Disrupting Autophagy Restores Peroxisome Function to an
Arabidopsis lon2 Mutant and Reveals a Role for the LON2
Protease in Peroxisomal Matrix Protein Degradation
Lisa M. Farmer,1Mauro A. Rinaldi, Pierce G. Young, Charles H. Danan, Sarah E. Burkhart, and Bonnie Bartel2
Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 77005
Peroxisomes house critical metabolic reactions that are essential for seedling development. As seedlings mature, metabolic
requirements change, and peroxisomal contents are remodeled. The resident peroxisomal protease LON2 is positioned to
degrade obsolete or damaged peroxisomal proteins, but data supporting such a role in plants have remained elusive.
Arabidopsis thaliana lon2 mutants display defects in peroxisomal metabolism and matrix protein import but appear to
degrade matrix proteins normally. To elucidate LON2 functions, we executed a forward-genetic screen for lon2 suppressors,
which revealed multiple mutations in key autophagy genes. Disabling core autophagy-related gene (ATG) products prevents
autophagy, a process through which cytosolic constituents, including organelles, can be targeted for vacuolar degradation.
We found that atg2, atg3, and atg7 mutations suppressed lon2 defects in auxin metabolism and matrix protein processing and
rescued the abnormally large size and small number of lon2 peroxisomes. Moreover, analysis of lon2 atg mutants uncovered
an apparent role for LON2 in matrix protein turnover. Our data suggest that LON2 facilitates matrix protein degradation during
peroxisome content remodeling, provide evidence for the existence of pexophagy in plants, and indicate that peroxisome
destruction via autophagy is enhanced when LON2 is absent.
Peroxisomes are single membrane–bound organelles that com-
partmentalize certain oxidative reactions in eukaryotes, including
fatty acid b-oxidation and hydrogen peroxide metabolism (re-
viewed in Hu et al., 2012). Peroxisomal proteins are imported
into the organelle from the cytosol; defects in human peroxisomal
biogenesis underlie the Zellweger syndrome spectrum disorders,
which often are fatal (reviewed in Wanders and Waterham, 2005).
Plant peroxisomes also are essential (reviewed in Hu et al.,
2012); viable plant mutants lacking peroxisomes have not been
Peroxisome formation and maintenance requires membrane
recruitment, matrix protein import, organelle division, and dis-
tribution in daughter cells. Peroxisome biogenesis in yeast
requires more than 30 peroxin (PEX) genes; about half of these
are conserved in plants and mammals (reviewed in Hu et al.,
2012; Nagotu et al., 2012). PEX5 and PEX7 are receptors that
translocate matrix proteins into peroxisomes by binding cargo
with peroxisome-targeting signals (PTSs) and docking with the
PEX13 and PEX14 membrane peroxins (Nagotu et al., 2012).
PEX5 recognizes proteins containing a PTS1 (McCollum et al.,
1993; Van der Leij et al., 1993), a variant of a C-terminal Ser-Lys-
Leu (SKL-COOH) motif. PEX7 recognizes proteins bearing a
PTS2 (Marzioch et al., 1994), a nine–amino acid sequence em-
bedded in an ;30–amino acid N-terminal presequence. This
presequence is removed upon peroxisome import in mammals
and plants (Swinkels et al., 1991; Helm et al., 2007). After cargo
translocation into the organelle, PEX5 and PEX7 are returned to
the cytosol for reuse (Collins et al., 2000; Dammai and Subramani,
2001; Nair et al., 2004). PEX5 recycling requires the ubiquitin-
conjugating enzyme PEX4, which is tethered to the peroxisome
membrane by PEX22 (Collins et al., 2000; Zolman et al., 2005) and
assisted by a complex of membrane peroxins with ubiquitin-
protein ligase activity (Platta et al., 2009; Kaur et al., 2013).
In young seedlings, peroxisomes house fatty acid b-oxidation
and the glyoxylate cycle (reviewed in Eastmond and Graham,
2001), which allow utilization of storage lipids for energy and
fixed carbon before photosynthesis is established. Peroxisome-
defective Arabidopsis thaliana mutants therefore often require
external fixed carbon for normal development (Hayashi et al.,
1998; Zolman et al., 2000). Peroxisomes also metabolize the
protoauxin indole-3-butyric acid (IBA) (reviewed in Strader and
Bartel, 2011) into the active auxin indole-3-acetic acid (IAA),
which plays multiple critical roles in development (reviewed in
Woodward and Bartel, 2005b). Mutants with dysfunctional per-
oxisomes often display impaired IBA-to-IAA conversion (Strader
et al., 2010) and dampened IBA responsiveness (Zolman et al.,
2000). IBA resistance provides an indirect measure of peroxi-
some function that facilitates the isolation and characterization
of peroxisome-defective mutants (Zolman et al., 2000, 2005;
Zolman and Bartel, 2004; Woodward and Bartel, 2005a; Ramón
and Bartel, 2010; Ratzel et al., 2011).
As seedlings establish photosynthesis several days after ger-
mination, peroxisomes are remodeled, photorespiration enzymes
1Current address: Department of Pediatrics, USDA/Agricultural Re-
search Service Children’s Nutrition Research Center, Baylor College of
Medicine, Houston, TX 77030.
2Address correspondence to firstname.lastname@example.org.
The author responsible for distribution of materials integral to the findings
presented in this article in accordance with the policy described in the
Instructions for Authors (www.plantcell.org) is: Bonnie Bartel (bartel@
CSome figures in this article are displayed in color online but in black and
white in the print edition.
WOnline version contains Web-only data.
The Plant Cell, Vol. 25: 4085–4100, October 2013, www.plantcell.org ã 2013 American Society of Plant Biologists. All rights reserved.
like hydroxypyruvate reductase (HPR) are synthesized, and ob-
solete glyoxylate cycle enzymes, such as isocitrate lyase (ICL)
and malate synthase (MLS), are degraded. Immunolabeling
experiments in greening cucurbit cotyledons directly demon-
strate the presence of both photorespiration enzymes and
glyoxylate cycle enzymes in the same peroxisomes (Titus and
Becker, 1985; Nishimura et al., 1986; Sautter, 1986). Moreover,
MLS is stable following in vitro import into early pumpkin
(Cucurbita sp Amakuri Nankin) seedling peroxisomes but de-
graded after import into transitional peroxisomes (Mori and
Nishimura, 1989). These data provide support for the one-
population hypothesis, in which individual peroxisomal proteins
are degraded and new enzymes are imported into existing
peroxisomes during seedling maturation, rather than a two-
population hypothesis in which entire peroxisomes are degraded
and resynthesized with new content during developmental tran-
sitions (reviewed in Nishimura et al., 1996).
