Autophagy-Related Proteins Are Required for Degradation ofPeroxisomes in Arabidopsis Hypocotyls duringSeedling GrowthC W

Jimi Kim,1 Heeeun Lee,1 Han Nim Lee, Soon-Hee Kim,2 Kwang Deok Shin, and Taijoon Chung3

Department of Biological Sciences, Pusan National University, Geumjeong-gu, Busan, 609-735, Republic of Korea

Plant peroxisomes play a pivotal role during postgerminative growth by breaking down fatty acids to provide fixed carbons forseedlings before the onset of photosynthesis. The enzyme composition of peroxisomes changes during the transition of theseedling from a heterotrophic to an autotrophic state; however, the mechanisms for the degradation of obsolete peroxisomalproteins remain elusive. One candidate mechanism is autophagy, a bulk degradation pathway targeting cytoplasmic constituents tothe lytic vacuole. We present evidence supporting the autophagy of peroxisomes in Arabidopsis thaliana hypocotyls during seedlinggrowth. Mutants defective in autophagy appeared to accumulate excess peroxisomes in hypocotyl cells. When degradation in thevacuole was pharmacologically compromised, both autophagic bodies and peroxisomal markers were detected in the wild-typevacuole but not in that of the autophagy-incompetent mutants. On the basis of the genetic and cell biological data we obtained, wepropose that autophagy is important for the maintenance of peroxisome number and cell remodeling in Arabidopsis hypocotyls.

INTRODUCTION

The early growth of seedlings and their survival under adverseenvironments depend on nutrients remobilized from seed storagereserves. Oil is a major seed storage reserve for many plant spe-cies including oil crops and the oilseed model species Arabidopsisthaliana. Upon germination of Arabidopsis seeds, triacylglycerolstored in seed oil bodies is hydrolyzed by lipases (Kelly et al., 2011)to generate fatty acids, which are transferred to peroxisomes forb-oxidation (reviewed by Theodoulou and Eastmond, 2012).Acetyl-CoA, produced from the peroxisomal b-oxidation of fattyacids, enters the glyoxylate cycle, which controls the carbon flowfrom fatty acid catabolism to organic acids. The organic acids areused as sources of carbon for gluconeogenesis and the citric acidcycle to provide sugars and energy for seedling growth.

Two enzymes involved in the glyoxylate cycle—isocitrate lyase(ICL) and malate synthase (MLS)—are located in the matrix ofperoxisomes. These two enzymes, which are found in varioustissues in addition to the oil-storing tissues of germinating seeds(reviewed by Pracharoenwattana and Smith, 2008), rapidly dis-appear during postgerminative growth (Nishimura et al., 1986;Lingard et al., 2009). The degradation of the enzymes of theglyoxylate cycle is concomitant with the transition of ICL-positiveseedling peroxisomes (formerly called glyoxysomes) to ICL-

negative leaf peroxisomes, which participate in photorespira-tion. Thus, ICL and MLS have been used as model polypeptidesto study the degradation of peroxisomal matrix proteins (Zolmanet al., 2005; Lingard et al., 2009; Burkhart et al., 2013).The mechanism of degradation of obsolete peroxisomal proteins

is not well understood in plant cells (reviewed by Hu et al., 2012).Matrix proteins like ICL and MLS may be degraded within perox-isomes, but the peroxisomal proteases responsible for their deg-radation are yet to be identified. An excess of peroxisomal proteinssuch as PEROXIN5 (PEX5), the receptor of peroxisomal targetingsignal type 1, could be polyubiquitylated and degraded by pro-teasomes, as in budding yeast (Saccharomyces cerevisiae)(Plattaet al., 2004). The degradation of peroxisomal proteins by protea-somes is supported by the recent identification of Arabidopsis ICLas a ubiquitylation target (Kim et al., 2013). A third possible path-way for peroxisomal degradation is pexophagy, autophagy that isselective for the degradation of peroxisomes (reviewed by Till et al.,2012). Autophagy directs various cytoplasmic constituents to bedegraded in lytic compartments: vacuoles in yeast and plant cells,and lysosomes in mammalian cells. Macroautophagy is the bestcharacterized form of autophagy (macroautophagy is hereafterreferred to as autophagy), wherein cytoplasmic cargoes for deg-radation are initially sequestered by a membrane cisterna calledthe phagophore. The phagophore expands, forms a sac-likestructure, and matures into an autophagosome, a cytoplasmiccompartment with a limiting double membrane. Autophagosomesin plant cells are targeted to the vacuole, possibly by fusion ofthe outer limiting membrane with the tonoplast. In the vacuole, theinner membrane of the autophagosome and its cargoes inside themembrane, called autophagic bodies, are rapidly degraded by acidhydrolases in the vacuolar lumen.Although pexophagy was first described in mammalian cells

(De Duve and Baudhuin, 1966), the mechanisms of pexophagyhave been better characterized in the methylotrophic yeastPichia pastoris. In both P. pastoris andMusmusculus (mouse), a set

1 These authors contributed equally to this work.2 Current address: Department of Biology Education, Pusan NationalUniversity, Geumjeong-gu, Busan, 609-735, Republic of Korea.3 Address correspondence to taijoon@pusan.ac.kr.The author responsible for distribution of materials integral to the findingspresented in this article in accordance with the policy described in theInstructions for Authors (www.plantcell.org) is: Taijoon Chung (taijoon@pusan.ac.kr).C Some figures in this article are displayed in color online but in black andwhite in the print edition.W Online version contains Web-only data.www.plantcell.org/cgi/doi/10.1105/tpc.113.117960

The Plant Cell, Vol. 25: 4956–4966, December 2013, www.plantcell.org ã 2013 American Society of Plant Biologists. All rights reserved.

of Autophagy-related (Atg) genes is required for nonselective au-tophagy and pexophagy. For example, ~70 to 80% of perox-isomes in mouse liver appear to be degraded by autophagy, whichwas estimated from an analysis of Atg7 conditional knockout mice(Iwata et al., 2006). Whether pexophagy occurs in plant cells is notclear (Hu et al., 2012), despite evidence supporting it. First, elec-tron microscopic observation suggested pexophagy in castorbean (Ricinus communis) endosperm (Vigil, 1970), although similarobservations have not been reported. Second, Atg genes en-coding core autophagic machineries are highly conserved in var-ious eukaryotes, including dicots (Hanaoka et al., 2002; Doellinget al., 2002) and monocots (Chung et al., 2009), and reverse ge-netic studies using Arabidopsis demonstrated their functions inplant autophagy (reviewed by Kim et al., 2012). Of four conservedcore autophagic complexes, the ATG8 conjugation system hasbeen the most extensively studied. ATG8, a ubiquitin-like protein,is an important marker for autophagy in yeast, metazoans, andplants. For targeting to the autophagic membrane, ATG8 is con-jugated to the membrane phospholipid phosphatidylethanolamine(PE) by the E1 (ubiquitin-activating enzyme)–like ATG7 and the E2(ubiquitin-conjugating enzyme)–like ATG3. ATG12, another ubiquitin-like protein, forms a conjugate with ATG5, and this conjugate isrequired for ATG8-PE conjugation in vitro (Fujioka et al., 2008)and in planta (Chung et al., 2010).

