expressed in seedlings where cER bodies are formed, whereas TSA1 is expressed in JA-treated leaves where iER bodies are formed. We found that the expression of NAI2 in seedlings and the JA inducibility of TSA1 are conserved across other Brassicaceae plants. The accumulation of NAI2 transcripts in Arabidopsis seedlings is dependent on the transcription factor NAI1, whereas the JA induction of TSA1 in rosette leaves is dependent on MYC2, MYC3 and MYC4. We discovered regions of microsynteny, including the NAI2/TSA1 genes, but the promoter regions are differentiated between TSA1 and NAI2 genes in Brassicaceae. This suggests that the divergence of function between NAI2 and TSA1 occurred immediately after WGD in ancestral Brassicaceae plants to differentiate the formation of iER and cER bodies. Our findings indicate that At-α WGD enabled diversification of defense strategies, which may have contributed to the massive diversification of Brassicaceae plants.

Involvement of Adapter Protein Complex 4 in Hypersensitive Cell Death Induced by Avirulent Bacteria.

Plant immunity to avirulent bacterial pathogens is associated with subcellular membrane dynamics including fusion between the vacuolar and plasma membranes, resulting in hypersensitive cell death. Here, we report that ADAPTOR PROTEIN COMPLEX-4 (AP-4) subunits are involved in plant immunity associated with hypersensitive cell death. We isolated a mutant with a defect in resistance to an avirulent strain of Pseudomonas syringae pv. tomato (Pto) DC3000 avrRpm1 from a vacuolar protein sorting mutant library of Arabidopsis (Arabidopsis thaliana). The mutant was identical to gfs4-1, which has a mutation in the gene encoding the AP-4 subunit AP4B. Thus, we focused on AP4B and another subunit, AP4E. All of the mutants (ap4b-3, ap4b-4, ap4e-1, and ap4e-2) were defective in hypersensitive cell death and resistance to Pto DC3000 with the type III effector AvrRpm1 or AvrRpt2, both of which are recognized on the plasma membrane, while they showed slightly enhanced susceptibility to the type-III-secretion-deficient P. syringae strain hrcC On the other hand, both ap4b-3 and ap4b-4 showed no defect in resistance to Pto DC3000 with the type III effector AvrRps4, which is recognized in the cytosol and does not induce hypersensitive cell death. Upon infection with Pto DC3000 avrRpt2, the ap4b-3 and ap4b-4 leaf cells did not show fusion between vacuolar and plasma membranes, whereas the wild-type leaf cells did. These results suggest that AP-4 contributes to cell death-associated immunity, possibly via membrane fusion, after type III effector-recognition on the plasma membrane.

Grana-Localized Proteins, RIQ1 and RIQ2, Affect the Organization of Light-Harvesting Complex II and Grana Stacking in Arabidopsis.

Grana are stacked thylakoid membrane structures in land plants that contain PSII and light-harvesting complex II proteins (LHCIIs). We isolated two Arabidopsis thaliana mutants, reduced induction of non-photochemical quenching1 (riq1) and riq2, in which stacking of grana was enhanced. The curvature thylakoid 1a (curt1a) mutant was previously shown to lack grana structure. In riq1 curt1a, the grana were enlarged with more stacking, and in riq2 curt1a, the thylakoids were abnormally stacked and aggregated. Despite having different phenotypes in thylakoid structure, riq1, riq2, and curt1a showed a similar defect in the level of nonphotochemical quenching of chlorophyll fluorescence (NPQ). In riq curt1a double mutants, NPQ induction was more severely affected than in either single mutant. In riq mutants, state transitions were inhibited and the PSII antennae were smaller than in wild-type plants. The riq defects did not affect NPQ induction in the chlorophyll b-less mutant. RIQ1 and RIQ2 are paralogous and encode uncharacterized grana thylakoid proteins, but despite the high level of identity of the sequence, the functions of RIQ1 and RIQ2 were not redundant. RIQ1 is required for RIQ2 accumulation, and the wild-type level of RIQ2 did not complement the NPQ and thylakoid phenotypes in riq1 We propose that RIQ proteins link the grana structure and organization of LHCIIs.