Although glyoxylate cycle enzymes and photorespiration
enzymes have not been directly colocalized in Arabidopsis
peroxisomes, these enzymes are present together in cotyledons
during the ;2-d window during seedling development when ICL
and MLS are degraded and HPR begins accumulating (Lingard
et al., 2009). Peroxisome entry is a prerequisite for efficient ICL
and MLS degradation; pex mutants with matrix protein import
defects stabilize ICL and MLS (Lingard et al., 2009; Burkhart
et al., 2013). Moreover, PEX4 and PEX22 promote ICL and MLS
turnover, hinting at a role for ubiquitination in matrix protein
degradation (Zolman et al., 2005; Lingard et al., 2009). However,
because PEX4 and PEX22 also are implicated in PEX5 recycling
and thus indirectly promote matrix protein import, this finding
does not provide a definitive mechanism for matrix protein
Several peroxisomal proteases have been examined for pos-
sible matrix protein degradation roles (Lingard and Bartel, 2009).
LON proteases were first characterized in bacteria (reviewed
in Tsilibaris et al., 2006) and contain an N-terminal substrate
binding domain, a central ATPase domain containing Walker A
and B ATP-binding motifs, and a C-terminal proteolytic domain
containing a Ser-Lys catalytic dyad (Tsilibaris et al., 2006). LON
proteases are predicted to form ring-shaped oligomers and
often degrade misfolded proteins (Tsilibaris et al., 2006).
Eukaryotic LON isoforms are found in mitochondria, chloro-
plasts, and peroxisomes (Kikuchi et al., 2004; Ostersetzer
et al., 2007; Lingard and Bartel, 2009). The peroxisomal Lon
from the methylotrophic yeast Hansenula polymorpha promotes
degradation of misfolded peroxisomally targeted dihydrofolate
reductase (Aksam et al., 2007), and in Penicillium chrysogenum,
peroxisomal Lon degrades oxidatively damaged catalase-
peroxidase (Bartoszewska et al., 2012). By contrast, mammalian
cells expressing a dominant-negative form of rat liver peroxi-
somal Lon mislocalize catalase to the cytosol (Omi et al.,
2008), indicating that peroxisomal LON function somehow
promotes matrix protein import. Arabidopsis encodes four LON
isoforms; LON2 is the only isoform bearing a canonical PTS1
and localized in peroxisomes (Ostersetzer et al., 2007; Eubel
et al., 2008; Reumann et al., 2009). All examined Arabidopsis
lon2 alleles are resistant to IBA-induced lateral root formation
but respond normally to auxins not requiring b-oxidation (Lingard
and Bartel, 2009; Burkhart et al., 2013), suggesting reduced
IBA-to-IAA conversion. Additionally, lon2 mutants display age-
dependent defects in PTS2 processing that are accompanied
by defects in peroxisomal matrix protein import (Lingard and
Bartel, 2009; Burkhart et al., 2013), indicating that LON2 is
necessary for sustained import of matrix proteins. This age-
dependent defect in matrix protein import contrasts with import
defects in pex14 mutants, which appear less severe as seedlings
age (Hayashi et al., 2000; Monroe-Augustus et al., 2011). In spite
of these defects in peroxisome physiology, disrupting Arabidopsis
LON2 does not appear to result in matrix protein stabilization
(Lingard and Bartel, 2009; Burkhart et al., 2013).
A second peroxisomal protease that has been tested for a role
in Arabidopsis matrix protein degradation is DEG15. DEG15
processes PTS2 proteins into their mature forms by removing
the PTS2-containing N-terminal region (Helm et al., 2007;
Schuhmann et al., 2008), but DEG15 is not required for ICL or
MLS degradation (Lingard and Bartel, 2009). Similarly, the per-
oxisomal M16 metalloprotease PXM16 is not required for deg-
radation of glyoxylate cycle enzymes; pxm16 and lon2 pxm16
mutants efficiently degrade ICL and MLS (Lingard and Bartel,
Beyond possible protease involvement in degrading individual
peroxisomal matrix proteins, yeast and mammals can degrade
peroxisomes using a specialized form of autophagy termed
pexophagy. Interestingly, Aspergillus nidulans ICL appears to be
degraded via pexophagy when cells are moved from fatty acid
to Glc carbon sources (Amor et al., 2000). Autophagy functions
in bulk degradation and recycling of cytosolic constituents, of-
ten in response to nutrient limitation or other stresses (reviewed
in Bassham, 2007; Xie and Klionsky, 2007; Reumann et al.,
2010; Li and Vierstra, 2012). Preautophagosomal structures are
formed by a subset of the AUTOPHAGY-RELATED (ATG) pro-
teins and assist an isolation membrane in engulfing cytoplasmic
constituents in autophagosomes. Autophagosomes can envelop
diverse substrates, including ribosomes, organelles, and protein
aggregates (reviewed in Xie and Klionsky, 2007; Li and Vierstra,
2012). Mature autophagosomes are delivered to the lytic vacuole
by fusion of the outer autophagosome membrane with the vac-
uolar membrane. Once in the vacuole, the inner membrane is
dissolved, and autophagosome contents are degraded by vacu-
olar hydrolases. Although the core autophagy machinery is con-
served in plants (reviewed in Bassham, 2007; Reumann et al.,
2010; Li and Vierstra, 2012), it is unclear whether peroxisome
turnover by pexophagy occurs in plants.