In this study, we tested the hypothesis that autophagy playsa role in eliminating obsolete peroxisomal proteins. We showedthat atg7 and atg5mutants accumulated markers for peroxisomalmatrix proteins. Consistent with our hypothesis, peroxisomalfluorescent markers were stabilized in the vacuole when vacuolardegradation was pharmacologically inhibited. Furthermore, weobserved puncta formed by the autophagic marker green fluo-rescent protein (GFP)–ATG8a, which partially overlapped withperoxisomal markers. Our data suggest a role for autophagy inthe degradation of peroxisomes during postgerminative growth.

RESULTS

Autophagy-Defective Mutants Accumulate PeroxisomalMarkers in Hypocotyl Cells

To investigate the potential role of core ATG genes in peroxisomedegradation, we crossed an Arabidopsis atg7-2 mutant (Chunget al., 2010) with a transgenic line overproducing a cyan fluo-rescent protein containing a canonical peroxisomal targetingsignal type 1, Ser-Lys-Leu, at its C terminus (CFP-SKL) (Nelsonet al., 2007). GFP-SKL, the green fluorescent variant of CFP-SKL,has been widely used as a peroxisomal marker (Mano et al., 2002;Zolman and Bartel, 2004). Arabidopsis ICL and MLS containsimilar tripeptides, Ser-Arg-Met and Ser-Arg-Leu, respectively, attheir C termini, and Lingard et al. (2009) observed that the patternof GFP-ICL fluorescence resembled GFP-SKL puncta in Arabi-dopsis seedlings. atg7-2 homozygous seedlings expressing theCFP-SKL transgene were isolated by genotyping from a segre-gating F2 population, and self-fertilized progenies from the ho-mozygotes were analyzed by confocal microscopy. Hypocotylsof atg7-2 seedlings expressing the transgene were comparedwith those of the homozygous wild-type (ATG7) CFP-SKL

transgenic line that was used for the genetic cross. In wild-typehypocotyls, the number of peroxisomes was significantly reducedafter 5 days of incubation (DOI) at 21°C (Figures 1A and 1B),consistent with a previous report (Mano et al., 2002). In contrast,atg7-2 hypocotyl cells accumulated more CFP-SKL signal after 5,7, and 9 DOI than did wild-type cells (Figures 1A and 1B). Thisphenotype was evident also in atg5-1 hypocotyls expressing theCFP-SKL transgene (Figure 1C; see Supplemental Figure 1 on-line), indicating that defective ATG8 conjugation is responsible forthe accumulation of CFP-SKL puncta. After 20 DOI, the accu-mulation of CFP-SKL puncta in the mutant hypocotyls was stillobserved (see Supplemental Figure 2A online). Furthermore, themutant cotyledons after 20 DOI appeared to accumulate slightlymore CFP-SKL puncta than did wild-type cotyledons (seeSupplemental Figure 2B online); however, less obvious pheno-typic differences were observed in cotyledons than in hypocotyls(compared with Supplemental Figures 2C and 2D online).The abundance of CFP-SKL puncta in the mutants likely results

from a difference in posttranscriptional processes because CFP-SKL transgene expression is driven by the constitutive cauliflowermosaic virus 35S promoter (Pro35S) (Nelson et al., 2007). Quanti-tative RT-PCR analysis indicated that the levels of CFP-SKL tran-scripts in atg7-2 and atg5-1mutants did not significantly differ fromthose in the wild type (Figure 1D). Immunoblot analysis of atg7-2and atg5-1 hypocotyl extracts revealed two protein bands withslightly different mobility, neither of which was detected in thenontransgenic control (Figure 1E). We could not determine whichband represented the unmodified, full-length CFP-SKL polypeptide.If CFP-SKL was covalently modified (e.g., phosphorylated), thebottom band would be the CFP-SKL. Alternatively, the top bandmight represent the full-length CFP-SKL, whereas the lower bandmight correspond to a shorter form of CFP-SKL that is partiallydegraded. When the intensities of both bands were considered, themutant hypocotyls contained similar levels of CFP-SKL proteinswhen compared with the wild-type extract (Figure 1E). Together,these data do not support the possibility that increased transcrip-tion of the CFP-SKL transgene in the mutants leads to accumula-tion of CFP-SKL puncta in the atg7-2 hypocotyls.We asked whether light is required for the ATG7-dependent re-

duction in peroxisome number. When seedlings were grown in thedark, the number of peroxisomes gradually decreased in wild-typeand atg7-2 and atg5-1 hypocotyls (see Supplemental Figure 3online). The accumulation of CFP-SKL puncta was not evident inthe mutant hypocotyls of etiolated seedlings, implying that lighttriggers ATG7-dependent reduction in peroxisome number inArabidopsis hypocotyls.

Endogenous Peroxisomal Proteins Accumulate inAutophagy-Defective Hypocotyls

The accumulation of CFP-SKL puncta in autophagy-defective mu-tants suggests a role for autophagy in the degradation of peroxisomalproteins. To test this hypothesis, we performed immunoblot analysisof nontransgenic hypocotyls, using antisera against ICL and MLS,peroxisomal glyoxylate cycle enzymes. Stabilization of ICL and MLSwas used to demonstrate the delayed degradation of peroxisomes invarious mutants (Lingard et al., 2009; Burkhart et al., 2013). As ex-pected, ICL and MLS rapidly disappeared in the wild-type seedlings

Degradation of Peroxisomes by Autophagy 4957

whenRubisco large subunits accumulated (Figure 2A). Time-dependentICL and MLS degradation was observed also in dissectedwild-type hypocotyls (Figures 2B and 2C). After 5 and 7 DOI,ICLwas barely detectable in wild-type hypocotyls, and a higher level ofICL was detectable in atg7-2 and atg5-1 hypocotyls (Figures 2B and2C). Delayed degradation of glyoxylate cycle enzymes in atg7-2 andatg5-1 hypocotyls was confirmed in the immunoblots of MLS (Figures2B and 2C). The apparent stabilization in the mutants was only tran-sient; ICL and MLS gradually disappeared in mutant hypocotyls.