Effect of mutation of C-terminal and heme binding region of Arabidopsis catalase on the import to peroxisomes.

We evaluated the import of Arabidopsis catalase to peroxisomes under homogenous transient expression. The amino acids at -11 to -4 from the C-terminus are necessary for catalase import. The results are in agreement with the previous work under stable expression. We first demonstrate that heme-binding sites are important for peroxisomal import, suggesting the importance of catalase folding. Abbreviations: AtCat: Arabidopsis catalase; PTS: peroxisomal targeting signal; PEX: Peroxin.

Re-evaluation of physical interaction between plant peroxisomes and other organelles using live-cell imaging techniques.

The dynamic behavior of organelles is essential for plant survival under various environmental conditions. Plant organelles, with various functions, migrate along actin filaments and contact other types of organelles, leading to physical interactions at a specific site called the membrane contact site. Recent studies have revealed the importance of physical interactions in maintaining efficient metabolite flow between organelles. In this review, we first summarize peroxisome function under different environmental conditions and growth stages to understand organelle interactions. We then discuss current knowledge regarding the interactions between peroxisome and other organelles, i.e., the oil bodies, chloroplast, and mitochondria from the perspective of metabolic and physiological regulation, with reference to various organelle interactions and techniques for estimating organelle interactions occurring in plant cells.

PYK10 myrosinase reveals a functional coordination between endoplasmic reticulum bodies and glucosinolates in Arabidopsis thaliana.

The endoplasmic reticulum body (ER body) is an organelle derived from the ER that occurs in only three families of the order Brassicales and is suggested to be involved in plant defense. ER bodies in Arabidopsis thaliana contain large amounts of ß-glucosidases, but the physiological functions of ER bodies and these enzymes remain largely unclear. Here we show that PYK10, the most abundant ß-glucosidase in A. thaliana root ER bodies, hydrolyzes indole glucosinolates (IGs) in addition to the previously reported in vitro substrate scopolin. We found a striking co-expression between ER body-related genes (including PYK10), glucosinolate biosynthetic genes and the genes for so-called specifier proteins affecting the terminal products of myrosinase-mediated glucosinolate metabolism, indicating that these systems have been integrated into a common transcriptional network. Consistent with this, comparative metabolite profiling utilizing a number of A. thaliana relatives within Brassicaceae identified a clear phylogenetic co-occurrence between ER bodies and IGs, but not between ER bodies and scopolin. Collectively, our findings suggest a functional link between ER bodies and glucosinolate metabolism in planta. In addition, in silico three-dimensional modeling, combined with phylogenomic analysis, suggests that PYK10 represents a clade of 16 myrosinases that arose independently from the other well-documented class of six thioglucoside glucohydrolases. These findings provide deeper insights into how glucosinolates are metabolized in cruciferous plants and reveal variation of the myrosinase-glucosinolate system within individual plants.

Sucrose Production Mediated by Lipid Metabolism Suppresses the Physical Interaction of Peroxisomes and Oil Bodies during Germination of Arabidopsis thaliana.

Physical interaction between organelles is a flexible event and essential for cells to adapt rapidly to environmental stimuli. Germinating plants utilize oil bodies and peroxisomes to mobilize storage lipids for the generation of sucrose as the main energy source. Although membrane interaction between oil bodies and peroxisomes has been widely observed, its underlying molecular mechanism is largely unknown. Here we present genetic evidence for control of the physical interaction between oil bodies and peroxisomes. We identified alleles of the sdp1 mutant altered in oil body morphology. This mutant accumulates bigger and more oil body aggregates compared with the wild type and showed defects in lipid mobilization during germination. SUGAR DEPENDENT 1 (SDP1) encodes major triacylglycerol lipase in Arabidopsis Interestingly, sdp1 seedlings show enhanced physical interaction between oil bodies and peroxisomes compared with the wild type, whereas exogenous sucrose supplementation greatly suppresses the interaction. The same phenomenon occurs in the peroxisomal defective 1 (ped1) mutant, defective in lipid mobilization because of impaired peroxisomal ß-oxidation, indicating that sucrose production is a key factor for oil body-peroxisomal dissociation. Peroxisomal dissociation and subsequent release from oil bodies is dependent on actin filaments. We also show that a peroxisomal ATP binding cassette transporter, PED3, is the potential anchor protein to the membranes of these organelles. Our results provide novel components linking lipid metabolism and oil body-peroxisome interaction whereby sucrose may act as a negative signal for the interaction of oil bodies and peroxisomes to fine-tune lipolysis.