We undertook a forward-genetic screen for lon2 suppressors
to elucidate the molecular functions and targets of LON2 in
Arabidopsis. We isolated multiple alleles of several pivotal ATG
genes that suppressed the suite of lon2 physiological and mo-
lecular defects. At the same time, disrupting autophagy revealed
an additional molecular defect in lon2; lon2 atg double mutants
failed to degrade obsolete glyoxylate cycle enzymes in a timely
fashion during seedling development. Our results imply that
LON2 normally functions in the removal of obsolete matrix
proteins during development. Moreover, the absence of LON2
accelerates pexophagy, which degrades peroxisomes that
otherwise would carry out important metabolic functions in de-
4086The Plant Cell
Isolating Suppressors of lon2 Defects
Because of the unusual assortment of defects in peroxisome
physiology that result when Arabidopsis LON2 is mutated, we
sought to identify LON2 regulators or substrates among LON2
genetic interactors. We screened for mutations that suppressed
the IBA resistance and PTS2-processing defects of lon2-2,
which contains a T-DNA insertion in the last exon of LON2 that
would remove the PTS1 from the protein and prevent its import
into the peroxisome (Lingard and Bartel, 2009). We mutagenized
lon2-2 seeds carrying a cauliflower mosaic virus 35S promoter–
driven version of green fluorescent protein (GFP) bearing an
N-terminal PTS2 peptide (35S:PTS2-GFP) (Woodward and
Bartel, 2005a) and screened ;80,000 M2 seedlings for sup-
pressors in a two-part screen. First, we identified M2 seedlings
that produced approximately wild-type numbers of lateral roots
in the presence of IBA, thereby rescuing the lon2-2 resistance to
IBA-induced lateral root formation. Approximately 800 putative
mutants were transferred to soil and allowed to self-fertilize, and
surviving M3 progeny were rescreened for IBA sensitivity. Next,
the 61 suppressors displaying the most robust IBA sensitivity
were examined by immunoblotting for suppression of the lon2
PTS2-processing defect, revealing nine strong suppressors of
both lon2 phenotypes from eight different M1 pools: 1-1, 4-114,
5-49, 8-8, 11-10, 13-3, 14-22, 14-36, and 16-24. Whereas 8-d-
old lon2 seedlings rarely made a lateral root after growth on 10 mM
IBA, IBA-induced lateral root numbers were comparable to
those of the wild type in these suppressors (Figure 1A), sug-
gesting rescued IBA-to-IAA conversion. Additionally, the sup-
pressors fully restored the ability of lon2-2 to process the PTS2
protein peroxisomal malate dehydrogenase (PMDH) from its
precursor into its mature form (Figure 1B), suggesting that these
suppressors also rescued the lon2-2 peroxisomal matrix protein
Mutations in the Autophagy Gene ATG7 Suppress
We used recombination mapping to localize the causal lesions in
four suppressors (1-1, 4-114, 5-49, and 16-24) to the bottom of
chromosome 5 between At5g42590 and At5g54950, near the
LON2 locus (At5g47040) (Figure 2A). This linkage complicated
our ability to identify the causal lesions by traditional mapping
methods. Therefore, we used whole-genome sequencing of
backcrossed suppressor lines to identify mutations in three
suppressors (4-114, 5-49, and 16-24). Among the homozygous
mutations present in these suppressors (Figures 3A to 3C; see
Supplemental Data Set 1 online), each carried an independent
mutation in ATG7 (At5g45900; Figure 2B). In addition, we
identified atg7 lesions in the 1-1 and 8-8 suppressors (Figure 2B)
following PCR amplification and sequencing of ATG7 genomic
DNA from these mutants (see Supplemental Table 1 online). All
five atg7 alleles identified in our lon2 suppressors are expected
to impair ATG7 function; three were nonsense alleles, which we
renamed atg7-4 (suppressor 5-49; Gln-12 to stop), atg7-5
(suppressor 1-1; Trp-119 to stop), and atg7-6 (suppressor 8-8;
Trp-344 to stop), while two changed conserved Gly residues
(see Supplemental Figure 1 online) to Asp residues, which we
renamed atg7-7 (suppressor 16-24; Gly-247 to Asp) and atg7-8
(suppressor 4-114; Gly-286 to Asp).
ATG7 is a 697–amino acid ATP-dependent ubiquitin-activating
(E1)-like enzyme that activates two ubiquitin-like modifiers act-
ing in the autophagy conjugation pathway, resulting in ATG8
lipidation with phosphatidylethanolamine (PE). ATG8-PE in the
inner autophagosomal membrane docks via adaptor proteins
with cytosolic components to be engulfed, and ATG8-PE in the
outer membrane is tethered to the microtubule-based transport
machinery to allow autophagosome delivery to the vacuole (re-
viewed in Li and Vierstra, 2012). To determine whether blocking
autophagy affected peroxisome physiology, we compared the
IBA responsiveness and PTS2 processing in atg7 null mutants
to two lon2 alleles and our lon2-2 atg7 suppressors. The atg7-2
and atg7-3 null alleles harbor T-DNA insertions in the 7th exon
and abolish autophagy (Hofius et al., 2009; Chung et al., 2010;
Lai et al., 2011; Wang et al., 2011). lon2-4 carries a splice site
mutation at the 39 end of intron 3 and generates a premature
stop codon, likely truncating the protein. Both atg7-2 and atg7-3
single mutants responded to IBA similarly to the wild type and
Figure 1. Suppressors of lon2 Are IBA Sensitive and Lack PTS2-
(A) Number of lateral roots per millimeter root length of 8-d-old wild-type
(Wt) 35S:PTS2-GFP, lon2-2 35S:PTS2-GFP, and lon2-2 suppressor
seedlings (M5 progeny of original isolates) that were grown under yellow-
filtered light on Suc-supplemented medium for 4 d and then transferred
to Suc-supplemented medium with or without 10 mM IBA for an addi-
tional 4 d. Error bars show SD (n $ 12).
(B) Extracts from 8-d-old wild-type 35S:PTS2-GFP, lon2-2 35S:PTS2-
GFP, and each suppressor line grown in yellow light on Suc- and IBA-
supplemented medium were processed for immunoblotting and serially
probed with antibodies raised against the indicated proteins. PMDH is
expressed as a precursor (p); the PTS2 region is cleaved to the mature
(m) form in the peroxisome. Protein loading was monitored by probing
with antibodies against HSC70. The positions of molecular mass mark-
ers (in kilodaltons) are shown on the right.
[See online article for color version of this figure.]
Peroxisomes Are Regulated by Autophagy4087
fully processed PMDH, whereas lon2-2 and lon2-4 mutants were
completely resistant to the promotive effects of IBA on lateral
root production and incompletely processed PMDH (Figures
2C and 2D).
All five of our atg7 alleles suppressed lon2-2 IBA resistance
(Figure 2C) and rescued lon2 defects in PMDH processing
(Figure 2D). We examined ATG7 protein levels in the suppressor
mutants and did not detect ATG7 protein in the atg7-4, atg7-5,
or atg7-6 nonsense alleles. Although the atg7-7 and atg7-8
missense alleles retained low levels of atg7 protein (Figure 2D),
this protein is likely dysfunctional as atg7-7 and atg7-8 sup-
pressed lon2-2 phenotypic defects as fully as the nonsense
alleles (Figures 2C and 2D). As ATG7 activity is essential for
autophagosome formation (reviewed in Li and Vierstra, 2012),
we concluded that an intact autophagy system is required for
lon2 deficiencies in IBA responsiveness and PTS2 processing to
Mutations in the Autophagy Gene ATG2 Suppress
We used recombination mapping to localize suppressors 11-10
and 13-3 to the northern arm of chromosome 3 between
At3g12560 and At3g23633 (Figure 4A). We sequenced the ge-
nome of a backcrossed line of suppressor 13-3, and among the
seven homozygous mutations in coding sequences in the
mapping interval (Figure 3D; see Supplemental Data Set 1 on-
line), we identified a nonsense mutation (Trp-429 to stop) in the
ATG2 locus (At3g19190), which we named atg2-3 (Figure 4B).