The increased number of peroxisomes and stabilization of per-oxisomal proteins may result from the developmental retardationof the mutants. This possibility was excluded by immunoblotanalysis with antibodies against peroxisomal hydroxypyruvate re-ductase (pHPR), another peroxisomal protein involved in photo-respiration. Because pHPR was shown to accumulate when ICLstarts to be degraded (Lingard et al., 2009), delayed transition ofseedling peroxisomes to leaf peroxisomes would be observed indevelopmentally delayed mutants. This was not the case in atg7-2and atg5-1 mutants, in which pHPR started to accumulate after 3to 5 DOI, similarly to the wild type (Figures 2B and 2C). In fact, the

pHPR levels in the mutants appeared to be higher than those inthe wild-type hypocotyl extracts (Figures 2B and 2C). As an in-ternal control, we performed anti–histone H3 immunoblot analysis,which did not show overaccumulation of the nuclear proteins inthe mutant hypocotyls (Figures 2B and 2C).To examine whether transcriptional regulation is responsible for

the differential accumulation of peroxisomal proteins in the mu-tant hypocotyls, we performed quantitative RT-PCR analysisusing wild-type and atg7-2 hypocotyls (Figures 2D to 2F). Theatg7-2 hypocotyls after 3 DOI contained a higher level of ICLtranscripts than did the wild-type hypocotyls. After 5 DOI, ICLexpression was markedly decreased in both wild-type and atg7-2hypocotyls (Figure 2D). However, we detected no significant dif-ference inMLS and pHPR transcript levels between wild-type andatg7-2 hypocotyls at any time point (Figures 2E and 2F). Thus,transcriptional regulation is an unlikely mechanism for the differ-ential accumulation of MLS and pHPR in the atg7-2 hypocotyls(Figure 2B). We cannot exclude the possibility that the increase(twofold) in ICL transcripts in atg7-2 hypocotyls after 3 DOI resultedin a higher level of ICL proteins, which remained until after 7 DOI.

Figure 1. Autophagy-Defective Mutant Seedlings Accumulated the Peroxisomal Marker CFP-SKL in Hypocotyl Cells.

(A) Confocal microscopy images of wild-type (ATG7, top panels) and atg7-2 (bottom panels) homozygous hypocotyl tissues expressing the Pro35S-CFP-SKL transgene. Transgenic seedlings were grown on MS agar medium for the indicated number of days. Insets show higher magnification imagesof the areas marked with white squares. Bars = 10 mm (insets, Bars = 2 mm). (B) and (C) Quantification of peroxisomes from confocal images of wild-type, atg7-2 (B), and atg5-1 (C) hypocotyl cells after 3, 5, 7, or 9 DOI at 21°C. A cytoplasm-rich area (50 3 200 mm) was selected from confocal images,and CFP-SKL dots were quantified with ImageJ using the Cell Counter Plugin. Error bars represent standard deviation (n = 4 seedlings or more). (D)Quantification of CFP-SKL transgene expression in wild-type (WT) and atg7-2 and atg5-1 mutant seedlings by real-time RT-PCR. RNA was extractedfrom whole seedlings grown on MS agar medium for 9 days. The amount of CFP-SKL transcript was normalized to that of UBC9 as a referencetranscript, and relative expression was calculated using the average of normalized values of wild-type samples. Error bars represent standard deviation(n = 3 seedling populations). (E) Immunoblot analysis of wild-type, atg7-2, and atg5-1 hypocotyl extracts after various DOI. Each lane contains proteinextract from 15 hypocotyl segments. Anti-GFP antibodies react with two polypeptide species of CFP-SKL with different mobility (arrowheads to topimages). In the bottom images, histone H3 protein bands are shown as a loading control. Representative images were chosen from at least threeindependent experiments. The molecular weight indicated on the right was estimated from protein size markers. NT, nontransgenic Col-0 extract.[See online article for color version of this figure.]

4958 The Plant Cell

Expression of ATG7 Is Induced in Hypocotyls but Notin Cotyledons

In contrast with the results we obtained with hypocotyl extracts,only slight stabilization of ICL and MLS was observed in whole-seedling extracts of atg7-2 and atg5-1 after 5 DOI (Figure 2A;

see Supplemental Figure 4A online). We also noticed that theatg7-2 mutation exerted a greater effect on peroxisome numberin hypocotyls than in cotyledons (compared with SupplementalFigures 2C and 2D online). These data imply that the ATG8conjugation system has at least a partial role in peroxisomaldegradation, mainly in hypocotyls. Thus, we hypothesized thatautophagy is induced in wild-type hypocotyls after ~5 DOI whenthe number of CFP-SKL puncta and the levels of ICL proteindecreased. We performed quantitative RT-PCR to analyze thetime course of core ATG expression in hypocotyls. As a control,we compared the expression pattern of ATG in cotyledons (seeSupplemental Figure 5 online). We focused on genes encodingthe ATG8 conjugation system because the expression of thesegenes is often regulated developmentally (van der Graaff et al.,2006; Chung et al., 2009). A preliminary search in the Gene-vestigator database (http://www.genevestigator.com) revealedthat ATG3, ATG8b, ATG8g, and ATG8i showed higher expres-sion levels in hypocotyls than in cotyledons. Our RT-PCR resultsfor selected ATG genes indicated that ATG7 and, to a lesserextent, ATG8b transcripts in hypocotyls increased after 5 DOIand then decreased to basal levels (see Supplemental Figures5A and 5D online, dark gray bars), whereas transcripts encodingother ATG8 isoforms and ATG5 did not significantly change.Although ATG3 transcripts gradually decreased in hypocotyls(see Supplemental Figure 5B online, dark gray bar), it is possiblethat an induction of ATG3 peaks before 4 DOI. It was impracticalto analyze the expression pattern before 2 DOI because the2-day-old seedlings were very short, which made it difficult todefine their hypocotyl region. Because ATG8-PE conjugationrequires both ATG7 and ATG3 activities, the data suggest thatATG8 conjugation is activated in hypocotyls at ~5 DOI. In con-trast, this induction was not observed in cotyledon samples, andno ATG3 transcript was detected after 5 DOI (see SupplementalFigure 5 online, light gray bars), consistent with our idea that thephenotypes of ATG7 and ATG5 were more highly associatedwith hypocotyls than with other organs.

Autophagy-Defective Seedlings Show NormalPeroxisome Functions

The mutant phenotypes may be explained by a previous obser-vation that atg7-2 and atg5-1 mutants are hypersensitive to oxi-dative stress (Xiong et al., 2007), which could result in stabilizationof peroxisomes with defective functions via unknown feedbackregulation. To determine whether peroxisomes of atg7-2 andatg5-1 mutants are functional, we investigated their sensitivityto indole-3-butyric acid (IBA), which is converted to auxin byb-oxidation. Mutants defective in peroxisomal b-oxidation are lesssensitive to exogenously applied IBA (Zolman et al., 2000). Theatg7-2 and atg5-1 mutant roots showed normal sensitivity to IBAwhen tested by a root growth assay (see Supplemental Figure 6online), indicating that b-oxidation in peroxisomes is functional inthe mutants. Our analysis of peroxisomal proteins also suggestednormal peroxisomal import in atg7-2 and atg5-1 seedlings be-cause we detected no precursor form of peroxisomal malatedehydrogenase proteins in these mutants (see SupplementalFigure 4B online). Furthermore, we found no difference in dia-minobenzidine staining between wild-type and mutant hypocotyls

Figure 2. Endogenous Peroxisomal Matrix Proteins Accumulated inAutophagy-Defective Mutant Hypocotyls.