Plastidial Folate Prevents Starch Biosynthesis Triggered by Sugar Influx into Non-Photosynthetic Plastids of Arabidopsis.

Regulation of sucrose-starch interconversion in plants is important to maintain energy supplies necessary for viability and growth. Arabidopsis mutants were screened for aberrant responses to sucrose to identify candidates with a defect in the regulation of starch biosynthesis. One such mutant, fpgs1-4, accumulated substantial amounts of starch in non-photosynthetic cells. Dark-grown mutant seedlings exhibited shortened hypocotyls and accumulated starch in etioplasts when supplied with exogenous sucrose/glucose. Similar starch accumulation from exogenous sucrose was observed in mutant chloroplasts, when photosynthesis was prevented by organ culture in darkness. Molecular genetic analyses revealed that the mutant was defective in plastidial folylpolyglutamate synthetase, one of the enzymes engaged in folate biosynthesis. Active folate derivatives are important biomolecules that function as cofactors for a variety of enzymes. Exogenously supplied 5-formyl-tetrahydrofolate abrogated the mutant phenotypes, indicating that the fpgs1-4 mutant produced insufficient folate derivative levels. In addition, the antifolate agents methotrexate and 5-fluorouracil induced starch accumulation from exogenously supplied sucrose in dark-grown seedlings of wild-type Arabidopsis. These results indicate that plastidial folate suppresses starch biosynthesis triggered by sugar influx into non-photosynthetic cells, demonstrating a hitherto unsuspected link between plastidial folate and starch metabolism.

Phosphorylation of the C Terminus of RHD3 Has a Critical Role in Homotypic ER Membrane Fusion in Arabidopsis.

The endoplasmic reticulum (ER) consists of dynamically changing tubules and cisternae. In animals and yeast, homotypic ER membrane fusion is mediated by fusogens (atlastin and Sey1p, respectively) that are membrane-associated dynamin-like GTPases. In Arabidopsis (Arabidopsis thaliana), another dynamin-like GTPase, ROOT HAIR DEFECTIVE3 (RHD3), has been proposed as an ER membrane fusogen, but direct evidence is lacking. Here, we show that RHD3 has an ER membrane fusion activity that is enhanced by phosphorylation of its C terminus. The ER network was RHD3-dependently reconstituted from the cytosol and microsome fraction of tobacco (Nicotiana tabacum) cultured cells by exogenously adding GTP, ATP, and F-actin. We next established an in vitro assay system of ER tubule formation with Arabidopsis ER vesicles, in which addition of GTP caused ER sac formation from the ER vesicles. Subsequent application of a shearing force to this system triggered the formation of tubules from the ER sacs in an RHD-dependent manner. Unexpectedly, in the absence of a shearing force, Ser/Thr kinase treatment triggered RHD3-dependent tubule formation. Mass spectrometry showed that RHD3 was phosphorylated at multiple Ser and Thr residues in the C terminus. An antibody against the RHD3 C-terminal peptide abolished kinase-triggered tubule formation. When the Ser cluster was deleted or when the Ser residues were replaced with Ala residues, kinase treatment had no effect on tubule formation. Kinase treatment induced the oligomerization of RHD3. Neither phosphorylation-dependent modulation of membrane fusion nor oligomerization has been reported for atlastin or Sey1p. Taken together, we propose that phosphorylation-stimulated oligomerization of RHD3 enhances ER membrane fusion to form the ER network.

A novel membrane fusion-mediated plant immunity against bacterial pathogens.