Because the 11-10 suppressor mapped to a similar region, we
amplified and sequenced ATG2 in this mutant and discovered
a second nonsense allele (Trp-1441 to stop), which we named
atg2-4 (Figure 4B; see Supplemental Table 1 online). In yeast,
ATG2 associates peripherally with preautophagosomal struc-
tures during expansion and closure of the vesicle through as-
sociation with ATG18 and the multi-membrane-spanning ATG9
(Suzuki et al., 2007; Xie and Klionsky, 2007).
We examined an atg2 null allele in assays of peroxisome
function and found that seedlings of the atg2-1 T-DNA allele
(Inoue et al., 2006; Yoshimoto et al., 2009) responded to IBA
similarly to the wild type (Figure 4C) and processed PTS2 pro-
teins normally (Figure 4D). Like our atg7 alleles, both atg2 non-
sense alleles recovered in our screen fully suppressed lon2-2,
Figure 2. Multiple atg7 Alleles Suppress lon2-2 Peroxisomal Defects.
(A) Several lon2 suppressors mapped near ATG7 and LON2 on the
southern arm of chromosome 5. The fractions of recombinant chromo-
somes identified at two mapping markers (K16E1 and PDC2) in the 4-114
(lon2-2 atg7-8) suppressor mapping population are shown.
(B) Diagram of the ATG7 gene. Boxes and lines denote protein coding
regions and introns, respectively. The ThiF-like adenylation domain is
indicated. Triangles mark the locations of previously described T-DNA
insertion alleles. Arrows indicate the ATG7 active-site Cys and the po-
sitions of the nonsense (atg7-4, atg7-5, and atg7-6) and missense (atg7-7
and atg7-8) mutations that were identified as lon2-2 suppressors. The
sequence of the atg7-8 missense allele relative to the wild-type ATG7
sequence is shown. The original isolation number of each mutation is
listed in parentheses. aa, amino acids.
(C) Number of lateral roots per millimeter root length of 8-d-old wild type
(Wt), lon2-2, lon2-4, lon2-2 atg7, atg7-2, atg7-3, pex7-2, and deg15-1
seedlings grown under yellow-filtered light on Suc-supplemented me-
dium for 4 d and then transferred to Suc-supplemented medium with or
without 10 mM IBA for an additional 4 d. Error bars show SD (n $ 8).
(D) Extracts prepared from 10-d-old seedlings grown in white light on
Suc-supplemented medium were processed for immunoblotting with
antibodies to the indicated proteins. PMDH is expressed as a precursor
(p); the PTS2 region is cleaved to a mature (m) form in the peroxisome.
pex7-2 is a control displaying reduced PMDH processing because of
reduced matrix protein import (Ramón and Bartel, 2010); deg15-1 dis-
plays reduced processing because it is a null allele of the PTS2 pro-
cessing protease (Schuhmann et al., 2008; Lingard and Bartel, 2009).
Membranes from duplicate gels were serially probed with the indicated
antibodies to obtain the top two and bottom three panels. Protein
loading was monitored by probing with antibodies against HSC70.
Numbers below bands indicate the ATG7/HSC70 or PEX7/HSC70 ratios
in the labeled lanes, with the wild-type ratio normalized to 1.0. The po-
sitions of molecular mass markers (in kilodaltons) are shown on the right.
[See online article for color version of this figure.]
4088The Plant Cell
Figure 3. Whole-Genome Sequencing Reveals atg7 and atg2 Lesions in lon2-2 Suppressors.
Genomic DNA prepared from pooled F3 seedlings from three backcrossed lines was sequenced and examined for homozygous single nucleotide
polymorphisms typical of EMS mutagenesis (G/C to A/T transitions) in splice sites and coding sequences (nonsynonymous changes). Locus identifiers
of mutated genes were placed to the right of their positions on the five Arabidopsis chromosomes using the Chromosome Map Tool at The Arabidopsis
Information Resource (www.Arabidopsis.org). Mutations within the mapping intervals are depicted in black, other lesions are depicted in gray,
and the LON2 locus is indicated. Mutations in ATG7 were found in mutants 5-49 (A), 16-24 (B), and 4-114 (C); a mutation in ATG2 was found in
suppressor 13-3 (D).
[See online article for color version of this figure.]
Peroxisomes Are Regulated by Autophagy 4089
producing similar numbers of lateral roots as the wild type when
treated with IBA (Figure 4C) and fully processing the PTS2
proteins PMDH and thiolase (Figure 4D). Our isolation of atg2 as
a suppressor of lon2 physiological and molecular defects sup-
ports the conclusion that autophagy is required to observe lon2
IBA resistance and PTS2-processing defects.
A Mutation in the Autophagy Gene ATG3 Suppresses
We used recombination mapping to localize suppressors 14-22
and 14-36 to the southern arm of chromosome 5 near the LON2
locus. We sequenced the genome of pooled backcrossed lines
of the 14-22 suppressor. Among the 12 homozygous mutations
on chromosome 5 (Figure 5A; see Supplemental Data Set 1
online), we identified a mutation in the ATG3 locus (At5g61500),
which we named atg3-1 (Figure 5B). This mutation changes the
G that is the first nucleotide of exon 2 to an A, which would
change Trp-54 to a stop codon if the mRNA were still spliced
correctly. We also sequenced a single backcrossed line of
the 14-36 mutant and found the same atg3-1 lesion (see
Supplemental Data Set 1 online). Because 14-22 and 14-36
arose from the same M2 pool and carried the same atg3 lesion,
we concluded that these isolates were likely siblings. ATG3
functions as the E2-like conjugating enzyme that acts with ATG7
to lipidate ATG8 (Phillips et al., 2008; Yamaguchi et al., 2012);
atg3 mutants are thus expected to display defects similar to
those of atg7 mutants. We found that ATG3 immunoreactivity
was reduced in the lon2-2 atg3-1 mutant, indicating that the
atg3-1 splicing or nonsense lesion impaired ATG3 accumulation
(Figure 5D). The atg3-1 nonsense allele fully suppressed lon2-2
defects; the lon2-2 atg3-1 double mutant induced wild-type
numbers of lateral roots in the presence of IBA (Figure 5C) and
fully processed the thiolase and PMDH PTS2 proteins (Figure 5D).