(A) to (C) Immunoblot analysis of whole-seedling (A) and hypocotyl (B)and (C) extracts after various DOI was performed using antisera againstICL, MLS, pHPR, or histone H3 (Histone) as a loading control. The proteinband corresponding to Rubisco large subunit (RbcL) was identified inmembrane stained with Ponceau S. Each lane contains 30 mg of totalproteins (A) or protein extract from 15 hypocotyl segments (B) and (C).Lanes on the left contain protein extracts from the wild type (Col-0), andlanes on the right contain protein extracts from atg7-2 (A) and (B) oratg5-1 (C). Molecular weight, indicated on the right, was estimated fromprotein size markers. Representative images were chosen from at leastthree independent experiments. (D) to (F) Quantitative RT-PCR analysis ofwild-type (dark gray bars) and atg7-2 (light gray bars) hypocotyls to detecttranscripts of ICL (D), MLS (E), and pHPR (F) genes. Hypocotyls weredissected from seedlings grown on MS agar medium for the indicatednumber of days. The amount of target transcript was normalized to that ofUBC9 as a reference transcript, and relative expression was calculatedusing the average of normalized values of wild-type samples after 3 DOI.Error bars represent standard deviation (n = 4 seedling populations). As-terisk indicates a significant difference (0.01 < P < 0.05; Student’s t test).

Degradation of Peroxisomes by Autophagy 4959

between 3 and 9 DOI (see Supplemental Figure 7 online), sug-gesting that hydrogen peroxide levels were not altered, in contrastwith a previous report that the hydrogen peroxide level is higher in8-week-old atg5 mutant leaves than in those of the wild type(Yoshimoto et al., 2009). Taken together, these data support theidea that atg7-2 and atg5-1 mutants retain normal peroxisomalfunctions at least until 9 DOI.

Peroxisomal Proteins Are Targeted to the Central Vacuolefor Degradation

To test the hypothesis that the mutant phenotypes are due todefective autophagy of peroxisomes, we examined the effects ofconcanamycin A (CA), an inhibitor of the vacuolar proton pump(Dröse et al., 1993; Matsuoka et al., 1997). CA inhibits degrada-tion in the vacuole by blocking the acidification of the vacuole andhas been used to stabilize autophagic cargoes in the vacuole(Yoshimoto et al., 2004; Wada et al., 2009; Liu et al., 2012). Ifperoxisomes are autophagic cargoes targeted into the vacuolefor degradation, CA may stabilize peroxisomes that would bedegraded otherwise. To study the effect of CA on peroxisomedegradation, we germinated CFP-SKL transgenic seeds in liquidmedium and found that hypocotyls of seedlings hydroponicallygrown for 5 to 7 days also exhibited a reduction in the number ofCFP-SKL puncta. At day 5 of incubation, CFP-SKL transgenicseedlings in either the wild-type or atg7-2 mutant background inliquid medium were treated with either CA or DMSO. Confocalmicroscopy of a series of 1 mm optical sections of hypocotyl cells(Figures 3A to 3D) revealed occasional CFP-SKL bodies inthe central portion of cells treated with CA (Figure 3B, yellowarrowheads). Note that typical hypocotyl cells, like mature rootcells, have a large central vacuole, which causes chloroplasts,peroxisomes, and most of the cytoplasm to be positioned in theperipheral region of cells. These CFP-SKL bodies, which weretypically less than 1 mm in diameter, may represent partially de-graded peroxisomes in the vacuole. The CFP-SKL bodies werenot observed in the center of wild-type hypocotyl cells treatedwith DMSO (Figure 3A) or in atg7-2 cells treated with DMSO(Figure 3C) or CA (Figure 3D), except in cells where cytoplasmicperoxisomes were found in transvacuolar strands (see below).

To verify the subcellular location of the partially degraded per-oxisomes, we performed a double-labeling experiment in whicha tonoplast marker, gamma tonoplast intrinsic protein (gTIP)-CFP,was compared with mCherry-SKL (Nelson et al., 2007). We gen-erated transgenic plants expressing both gTIP-CFP andmCherry-SKL, hydroponically grew the transgenic seedlings, and treatedthem with either CA or DMSO. We searched for any stabilizedmCherry-SKL bodies in the central portion of the cells (Figures 3Eand 3F) and indeed found mCherry-SKL bodies in the center ofhypocotyl cells treated with CA (Figure 3F, yellow arrowheads).These mCherry-SKL signals in the center were distinct from thevacuolar boundary decorated by gTIP-CFP. In both the DMSOcontrol and CA-treated cells, simultaneous labeling of mCherry-SKL and gTIP-CFP signals was occasionally detected in thecenter of hypocotyl cells (Figure 3E, yellow arrows), indicating thatthese signals were derived from cytoplasmic peroxisomes intransvacuolar strands. On the basis of the data shown in Figure 3,we conclude that CA stabilizes peroxisomal proteins in the

vacuole and that small CFP-SKL and mCherry-SKL bodies in thecenter of the cells represent peroxisomal proteins stabilized in thevacuole.

Vacuolar Targeting of Peroxisomal Proteins Requires ATG7

Our data indicated that ATG7 and ATG5 are required for thedegradation of peroxisomes in the vacuole of hypocotyl cellsduring postgerminative growth (Figures 1 and 3). Because thesetwo genes are essential for the conjugation of ATG8 to PE, wespeculated that ATG8-PE plays a role in targeting peroxisomesto the vacuole. Specifically, ATG8-positive autophagic vesicles

Figure 3. Peroxisomal Protein Markers Are Degraded in the Vacuole ofWild-Type Hypocotyl Cells, but Not in the Vacuole of Autophagy-DefectiveMutant Cells.

Seedlings were grown in liquid MS medium for 6 days and treated witheither DMSO (A), (C), and (E) or 1 mM CA (B, D, and F) for 17 h beforeimage acquisition. (A) to (D) Confocal Z-series of wild-type (A) and (B) oratg7-2 (C) and (D) transgenic hypocotyls expressing the Pro35S-CFP-SKL transgene. Optical sections were acquired at 1-mm intervals. Yellowarrowheads with numbers indicate three vacuolar CFP-SKL punctastabilized by CA. Magenta dotted lines were added to images in the firstcolumn to highlight the boundary of each cell. (E) and (F) Confocal im-ages of wild-type transgenic hypocotyls expressing both the Pro35S-mCherry-SKL and Pro35S-g-TIP-CFP transgenes. Transvacuolar strandscan be seen in the top cells at g-TIP-CFP images. Magenta dotted lineswere added to images in the middle column. In the merged images onthe right, the signals of mCherry-SKL and gTIP-CFP are pseudocoloredmagenta and green, respectively. Two yellow arrows (E) label cyto-plasmic peroxisomes in the transvacuolar strand. Two yellow arrow-heads (F) indicate vacuolar mCherry-SKL bodies lacking gTIP-CFPsignal. Representative images were chosen from at least four biologicalreplicates. Bars =5 mm (A) to (D) or 2 mm (E) and (F).