Plants have developed their own defense strategies because they have no immune cells. A common plant defense strategy involves programmed cell death (PCD) at the infection site, but how the PCD-associated cell-autonomous immunity is executed in plants is not fully understood. Here we provide a novel mechanism underlying cell-autonomous immunity, which involves the fusion of membranes of a large central vacuole with the plasma membrane, resulting in the discharge of vacuolar antibacterial proteins to the outside of the cells, where bacteria proliferate. The extracellular fluid that was discharged from the vacuoles of infected leaves had both antibacterial activity and cell death-inducing activity. We found that a defect in proteasome function abolished the membrane fusion associated with both disease resistance and PCD in response to avirulent bacterial strains but not to a virulent strain. Furthermore, RNAi plants with a defective proteasome subunit PBA1 have reduced DEVDase activity, which is an activity associated with caspase-3, one of the executors of animal apoptosis. The plant counterpart of caspase-3 has not yet been identified. Our results suggest that PBA1 acts as a plant caspase-3-like enzyme. Thus, this novel defense strategy through proteasome-regulating membrane fusion of the vacuolar and plasma membranes provides plants with a mechanism for attacking intercellular bacterial pathogens.

HSP90 Stabilizes Auxin-Responsive Phenotypes by Masking a Mutation in the Auxin Receptor TIR1.

Heat shock protein 90 (HSP90) is a molecular chaperone that is required for the function of various substrate proteins, also known as client proteins. It is proposed that HSP90 buffers or hides phenotypic variations in animals and plants by masking mutations in some of its client proteins. However, none of the client proteins with cryptic mutations has been identified to date. Here, we identify the first client protein example by which HSP90 buffers a mutation: the auxin receptor transport inhibitor response 1 (TIR1). TIR1 interacts with HSP90 in the nucleus. An HSP90-specific inhibitor abolished the nuclear localization of TIR1 and the auxin-induced degradation of a TIR1-substrate, indicating that TIR1 is an HSP90 client protein. Plants with a null mutation in the TIR1 gene had a defect in auxin response, whereas plants with a point mutation in the TIR1 gene responded to auxin treatment in young seedlings, but a cryptic defect in its auxin response was exposed with HSP90 inhibitor treatment. These results demonstrate that HSP90 masks a point mutation in the auxin receptor TIR1 and thereby buffers auxin-responsive phenotypes.

Organ-specific quality control of plant peroxisomes is mediated by autophagy.

Peroxisomes are essential organelles that are characterized by the possession of enzymes that produce hydrogen peroxide (H2O2) as part of their normal catalytic cycle. During the metabolic process, peroxisomal proteins are inevitably damaged by H2O2 and the integrity of the peroxisomes is impaired. Here, we show that autophagy, an intracellular process for vacuolar degradation, selectively degrades dysfunctional peroxisomes. Marked accumulation of peroxisomes was observed in the leaves but not roots of autophagy-related (ATG)-knockout Arabidopsis thaliana mutants. The peroxisomes in leaf cells contained markedly increased levels of catalase in an insoluble and inactive aggregate form. The chemically inducible complementation system in ATG5-knockout Arabidopsis provided the evidence that these accumulated peroxisomes were delivered to vacuoles for degradation by autophagy. Interestingly, autophagosomal membrane structures specifically recognized the abnormal peroxisomes at the site of the aggregates. Thus, autophagy is essential for the quality control of peroxisomes in leaves and for proper plant development under natural growth conditions.

Extension of oil biosynthesis during the mid-phase of seed development enhances oil content in Arabidopsis seeds.

Regulation of oil biosynthesis in plant seeds has been extensively studied, and biotechnological approaches have been designed to increase seed oil content. Oil and protein synthesis is negatively correlated in seeds, but the mechanisms controlling interactions between these two pathways are unknown. Here, we identify the molecular mechanism controlling oil and protein content in seeds. We utilized transgenic Arabidopsis thaliana plants overexpressing WRINKLED1 (WRI1), a master transcription factor regulating seed oil biosynthesis, and knockout mutants of major seed storage proteins. Oil and protein biosynthesis in wild-type plants was sequentially activated during early and late seed development, respectively. The negative correlation between oil and protein contents in seeds arises from competition between the pathways. Extension of WRI1 expression during mid-phase of seed development significantly enhanced seed oil content. This study demonstrates that temporal activation of genes involved in oil or storage protein biosynthesis determines the oil/protein ratio in Arabidopsis seeds. These results provide novel insights into potential breeding strategies to generate crops with high oil contents in seeds.