Disrupting Autophagy Reduces lon2 Defects in Peroxisome
Size and Abundance
Enlarged peroxisomes can reflect defects in peroxisome division
or metabolic defects in the organelle. For example, mutants
disrupted in various peroxisome division factors (Mano et al.,
Figure 4. Multiple atg2 Alleles Suppress lon2-2 Peroxisomal Defects.
(A) Two lon2-2 suppressors (11-10 and 13-3) mapped near ATG2 on the
northern arm of chromosome 3. The fraction of recombinant chromo-
somes identified at three mapping markers (T2E22, MXL8, and LCS341)
in the 11-10 (lon2-2 atg2-4) suppressor mapping population are shown.
(B) Diagram of the ATG2 gene. Boxes and lines denote protein coding
regions and introns, respectively. The locations of the previously char-
acterized T-DNA insertion (atg2-1) and nonsense (atg2-2) alleles are
shown. The sequences generated by the atg2-3 and atg2-4 nonsense
mutations identified as lon2-2 suppressors are shown. The original
number of each mutation is listed. aa, amino acids.
(C) Number of lateral roots per millimeter root length of 8-d-old wild-type
(Wt), lon2-2, lon2-4, lon2-2 atg2, atg2-1, and pex7-2 seedlings that were
grown under yellow-filtered light on Suc-supplemented medium for
4 d and then transferred to Suc-supplemented medium with or without
10 mM IBA for an additional 4 d. Error bars show SD (n $ 8). This ex-
periment was conducted at the same time as the Figure 2C experiment;
control data are duplicated on the two graphs for clarity.
(D) Extracts from 10-d-old seedlings grown in white light on Suc-
supplemented medium were processed for immunoblotting. The mem-
brane was serially probed with antibodies to the indicated proteins.
Thiolase and PMDH are synthesized as precursors (p) that are processed
in the peroxisome to mature forms (m) lacking the PTS2 region. Protein
loading was monitored by probing with antibodies against HSC70.
Numbers below bands (or above for the PMDH and thiolase precursor
bands) indicate the ratio of the band to HSC70, normalized such that the
ratio from the wild type was set to 1.0. The positions of molecular mass
markers (in kilodaltons) are shown on the right.
[See online article for color version of this figure.]
4090 The Plant Cell
2004; Lingard et al., 2008; Zhang and Hu, 2009), the peroxi-
somal NAD+and CoA transporter (PXN) (Mano et al., 2011;
Agrimi et al., 2012; Bernhardt et al., 2012), or the fatty acid
b-oxidation enzyme 3-ketoacyl-CoA thiolase (KAT2/PED1)
(Hayashi et al., 1998) have enlarged peroxisomes. Similarly, lon2
mutants carrying a peroxisomally targeted GFP derivative dis-
play larger fluorescent puncta than the wild type (Burkhart et al.,
2013). We observed that the size of lon2-2 peroxisomes marked
by GFP-PTS1 (Zolman and Bartel, 2004) resembled wild-type
peroxisomes in epidermal and mesophyll cells of 4-d-old
seedling cotyledons (Figures 6A and 6B). As the seedlings aged,
however, lon2-2 puncta appeared larger and less abundant in
both mesophyll and epidermal cells (Figure 6B). In addition,
some cytosolic GFP-PTS1 became apparent in lon2-2 cotyle-
don cells (Figure 6B), concomitant with the appearance of PTS2-
processing defects as lon2 seedlings age (Lingard and Bartel,
2009). In contrast with the enlarged GFP-PTS1 puncta, chloroplasts
appeared similar to the wild type in the lon2-2 mutant (Figure
6B). Like lon2-2 expressing 35S:GFP-PTS1 (Figure 6B), cotyle-
dons of 8-d-old lon2-2 seedlings expressing 35S:PTS2-GFP
Figure 5. An atg3 Mutant Suppresses lon2-2 Peroxisomal Defects.
(A) Whole-genome sequencing data for the backcrossed 14-22 suppressor was analyzed as described in the legend to Figure 3. A G-to-A mutation in
the first nucleotide of ATG3 exon 2 was found. The same lesion was present in whole-genome sequencing data from the 14-36 suppressor (see
Supplemental Data Set 1 online).
(B) The position of the atg3 mutation present in the 14-22 and 14-36 suppressors is shown on a diagram of the ATG3 gene along with the location of the
ATG7 active-site Cys. Boxes and lines denote protein coding regions and introns, respectively. aa, amino acids.
(C) Number of lateral roots per millimeter root length of 8-d-old wild-type (Wt), lon2-2, lon2-2 atg3-1 (two isolates), pex7-2, and deg15-1 seedlings that
were grown under white light on Suc-supplemented medium for 5 d and then transferred to Suc-supplemented medium with or without 10 mM IBA and
grown under yellow-filtered light for an additional 3 d. Error bars show SD (n $ 5).
(D) Extracts from 10-d-old seedlings grown in white light on Suc-supplemented medium were processed for immunoblotting. Triplicate membranes
were serially probed with antibodies to the indicated proteins. Thiolase and PMDH are synthesized as precursors (p) that are processed to a mature
form (m) lacking the PTS2 region in the peroxisome. Protein loading was monitored by probing with antibodies against HSC70. Numbers above or
below bands indicate the ratio of the band to HSC70, normalized such that the ratio from the wild type was set to 1.0. The positions of molecular mass
markers (in kilodaltons) are shown on the right.
[See online article for color version of this figure.]
Peroxisomes Are Regulated by Autophagy4091
displayed large puncta that appeared to be less abundant than
wild-type seedling peroxisomes (Figures 7A and 7B). Because
older lon2 seedlings appeared to possess fewer peroxisomes
than the wild type, we used immunoblotting to examine levels of
several peroxins in 10-d-old lon2 seedlings. We observed that
lon2 mutants displayed apparently normal levels of the PTS1
receptor PEX5 but slightly reduced levels of the PTS2 receptor
PEX7 (Figure 4D) and the membrane peroxin PEX14 (Figures 4D
and 5D), consistent with our observation that peroxisomes were
less abundant in older lon2 seedlings.
In contrast with the large puncta observed in 8-d-old lon2-2
seedlings, the lon2-2 atg2, lon2-2 atg3, and lon2-2 atg7 mutants
had peroxisomes that more closely resembled wild-type per-
oxisomes in both size and abundance; we no longer detected
enlarged puncta in the suppressors (Figures 7C to 7H). More-
over, PEX14 levels were no longer reduced in lon2 atg2 (Figure
4D) or lon2 atg3 (Figure 5D) mutants compared with the wild
type. Because ATG2, ATG3, and ATG7 are essential for auto-
phagosome formation and closure, we concluded that muta-
tions in these loci prevented deposition of lon2 peroxisomes into
the lytic vacuole.