4960 The Plant Cell

may sequester and deliver peroxisomes to the vacuole. To ex-amine this possibility, we designed double-labeling experimentsin which GFP-ATG8a, a marker for various autophagic struc-tures (Thompson et al., 2005), was monitored with mCherry-SKLbodies used as reference (Figure 4). GFP-ATG8a fluorescence inplant cells displays either in a diffuse or in a punctate pattern.The former is presumed to represent a nonconjugated, solubleform of GFP-ATG8a, whereas the latter is likely a membrane-associated form of GFP-ATG8a-PE (Chung et al., 2010).

Round GFP-ATG8a puncta in the cytoplasm, possibly repre-senting autophagosomes (Figure 4A), were detected after 6 DOIin the hypocotyl cells of wild-type seedlings expressing both theGFP-ATG8a and mCherry-SKL transgenes. The GFP-ATG8apuncta were 2 mm in diameter or smaller, with one overlapping withcytoplasmic mCherry-SKL signal (Figure 4A, yellow arrow). Similarresults were obtained from mCherry-SKL transgenic plants coex-pressing a GFP-ATG8a transgene under the control of the poly-ubiquitin gene promoter (ProUBQ10) (Suttangkakul et al., 2011) (seeSupplemental Figure 8A online, yellow arrows). In addition to theputative autophagosomes sequestering peroxisomes, we detectedphagophore-like GFP-ATG8a puncta partially overlapping witha cytoplasmic mCherry-SKL structure (Figure 4B, yellow arrow).

We also performed double-labeling experiments using atg7-2mutant seedlings expressing both the GFP-ATG8a and mCherry-SKL transgenes (see Supplemental Figure 8B online). As pre-viously reported in autophagy-defective mutants (Yoshimotoet al., 2004; Suttangkakul et al., 2011), atg7-2 mutant cells ac-cumulate diffuse GFP-ATG8a signal in the cytoplasm, indicatingits lack of delivery to the vacuole. In addition, the mutant cellsoccasionally contained cytoplasmic GFP-ATG8a foci that weretypically brighter than GFP-ATG8a puncta in wild-type cells (seeSupplemental Figure 8B online, green arrows) (Yoshimoto et al.,2004; Kim et al., 2012). Unlike in wild-type cells, GFP-ATG8a fociin the mutant cells barely overlap with mCherry-SKL puncta.

CA stabilizes GFP-ATG8a puncta in the vacuole of hypocotylcells (Thompson et al., 2005), and GFP-ATG8, in combinationwith CA, has also been used as a marker for autophagic bodiesin the vacuole. To determine whether mCherry-SKL bodies thatare stabilized in the vacuole (Figure 3) overlap with GFP-ATG8a–positive autophagic bodies, we incubated the transgenic seedlingsexpressing both Pro35S-mCherry-SKL and Pro35S-GFP-ATG8ain medium containing CA (Figure 4C). Analysis of confocaloptical sections revealed fewer vacuolar mCherry-SKL bodies(Figure 4C, magenta and yellow arrowheads) than GFP-ATG8aautophagic bodies. We observed colocalization of the twosignals in the vacuole (Figure 4C, yellow arrowheads), althoughtypical mCherry-SKL bodies in the vacuole did not exhibitGFP-ATG8a signals (Figure 4C, magenta arrowheads). Asa negative control, we observed hypocotyls of transgenic plantsexpressing only Pro35S-GFP-ATG8a (Figure 4D) and did notdetect any mCherry signal.

To investigate whether ATG7 is needed for colocalization, weanalyzed the effect of DMSO and CA on wild-type and atg7-2hypocotyls expressing both the ProUBQ10-GFP-ATG8a andPro35S-mCherry-SKL transgenes (Figures 4E to 4J). Comparedwith the strong induction by Pro35S, the ProUBQ10 only mod-erately induced GFP-ATG8a transgene expression. Nevertheless,two fluorescent proteins showing similar localization patterns

were observed among the wild-type hypocotyl cells (Figures 4Eto 4H). In addition to globular GFP-ATG8a signals, we also ob-served GFP-ATG8a puncta that apparently represent phago-phores that are either overlapping or juxtaposed with cytoplasmicperoxisomes in wild-type hypocotyl cells (yellow arrows in Figures4E and 4F). The effects of CA treatment were reproducible in the

Figure 4. Peroxisomal Markers Partially Overlap with AutophagicMarkers in Wild-Type Hypocotyls, but Not in Autophagy-Defective Mu-tant Hypocotyls.

(A) to (C) Confocal images of wild-type transgenic hypocotyls expressingboth the Pro35S-mCherry-SKL and Pro35S-GFP-ATG8a transgenes. (D)Confocal image of hypocotyls expressing only the Pro35S-GFP-ATG8atransgene. (E) to (J) Confocal images of wild-type (E) to (H) or atg7-2 (I) and(J) transgenic hypocotyls expressing both the Pro35S-mCherry-SKL andProUBQ10-GFP-ATG8a transgenes. The transgenic seedlings were grown inliquid MS medium for 6 days and treated with either DMSO (A), (B), (E), (F),and (I) or 1 mM CA (C), (D), (G), (H), and (J) for 17 h before analysis. Rep-resentative images were chosen from at least five biological replicates. In themerged images, signals of mCherry-SKL and GFP-ATG8a are pseudocol-ored magenta and green, respectively. Bars =5 mm. (A) Yellow arrows in-dicate an autophagosome-like signal sequestering a peroxisome. (B), (E), (F),and (H) Yellow arrows mark phagophore-like signals partially overlapping orjuxtaposed with a peroxisome. (C), (G), and (H) Yellow arrowheads labelautophagic bodies overlapping a peroxisomal signal, and magenta arrow-heads indicate vacuolar peroxisomal puncta lacking GFP-ATG8a signal.

Degradation of Peroxisomes by Autophagy 4961

ProUBQ10-GFP-ATG8a transgenic line in the wild-type back-ground; we observed the colocalization of autophagic bodies witha peroxisomal signal (Figures 4G and 4H; yellow arrowheads) and,more frequently, GFP-ATG8a–negative mCherry-SKL signal in thevacuole (Figure 4H, magenta arrowheads). In contrast, DMSO-treated atg7-2 mutant hypocotyl cells had numerous mCherry-SKL puncta and diffuse GFP-ATG8a signals in the cytoplasm(Figure 4I), consistent with the data shown above (Figure 1A; seeSupplemental Figure 8B online). Treatment of the mutant with CAhad little effect on the localization of mCherry-SKL and GFP-ATG8a, and the mutant cells lacked autophagic bodies and vac-uolar mCherry-SKL bodies (Figure 4J). Taken together, these dataindicate that GFP-ATG8a–positive phagophores and autophago-somes partially colocalized with cytoplasmic peroxisomes, andthat ATG7 is required for the degradation of peroxisomal proteinsin the vacuole.