Spatiotemporal secretion of PEROXIDASE36 is required for seed coat mucilage extrusion in Arabidopsis.

The epidermal cells of the Arabidopsis thaliana seed coat, which correspond to the second layer of the outer integument (oi2), contain large quantities of a pectic polysaccharide called mucilage within the apoplastic space beneath the outer periclinal cell wall. Immediately after seed imbibition, the mucilage is extruded and completely envelops the seed in a gel-like capsule. We found that a class III peroxidase family protein, PEROXIDASE36 (PER36), functions as a mucilage extrusion factor. Expression of PER36 occurred only in oi2 cells for a few days around the torpedo stage. A PER36-green fluorescent protein fusion was secreted into the outer cell wall in a polarized manner. per36 mutants were defective in mucilage extrusion after seed imbibition due to the failure of outer cell wall rupture, although the mutants exhibited normal monosaccharide composition of the mucilage. This abnormal phenotype of per36 was rescued by pectin solubilization, which promoted cell wall loosening. These results suggest that PER36 regulates the degradation of the outer cell wall. Taken together, this work indicates that polarized secretion of PER36 in a developmental stage-dependent manner plays a role in cell wall modification of oi2 cells.

MAIGO5 functions in protein export from Golgi-associated endoplasmic reticulum exit sites in Arabidopsis.

Plant cells face unique challenges to efficiently export cargo from the endoplasmic reticulum (ER) to mobile Golgi stacks. Coat protein complex II (COPII) components, which include two heterodimers of Secretory23/24 (Sec23/24) and Sec13/31, facilitate selective cargo export from the ER; however, little is known about the mechanisms that regulate their recruitment to the ER membrane, especially in plants. Here, we report a protein transport mutant of Arabidopsis thaliana, named maigo5 (mag5), which abnormally accumulates precursor forms of storage proteins in seeds. mag5-1 has a deletion in the putative ortholog of the Saccharomyces cerevisiae and Homo sapiens Sec16, which encodes a critical component of ER exit sites (ERESs). mag mutants developed abnormal structures (MAG bodies) within the ER and exhibited compromised ER export. A functional MAG5/SEC16A-green fluorescent protein fusion localized at Golgi-associated cup-shaped ERESs and cycled on and off these sites at a slower rate than the COPII coat. MAG5/SEC16A interacted with SEC13 and SEC31; however, in the absence of MAG5/SEC16A, recruitment of the COPII coat to ERESs was accelerated. Our results identify a key component of ER export in plants by demonstrating that MAG5/SEC16A is required for protein export at ERESs that are associated with mobile Golgi stacks, where it regulates COPII coat turnover.

Highly oxidized peroxisomes are selectively degraded via autophagy in Arabidopsis.

The positioning of peroxisomes in a cell is a regulated process that is closely associated with their functions. Using this feature of the peroxisomal positioning as a criterion, we identified three Arabidopsis thaliana mutants (peroxisome unusual positioning1 [peup1], peup2, and peup4) that contain aggregated peroxisomes. We found that the PEUP1, PEUP2, and PEUP4 were identical to Autophagy-related2 (ATG2), ATG18a, and ATG7, respectively, which are involved in the autophagic system. The number of peroxisomes was increased and the peroxisomal proteins were highly accumulated in the peup1 mutant, suggesting that peroxisome degradation by autophagy (pexophagy) is deficient in the peup1 mutant. These aggregated peroxisomes contained high levels of inactive catalase and were more oxidative than those of the wild type, indicating that peroxisome aggregates comprise damaged peroxisomes. In addition, peroxisome aggregation was induced in wild-type plants by exogenous application of hydrogen peroxide. The cat2 mutant also contained peroxisome aggregates. These findings demonstrate that hydrogen peroxide as a result of catalase inactivation is the inducer of peroxisome aggregation. Furthermore, an autophagosome marker, ATG8, frequently colocalized with peroxisome aggregates, indicating that peroxisomes damaged by hydrogen peroxide are selectively degraded by autophagy in the wild type. Our data provide evidence that autophagy is crucial for quality control mechanisms for peroxisomes in Arabidopsis.