Because disrupting autophagy suppressed the size and
abundance defects of lon2 peroxisomes (Figure 7), we also
observed the effects of autophagy defects on peroxisome
morphology in seedlings with wild-type LON2. We found that
GFP-PTS1 fluorescence appeared similar to the wild type in
cotyledon cells of 4- and 8-d-old atg7-3 seedlings, which both
Figure 6. GFP-PTS1 Localization Reveals Import and Morphology
Defects in lon2-2 and Normal Appearance of atg7-3 Peroxisomes.
Cotyledon mesophyll and epidermal cells in 4- and 8-d-old wild-type (Wt)
(A), lon2-2 (B), and atg7-3 (C) seedlings expressing 35S:GFP-PTS1
were imaged for GFP fluorescence (green) using confocal microscopy.
Mesophyll cells also were imaged for chlorophyll autofluorescence
(magenta) to visualize chloroplasts. Epidermal cells from 8-d-old seed-
lings were imaged at both normal (left panels) and heightened (right
panels) gain settings to allow clear visualization of the GFP-PTS1 import
defect of the lon2-2 mutant. Bar = 20 mm.
Figure 7. atg2, atg3, and atg7 Mutations Suppress lon2-2 Defects in
Cotyledon epidermal cells in 8-d-old wild-type (Wt), lon2-2, and lon2-2
suppressor seedlings expressing 35S:PTS2-GFP were imaged for GFP
fluorescence (white) using confocal microscopy. Bar = 20 mm.
4092 The Plant Cell
displayed abundant small peroxisomes (Figure 6C). Our ob-
servations that disrupting autophagy did not notably impair
peroxisome morphology or abundance in these cells (Figure 6C)
and that atg2 and atg7 mutant seedlings responded like the wild
type to IBA (Figures 2C and 4C) are consistent with the possi-
bility that autophagy does not dramatically limit peroxisome
function in wild-type seedlings, at least in the tissues that we
Disrupting Autophagy Stabilizes Several Peroxisomal Matrix
Proteins in lon2 Mutants
During early seedling development, the metabolism and utilization
of stored fatty acids for energy is a critical function of peroxisomes.
The contents of seedling peroxisomes shift 4 to 5 d after ger-
mination when the glyoxylate cycle becomes obsolete as oil
stores are depleted and photosynthesis begins. During this
remodeling, the glyoxylate cycle enzymes ICL and MLS are
degraded, and photorespiration enzymes, including HPR, are
synthesized. Ubiquitin-dependent degradation is implicated in
peroxisome remodeling as mutations in both PEX4 and PEX22
result in partial ICL and MLS stabilization (Zolman et al., 2005;
Lingard et al., 2009). By contrast, resident peroxisomal pro-
teases have not been implicated in matrix protein turnover; ICL
and MLS are not notably stabilized in deg15 or lon2 mutants
(Lingard and Bartel, 2009; Burkhart et al., 2013).
To determine whether autophagy plays a role in peroxisome
remodeling via protein turnover during seedling maturation, we
examined ICL levels in maturing seedlings carrying the atg7-3
and atg2-1 null alleles. We found that ICL appeared to be de-
graded similarly to the wild type in both mutants (Figures 8A and
8B), suggesting that autophagy is not a major pathway through
which obsolete ICL is degraded. Moreover, we confirmed that
ICL was not stabilized in the lon2-2 mutant (Figures 8A and 8B),
consistent with previous reports (Lingard and Bartel, 2009;
Burkhart et al., 2013). Surprisingly, however, we observed that
ICL was markedly stabilized in our lon2-2 suppressors, including
lon2-2 atg2-3, lon2-2 atg3-1, and several lon2-2 atg7 mutants
(Figures 8A and 8B). These results suggest that autophagy must
be blocked to observe matrix protein stabilization in a lon2
We also examined degradation of MLS during seedling mat-
uration. As previously reported (Lingard et al., 2009), MLS was
similarly unstable in wild-type and lon2 seedlings (Figures 8C
and 8D). Like ICL, MLS was dramatically stabilized in lon2-2 atg
double mutants (Figures 8C and 8D). In addition, we found that
MLS was slightly stabilized in the atg7-3 and atg2-1 single
mutants (Figures 8C and 8D). These results suggest that both
LON2 and autophagy contribute to MLS turnover in wild-type
To examine whether LON2 substrates might extend beyond
the ICL and MLS glyoxylate cycle enzymes, we monitored thi-
olase levels during seedling development. Although not as
dramatic as the complete disappearance of ICL and MLS during
seedling maturation, thiolase levels also decline as seedlings
mature (Lingard and Bartel, 2009; Lingard et al., 2009). This
Figure 8. Several Peroxisomal Enzymes Are Stabilized in lon2-2 atg Mutants.
Extracts from 4-, 6-, and 8-d-old wild-type (Wt), lon2, atg, and lon2 atg seedlings grown in white light were processed for immunoblotting and serially
probed with antibodies against the indicated proteins. Protein loading was monitored by probing with antibodies against HSC70. Numbers below bands
(or above for the PMDH precursor band) indicate the ratio of the band to HSC70, normalized such that the ratio from 4-d-old wild type was set to 1.0.
The positions of molecular mass markers (in kilodaltons) are shown on the right.
[See online article for color version of this figure.]
Peroxisomes Are Regulated by Autophagy4093
decline is accelerated in lon2 mutants (Lingard and Bartel, 2009)
(Figure 8A). As with MLS, we found that the rate of thiolase
degradation was somewhat reduced in an atg7 mutant and
further delayed in a lon2 atg7 mutant (Figure 8A).