DISCUSSION

Recent studies have shown that a few types of organelles in plantcells are degraded by autophagy. ATG5 and other core ATGgenes are involved in the degradation of chloroplasts (Ishidaet al., 2008; Wada et al., 2009), amyloplasts (Nakayama et al.,2012), and endoplasmic reticulum (ER) (Liu et al., 2012). However,whether the core ATG genes are also required for the degradationof other types of organelles, such as peroxisomes, mitochondria,and oil bodies, has not been clear. Here, we presented evidencesupporting ATG7- and ATG5-dependent autophagy of perox-isomes in Arabidopsis hypocotyls. Two independent mutants,atg7-2 and atg5-1, both defective in ATG8 conjugation to PE (Chunget al., 2010), accumulated peroxisomal markers in hypocotyls—CFP-SKL puncta, ICL, and MLS (Figures 1 and 2). The accumulationof CFP-SKL puncta in the mutants is unlikely owing to ectopictransgene overexpression because endogenous ICL and MLSlevels were also affected by the mutations. The inhibition of ICLand MLS degradation by the mutations was only transient(Figures 2B and 2C), implying an ATG7- and ATG5-independentmechanism for the degradation of peroxisomal proteins. ICL andMLS proteins in Arabidopsis hypocotyls may be degraded ei-ther by peroxisomal matrix proteases or by other autophagy-independent degradation mechanisms, possibly facilitating theautophagic degradation of peroxisomes (Figure 5).

It has been proposed that degradation of peroxisomal proteinsin plant cells depends on proteolytic activities in three subcellularlocations—peroxisome matrix, cytosol, and the vacuole (Zolmanet al., 2005; Lingard et al., 2009; Burkhart et al., 2013; Figure 5).One model for extraperoxisomal degradation of matrix proteinsby the 26S proteasome is peroxisome-associated protein deg-radation (PexAD) (Lingard et al., 2009; Burkhart et al., 2013),which is analogous to the well-defined process of ER–associatedprotein degradation. Prerequisites for PexAD are polyubiquityla-tion and retranslocation of matrix proteins, whereas pexophagy isassumed to deliver peroxisomes to vacuoles for degradation. Toexamine the possible degradation of ICL and MLS in the perox-isomes, Lingard and Bartel (2009) performed a genetic analysis ofgenes encoding three putative peroxisomal proteases in Arabi-dopsis, namely, Lon-related protease 2 (LON2), Deg/HtrA protease

15 (DEG15), and peroxisomal M16 protease (PXM16), but foundthat mutations in these genes and double mutations of lon2 deg15and lon2 pxm16 did not result in the stabilization of ICL and MLS.Instead, lon2 mutants are defective in the import of peroxisomalproteins and the processing of peroxisomal targeting signal type 2(Lingard and Bartel, 2009).While this work was being revised, Farmer et al. (2013) reported

that atg7, atg3, and atg2 mutations were isolated from a forward-genetic screen for lon2 suppressors, providing independentevidence for pexophagy in young Arabidopsis seedlings. Ourobservation of pexophagy in Arabidopsis seedlings supportstheir model that defective autophagy in the absence of LON2proteases leads to the persistence of small peroxisomes, whichenables the suppression of various lon2 phenotypes.Several mutations have been shown to stabilize the enzymes

of the glyoxylate cycle: a pex4 pex22 double mutation (Zolmanet al., 2005) and single mutations of pex14, pex6, and ped1(Burkhart et al., 2013). PEX4 is a ubiquitin-conjugating enzymeand PEX22 is a peroxisomal membrane protein that interactswith PEX4. Although the ubiquitylation targets of PEX4 have yetto be identified in Arabidopsis, PEX4 was proposed to be in-volved in PEX5 recycling and/or PexAD (Hu et al., 2012; Lingardet al., 2009). PEX14, presumed to form a docking point for PEX5,is another peroxisomal membrane protein that is important forthe import of proteins containing a peroxisomal targeting signal

Figure 5. A Model for the Degradation of Peroxisomal Proteins in Wild-Type and Autophagy-Defective Hypocotyls.

Three possible mechanisms of peroxisomal degradation are shown in themodel: degradation by resident proteases, PexAD, and autophagy. Au-tophagy appeared to be activated in wild-type hypocotyls (A) after ~5DOI but was not functional in autophagy-defective atg5 and atg7 (B).Three types of peroxisomal proteins are depicted. (i) Glyoxylate cycleenzymes such as ICL and MLS were found in seedling peroxisomes atday 3 of incubation but were rapidly degraded at approximately day 5and not detected after 7 DOI. (ii) Photorespiratory enzymes like pHPRwere imported after 3 DOI and accumulated in peroxisomes afterward.(iii) Common peroxisomal enzymes, such as those involved in oxidation,were present in peroxisomes throughout all stages. In this study, CFP-SKL and mCherry-SKL may be considered common peroxisomal pro-teins. The types of peroxisomal proteins selected by each possiblemechanism of degradation are not clear.[See online article for color version of this figure.]

4962 The Plant Cell

(Hayashi et al., 2000). A yeast homolog of PEX6 is an ATPasetethered to the peroxisomal membrane and mediates the re-cycling of PEX5 back to the cytosol. Finally, PED1 is a peroxi-somal thiolase involved in b-oxidation of fatty acids.

Given the potential role of autophagy in peroxisome degra-dation, the involvement of PEX14 and PEX4 in peroxisomalprotein degradation is intriguing. Pex14p in a methylotrophicyeast Hansenula polymorpha was implicated in the selectiveautophagy of peroxisomes (Bellu et al., 2001). MammalianPex14p interacts with LC3, a mammalian homolog of ATG8,which is required for peroxisomal protein degradation (Hara-Kuge and Fujiki, 2008). The involvement of PEX4 in peroxisomaldegradation underscored the role of ubiquitylation, which in-duces the autophagy of mammalian peroxisomes (Kim et al.,2008). Thus, our report of autophagy in Arabidopsis hypocotylsprovides an explanation for the genetic data published pre-viously (Zolman et al., 2005; Burkhart et al., 2013). WhetherPEX4-dependent ubiquitylation mediates PexAD, pexophagy, orboth, needs further investigation.