Because the HPR photorespiration enzyme begins accumu-
lating around 4 d after germination (Lingard et al., 2009), we also
assessed HPR levels in these experiments to examine whether
delayed development might contribute to the apparent stabili-
zation of ICL, MLS, and thiolase in lon2 atg or atg seedlings. We
found that HPR accumulated similarly to the wild type in these
mutants (Figures 8B and 8D). Like HPR, the PMDH matrix pro-
tein was present at wild-type levels in lon2, atg7, and lon2 atg7
mutants (Figure 8A). We concluded that the prolonged presence
of glyoxylate cycle and b-oxidation enzymes in lon2 atg
mutants was due to a degradation defect rather than delayed
Arabidopsis LON2 is a peroxisomal protease that is neces-
sary for sustained import of matrix proteins into peroxisomes
(Lingard and Bartel, 2009). We screened for lon2 suppressors
and found that the lon2 peroxisome-related defects, IBA re-
sistance and inefficient PTS2 processing (Figures 1, 2, 4, and
5) as well as large peroxisomes (Figure 7), were suppressed by
genetically preventing autophagy. Moreover, impairing autophagy
in the lon2 background revealed an additional phenotype: clear
stabilization of the b-oxidation enzyme thiolase and the glyox-
ylate cycle enzymes ICL and MLS (Figure 8). Although not as
dramatic, we also observed slight stabilization of thiolase and
MLS in atg single mutants (Figure 8), suggesting that some
peroxisomes are degraded via autophagy in wild-type Arabi-
dopsis seedlings. Our data are consistent with a model in
which Arabidopsis LON2 assists in the degradation of obsolete
matrix proteins (Figure 9A). When autophagy is prevented in
atg mutants, some matrix proteins (e.g., MLS and thiolase)
appear to be degraded slightly more slowly (Figure 9B). When
matrix protein degradation is impaired in lon2 mutants, perox-
isomes may be recognized as abnormal and targeted for de-
struction via autophagy at increased rates (Figure 9C). As lon2
cells age and peroxisome numbers decline, newly synthesized
matrix proteins appear to be inefficiently imported, presumably
leading to the observed lon2 defects in IBA-responsive lateral
rooting and processing of PTS2 proteins. When key autophagy
genes (e.g., ATG2, ATG3, or ATG7) also are nonfunctional, au-
tophagy is prevented, allowing lon2 peroxisomes to persist and
continue importing and processing matrix proteins and metabo-
lizing IBA, despite the continued presence of obsolete matrix
proteins in these peroxisomes (Figure 9D).
The model predicts that proteins delivered to the peroxisome
matrix in lon2 mutants will be degraded via pexophagy whereas
proteins synthesized after lon2 cells have few normal peroxisomes
will be predominantly cytosolic. Whether the latter matrix proteins
are stable or unstable in the cytosol can vary depending on the
protein. For example, HPR and PMDH are abundant in the cytosol
of 8-d-old lon2 cotyledon cells (Lingard and Bartel, 2009) and are
present at wild-type levels in lon2 seedling extracts (Figure 8). By
contrast, thiolase levels are reduced in lon2 seedlings (Figure 8)
(Lingard and Bartel, 2009), implying that thiolase is degraded when
not appropriately compartmentalized in the peroxisome.
Peroxisomal LON isoforms are found in plants (Ostersetzer
et al., 2007; Lingard and Bartel, 2009), mammals (Kikuchi et al.,
2004; Omi et al., 2008; Okumoto et al., 2011), and a subset of
lower eukaryotes, including H. polymorpha (Aksam et al., 2007)
and P. chrysogenum (Bartoszewska et al., 2012), but not yeast
(Saccharomyces cerevisiae) or fruit fly (Drosophila melanogaster)
(Lingard and Bartel, 2009). Unlike Arabidopsis lon2 mutants,
mutants lacking the H. polymorpha peroxisomal LON isoform
(Pln) display slightly more numerous peroxisomes than the
wild type (Aksam et al., 2007). P. chrysogenum pln mutants
Figure 9. A Working Model for LON2 Action during Maturation of
(A) As wild-type seedlings mature, LON2 acts to degrade obsolete matrix
proteins, including glyoxylate cycle enzymes ICL and MLS and the
b-oxidation enzyme thiolase. In addition, autophagy acts to remove
some peroxisomes, contributing to matrix protein degradation. As pho-
tosynthesis is established, peroxisomal photorespiratory enzymes, ex-
emplified by HPR, are synthesized and imported into peroxisomes.
(B) When autophagy is prevented, seedling peroxisomes function nor-
mally, but reduced peroxisome turnover results in slight stabilization of
certain matrix proteins (MLS and thiolase).
(C) In lon2 seedlings, autophagy of peroxisomes (pexophagy) is trig-
gered, perhaps due to a modification of the peroxisome (X). Although
obsolete matrix proteins are no longer degraded by LON2, peroxisomes
and their contents are degraded via pexophagy, resulting in ICL, MLS,
and thiolase disappearance. As pexophagy continues, the number of
import-competent peroxisomes declines, and cytosolic accumulation of
newly synthesized matrix proteins, such as HPR, becomes apparent.
(D) In lon2 atg double mutants, autophagy is absent and lon2 perox-
isomes are no longer subject to pexophagy. The continued import of
matrix proteins into lon2 peroxisomes allows peroxisome functions to
continue, restoring PTS2 processing and IBA-to-IAA conversion.
[See online article for color version of this figure.]
4094The Plant Cell
accumulate inactive catalase-peroxidase, implying that Pln
normally functions in the degradation of oxidatively damaged
proteins (Bartoszewska et al., 2012). The apparently normal per-
oxisome numbers in P. chrysogenum pln mutants, combined with
the normal peroxisomal GFP-PTS1 localization in P. chrysogenum
and H. polymorpha pln mutants (Aksam et al., 2007; Bartoszewska
et al., 2012), suggests that fungal peroxisomes lacking their resi-
dent LON protease are not targeted for pexophagy.
Peroxisomal LON (pLon) isoforms also are present in mam-
mals (Kikuchi et al., 2004; Omi et al., 2008; Okumoto et al.,
2011). RNA interference–mediated reduction of pLon levels in
mammalian cells via transient transfection does not noticeably
alter peroxisomal morphology or matrix protein import (Okumoto
et al., 2011). By contrast, expressing a dominant-negative pLon
derivative in tissue culture cells impairs peroxisomal matrix protein
import, and overexpressing a tagged pLon derivative results in
reduced numbers of enlarged peroxisomes (Omi et al., 2008),
similar to our observations in the Arabidopsis lon2 mutant. It would
be interesting to examine a role for pexophagy in these changes.
Our lon2 suppressor screen identified atg2, atg3, and atg7
alleles. Three atg7 null alleles (Figure 2B) have previously been
characterized using reverse-genetic approaches: atg7-1 (apg7-1)
is a T-DNA insertion in the 10th intron and displays premature
senescence (Doelling et al., 2002; Thompson et al., 2005), atg7-2
is a T-DNA insertion in the 7th exon that lacks ATG8-PE ad-
ducts and confers hypersensitivity to darkness-induced carbon
starvation (Hofius et al., 2009; Chung et al., 2010), and atg7-3 is
a T-DNA insertion in the 7th exon that confers powdery mildew
(Golovinomyces cichoracearum) resistance (Wang et al., 2011)
and hypersusceptibility to necrotrophic fungal pathogens (Lai
et al., 2011). Previously reported Arabidopsis ATG2 alleles in-
clude atg2-1, a T-DNA insertion in the 5th exon that disrupts
autophagy and induces early senescence and excessive pro-
grammed cell death under nutrient-rich conditions (Inoue et al.,
2006; Yoshimoto et al., 2009), and atg2-2, a nonsense mutation
(Gln-803 to stop) that blocks autophagy, displays early senes-
cence, and exhibits enhanced powdery mildew resistance via
increased cell death and constitutive defense-related gene ex-
pression (Wang et al., 2011). Our lon2-suppressing atg7 alleles
include three nonsense alleles, and all five alleles reduce ATG7
protein accumulation (Figures 2B and 2D). Similarly, our atg2
and atg3 alleles are all nonsense alleles (Figures 4B and 5B).