Our cell biological data indicated that peroxisomes in Arabi-dopsis hypocotyl cells are delivered to the vacuole via the ATG7-dependent autophagic pathway. CA stabilized peroxisomalmarkers in the vacuole (Figure 3), and ATG7 was required forvacuolar targeting of mCherry-SKL bodies (Figure 4). We de-tected colocalization of autophagosomes and phagophores withperoxisomes in the wild-type cytoplasm (Figures 4,A, 4B, 4E, 4F,and 4H). In addition, we observed two pools of mCherry-SKLbodies in the vacuole when wild-type hypocotyls were treatedwith CA. Some of the mCherry-SKL bodies emitted GFP-ATG8asignals (i.e., autophagic bodies) (Figure 4, yellow arrowheads in4C, 4G, and 4H), whereas others did not (Figures 4C and 4H;magenta arrowheads). It appears that plant organelles deliveredto the vacuole by autophagy either contain or lack ATG8 markers.We compared our colocalization images with similar data thatother research groups recently published to investigate the au-tophagy of plant organelles (Ishida et al., 2008; Liu et al., 2012).When plant cells undergo ER stress, a punctate pattern of the ERmarker GFP-HDEL was found in the vacuole of leaf protoplaststreated with CA (Liu et al., 2012). Some of the GFP-HDEL punctawere colocalized with an autophagic marker, Cerulean-ATG8e,but others were not (Liu et al., 2012), similar to our data (Figures4C and 4H). Likewise, a small fraction of Rubisco-containingbodies lacking GFP-ATG8 signals were evident when mesophyllcells were treated with CA (Ishida et al., 2008).

The reason for the presence of the vacuolar mCherry-SKLbodies lacking GFP-ATG8a signals is not clear. Peroxisomesmay be released from autophagic bodies in the vacuole, and/orGFP-ATG8, attached to autophagic bodies sequestering per-oxisomes, may be removed prior to the degradation of perox-isomes that contains mCherry-SKL proteins in the matrix.Alternatively, peroxisomes may be delivered to the vacuole alsovia unknown autophagic routes that are not associated withATG8 but still require ATG7 (Figures 3D and 4J). One of thepossible routes is protrusion microautophagy involving a directengulfment of whole organelles by the tonoplast (Bassham et al.,2006). If protrusion microautophagy of peroxisomes is a majorautophagic route for peroxisomal degradation, peroxisomesstabilized by CA should be enclosed by the tonoplast. However,

we failed to observe gTIP-CFP signals enclosing mCherry-SKLbodies in the vacuole (Figure 3F).We did not investigate the possibility that ATG8 isoforms other

than ATG8a have a higher affinity to peroxisomes. Nine Arabi-dopsis genes encode ATG8 isoforms, all of which can be conju-gated to PE in vitro (Fujioka et al., 2008). No specific biochemicaland genetic roles have been assigned to any of these ATG8genes, although their gene expression pattern varies (Yoshimotoet al., 2004; Sláviková et al., 2005; Thompson et al., 2005). It is notknown whether ATG8 isoforms show specific subcellular local-izations. To test the possibility of differential affinities to perox-isomes, fluorescent markers fused to various ATG8 isoforms maybe used for colocalization with peroxisomal markers.The transition of seedling peroxisomes to leaf peroxisomes

appears to be a gradual process, as several reports describeda transitional form of peroxisomes containing both glyoxylatecycle and photorespiratory enzymes (Titus and Becker, 1985;Nishimura et al., 1986; Sautter, 1986). We do not know which ofthese three types of peroxisomes is subjected to autophagy(Figure 5). However, our data suggest that autophagy regulatedby ATG7 and ATG5 generally affects the homeostasis of multi-ple types of peroxisomes. We observed higher levels of ICL,MLS, and pHPR proteins in the atg7 and atg5mutants (Figure 2).Interestingly, Guiboileau et al. (2013) reported that catalase,another peroxisomal protein, overaccumulated in 30-day-oldand 60-day-old atg5 mutants, suggesting that peroxisomalproteins are degraded by autophagy in mature plants. Futureexperiments using a quantitative assay are necessary to assessthe selectivity of autophagy in Arabidopsis hypocotyls.What are the biological functions of autophagy during hypocotyl

development? During postgerminative growth, seedling perox-isomes containing glyoxylate cycle enzymes are converted to leafperoxisomes containing photorespiratory enzymes. ICL-positiveperoxisomes become obsolete organelles and are rapidly elimi-nated by autophagy. In addition, autophagy may play a role in thequality control of damaged and potentially toxic components ofcells. Glyoxylate cycle enzymes like ICL and MLS were found to behighly sensitive to oxidative damage (Yanik and Donaldson, 2005;Anand et al., 2009). Autophagy of peroxisomes containing dam-aged enzymes would allow hypocotyl cells to carry out normalfunctions. Supporting this view, our results revealed that ATG7was induced in hypocotyls after ~5 DOI, but not in cotyledons (seeSupplemental Figure 5 online). This organ-specific induction is alsoconsistent with the observation that ICL and MLS stabilization ismore clearly detected in hypocotyls than in whole seedlings (Figure2), which consist of cotyledons, true leaves, hypocotyls, and roots.Autophagy is spatiotemporally fine tuned during cell remodeling anddifferentiation of multicellular organisms (reviewed by Mizushimaand Komatsu, 2011), and the local induction of autophagy inArabidopsis hypocotyls can be a good model to study the reg-ulation of autophagy by a developmental program in plants.

METHODS

Mutants and Transgenic Plants

Arabidopsis thaliana T-DNA insertional mutants atg5-1 and atg7-2 werepreviously described (Thompson et al., 2005; Chung et al., 2010). The

Degradation of Peroxisomes by Autophagy 4963

pex5-1 mutant seeds (Zolman et al., 2000), transgenic seeds expressingPro35S-CFP-SKL and Pro35S-gTIP-CFP, and a binary vector for ex-pressing Pro35S-mCherry-SKL (Nelson et al., 2007) were obtained fromThe Arabidopsis Information Resource. To generate transgenic Arabi-dopsis plants expressing GFP-ATG8a under the control of ProUBQ10,Gateway LR Clonase II (Invitrogen) reaction was used, in which an entryclone of the full-length AtATG8a cDNA was inserted into the destinationvector pMDC99-AtUBQ10p-GFP (Suttangkakul et al., 2011). The resultingexpression clone pMDC99-AtUBQ10p-GFP-AtATG8a was introducedinto Agrobacterium tumefaciens strain GV3010, and an Agrobacteriumtransformant was used to infect Col-0 plants by the floral-dip method(Clough and Bent, 1998). After selection by hygromycin resistance,multiple T1 transgenic lines were analyzed by confocal microscopy toconfirm a common pattern of subcellular GFP-ATG8a distribution, whichwas similar to that of the Pro35S-GFP-ATG8a marker line describedpreviously (Thompson et al., 2005). We chose a ProUBQ10-GFP-ATG8atransgenic line showing a 3:1 segregation ratio for hygromycin resistancein the T2 population. The transgenic line exhibited normal plant mor-phology in T3 homozygous populations. For colocalization with mCherry-SKL, the representative ProUBQ10-GFP-ATG8a transgenic line wascrossed with atg7-2mutants, and the resulting F1 hybrids were transformedwith the binary vector containing Pro35S-mCherry-SKL. Kanamycin-resistant seedlings were selected and genotyped for the mCherry-SKLand GFP-ATG8a transgenes and for atg7-2.

Plant Growth Conditions

Arabidopsis seeds were surface sterilized in 50% (v/v) bleach for 20 minand washed three times with sterile water. The seeds were sown on solidMurashige and Skoog (MS) medium (13 MS macronutrient salt withvitamins, 1% [w/v] Suc, 0.7% [w/v] agar, pH 5.7) and stored at 4°C for2 days. The plates containing the seeds were incubated at 20 to 22°Cunder a long-day photoperiod (16 h light and 8 h dark). Uniform germi-nation wasmonitored after 2 DOI at 21°C. The IBA resistance assay and invivo detection of hydrogen peroxide were performed as described byZolman et al. (2000) and Thordal-Christensen et al. (1997), respectively.For liquid culture, the surface-sterilized seeds were placed in 24-wellplates containing MS liquid medium (13 MS macronutrient salt with vi-tamins, 1% [w/v] Suc, pH 5.7). Ten seeds per well were added to 1 mL ofMS liquid medium. The 24-well plates were stored at 4°C for 2 days, andincubated with shaking (100 rpm) at 20 to 22°C under a long-day pho-toperiod (16 h light and 8 h dark). When needed, CA (Wako Chemicals) oran equal volume of DMSO was added to the liquid medium and thesamples were further incubated for 17 h before analysis.

RNA Analysis

Total RNA was extracted from whole seedlings, hypocotyls, or cotyle-dons. For cDNA synthesis, 1 mg of total RNA was added to the reversetranscriptase reaction using oligo-dT primers (Thermo Scientific).Quantitative real-time PCR was performed using SYBR Green (Qiagen)and the gene-specific primers listed in Supplemental Table 1 online.Primers were designed using PrimerQuest (Integrated DNA Technolo-gies), with a typical amplicon size of ~100 bp. Primer efficiencies were 98to 100%.WithUBIQUITIN CONJUGATING ENZYME9 (UBC9) cDNA as aninternal reference (Czechowski et al., 2005), relative quantification oftransgene expression was calculated from threshold cycle values (Livakand Schmittgen, 2001).

Immunoblot Analysis

Whole seedlings or dissected hypocotyls were homogenized in Laemmlibuffer. Protein sampleswere separated by SDS-PAGEand transferred ontoImmobilon-P polyvinylidene fluoride membrane (Millipore). Immunoblots

were incubated in a blocking solution (PBS containing 0.1% [v/v] TritonX-100 and either 5% [w/v] nonfat dry milk or 3% [w/v] BSA). Primaryantibodies were diluted in blocking solution as follows: 1:2000 for anti-ICL(Ettinger and Harada, 1990) and 1:1000 for anti-GFP (Roche AppliedScience), anti-MLS (Ettinger and Harada, 1990), anti-pHPR (Agrisera)(Kleczkowski et al., 1986), anti–peroxisomal malate dehydrogenase(Pracharoenwattana et al., 2007), and anti–histone H3 (Abcam). Chem-iluminescence was detected using horseradish peroxidase–conjugatedsecondary antibodies and SuperSignal West Pico Chemiluminescent Kit(Thermo Scientific).

Microscopy

Images of hypocotyl cells were acquired using a Zeiss 510 laser scanningconfocal microscope (Carl Zeiss). Emission filters BP480-520IR, BP500-530IR, and BP565-615IR were used to detect signals of CFP, GFP, andmCherry, respectively. Raw images were processed using an LSM ImageViewer (Carl Zeiss) and Adobe Photoshop CS5 (Adobe Systems). Per-oxisome number was estimated from CFP-SKL fluorescence images withImageJ (National Institutes of Health) using the Cell Counter Plugin.

Accession Numbers

Sequence data from this article can be found in the Arabidopsis GenomeInitiative or GenBank/EMBL databases under the following accessionnumbers: ATG3 (At5g61500), ATG5 (At5g17290), ATG7 (At5g45900),ATG8a (At4g21980), ATG8b (At4g04620), ATG8d (At2g05630), ATG8g(At3g60640), ATG8i (At3g15580), ICL (At3g21720), MLS (At5g03860),pHPR/HPR1 (At1g68010), and UBC9 (At4g27960).

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure 1. Confocal Microscopy Images of Wild-Typeand atg5-1 Hypocotyl Tissues Expressing the Pro35S-CFP-SKLTransgene.

Supplemental Figure 2. Peroxisome Number after 20 DOI in Wild-Type, atg7-2, and atg5-1 Seedlings Expressing the Pro35S-CFP-SKLTransgene.

Supplemental Figure 3. Peroxisome Number in the Hypocotyls ofEtiolated Wild-Type, atg7-2, and atg5-1 Seedlings Expressing thePro35S-CFP-SKL Transgene.

Supplemental Figure 4. Determination of Peroxisomal Protein Levelsin Whole Seedlings of Autophagy-Defective Mutants.

Supplemental Figure 5. Expression of ATG7 Is Induced in Hypocotylsduring PostGerminative Growth.

Supplemental Figure 6. Sensitivity of Col-0, atg7-2, and atg5-1 Rootsto IBA.

Supplemental Figure 7. In Situ Detection of Hydrogen Peroxide inWild-Type and Autophagy-Defective Hypocotyls.

Supplemental Figure 8. ATG7 Is Required for the Formation ofAutophagic Vesicles Overlapping with Peroxisomal Markers.

Supplemental Table 1. Primers Used in this Study.

ACKNOWLEDGMENTS

We thank John Harada (anti-ICL and anti-MLS), Steven Smith, and JohnBussell (anti–peroxisomal malate dehydrogenase) for kindly providingantisera. We thank Richard Vierstra for advice on the preparation of the

4964 The Plant Cell

article and for generously sharing genetic stocks, atg7-2, atg5-1, andtransgenic lines expressing GFP-ATG8a. We appreciate technicalsupport by Sang Sook Lee and Hye Sun Cho. This work was supportedby the Basic Science Research Program through the National ResearchFoundation of Korea funded by the Ministry of Education, Science, andTechnology (2011-0010683) and by a grant from the Next-GenerationBioGreen 21 Program (No. PJ009004), Rural Development Administration,Republic of Korea.

AUTHOR CONTRIBUTIONS

J.K., H.L., H.N.L., S.K., and T.C. designed the research. J.K., H.L., H.N.L.,S.K., K.D.S., and T.C. performed the research. J.K., H.L., H.N.L., S.K., K.D.S.,and T.C. analyzed the data. T.C. wrote the article. J.K., H.L., H.N.L., S.K., andT.C. revised and edited the article.

Received September 23, 2013; revised November 8, 2013; acceptedNovember 29, 2013; published December 24, 2013.

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4966 The Plant Cell

DOI 10.1105/tpc.113.117960; originally published online December 24, 2013; 2013;25;4956-4966Plant Cell

Jimi Kim, Heeeun Lee, Han Nim Lee, Soon-Hee Kim, Kwang Deok Shin and Taijoon ChungHypocotyls during Seedling Growth

ArabidopsisAutophagy-Related Proteins Are Required for Degradation of Peroxisomes in

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