Because ATG2 functions in a different step of the autophagy
pathway than ATG3 and ATG7 (reviewed in Li and Vierstra,
2012), it is likely that any mutation abolishing autophagy would
suppress lon2 defects. Our recovery of multiple mutations in
ATG2 and ATG7 may reflect the fact that both are single-copy
genes in Arabidopsis that are above average in size, thereby
presenting unusually large targets for mutagenesis. ATG3 also is
a single-copy gene in Arabidopsis but had not previously
emerged from forward genetic screens. As atg3 alleles are not
present in publically available T-DNA collections, the atg3-1
nonsense allele described here will provide a useful tool for
analyzing ATG3 function in Arabidopsis. Moreover, we anticipate
that further screening for lon2 suppressors will uncover addi-
tional factors required for efficient autophagy in seedlings, as
well as genes that might be specifically required for pexophagy
The reduced numbers of peroxisomes in lon2 mutants and the
suppression of lon2 defects by blocking autophagy are consistent
with the possibility that pexophagy is induced in lon2 mutants
(Figure 9). What might trigger pexophagy in lon2? Autophagy in
plants is generally used under stress conditions including star-
vation and pathogen attack, as well as during programmed cell
death (reviewed in Bassham, 2007; Reumann et al., 2010; Li and
Vierstra, 2012). Perhaps metabolic stress in lon2 mutants trig-
gers pexophagy as reactive oxygen species (ROS) are produced
by peroxisomal metabolism. P. chrysogenum pln mutants dis-
play increased oxidative stress (Bartoszewska et al., 2012), and
elevated ROS is accompanied by increased pexophagy in
H. polymorpha (van Zutphen et al., 2011). However, H. polymorpha
peroxisomal LON mutants display both increased ROS levels
and more peroxisomes than the wild type, so a positive re-
lationship between ROS and pexophagy is not universal (Aksam
et al., 2007). H. polymorpha pexophagy also can be triggered by
degradation of Pex3p, a membrane peroxin acting in early per-
oxisome biogenesis (van Zutphen et al., 2011), suggesting that
alterations to peroxisome membrane proteins can trigger pex-
ophagy. We detected reduced levels of the membrane peroxin
PEX14 in lon2 seedlings, and it will be interesting to learn
whether this alteration is a cause or consequence of the in-
crease in pexophagy observed in lon2 mutants. Finally, accu-
mulation of damaged or misfolded organellar proteins can
trigger autophagy of the affected organelle. For example,
treating Arabidopsis seedlings with tunicamycin, which blocks
protein glycosylation and triggers the unfolded protein re-
sponse, promotes autophagy of the endoplasmic reticulum (Liu
et al., 2012). In addition, accumulation of protein aggregates in
the peroxisome matrix is accompanied by heightened pex-
ophagy in H. polymorpha (Manivannan et al., 2013). It is
tempting to speculate that the oxidized or obsolete matrix pro-
teins that accumulate as lon2 seedlings age trigger pexophagy.
Regardless of the specific pexophagy trigger in lon2, it does
not seem that undegraded matrix proteins cause the apparent
peroxisome expansion observed in lon2 as lon2 atg double
mutants overaccumulate ICL, MLS, and thiolase (Figure 8) but
have normally sized peroxisomes (Figure 7). This observation
raises the question of whether the large spherical structures
accumulating PTS2-GFP or GFP-PTS1 in older lon2 cells are in
fact peroxisomes or whether these structures might be pex-
ophagy intermediates. Future studies of seedlings in which au-
tophagosome markers have been introduced into lon2 and lon2
atg mutants are needed to address this question.
Specialized forms of autophagy, such as pexophagy, require
receptors targeting cargo destined for destruction to the inner
isolation membrane. For example, Pichia pastoris Atg30 local-
izes to the peroxisomal membrane through interactions with the
membrane peroxins Pex3 and Pex14, where phosphorylated
Atg30 can recruit Atg11 and Atg17 and induce pexophagy (Farré
et al., 2008). In S. cerevisiae, Atg36 binds to Pex3 and functions
as a pexophagy receptor (Motley et al., 2012). However, Atg30
and Atg36 homologs are not found outside of fungi (Farré et al.,
2008; Motley et al., 2012). A more widespread class of selective
autophagy receptors is represented by p62 and NBR1, adaptors
that tether ubiquitinated cargo to orthologs of the ATG8 ubiquitin-
like protein on the isolation membrane (Bjørkøy et al., 2005;
Peroxisomes Are Regulated by Autophagy4095
Kirkin et al., 2009). NBR1 functions as a pexophagy receptor in
mammals (Deosaran et al., 2013). It will be interesting to learn
whether Arabidopsis NBR1 (Svenning et al., 2011; Zientara-
Rytter et al., 2011), which functions in autophagy of insoluble
ubiquitinated aggregates (Zhou et al., 2013), similarly targets
peroxisomes for pexophagy in Arabidopsis and whether any
ubiquitinated proteins in the peroxisome membrane are required
for pexophagy. It is noteworthy that several peroxisomal pro-
teins are found in a recent proteomic analysis of ubiquitinated
Arabidopsis proteins (Kim et al., 2013).
Three pathways by which obsolete or damaged peroxisomal
matrix proteins might be turned over in Arabidopsis have been
suggested (Burkhart et al., 2013): (1) degradation within the
organelle by a resident protease, (2) degradation of the entire
organelle by autophagy, and (3) retrotranslocation and ubiquiti-
nation for proteasomal degradation in the cytosol. The first and
third pathways are consistent with the one-population hypoth-
esis and detection of transitional peroxisomes containing both
glyoxylate cycle and photorespiration enzymes (Titus and
Becker, 1985; Nishimura et al., 1986; Sautter, 1986). Previous
studies in Arabidopsis provide evidence for the third pathway
(Zolman et al., 2005; Lingard et al., 2009; Burkhart et al., 2013),
and our current data provide evidence for the first two pathways.
Is it possible that all three mechanisms are in play? Peroxisomal
protein import is unusual in that matrix proteins can be imported
as fully folded, oligomeric complexes (McNew and Goodman,
1994; Lee et al., 1997). Rather than fully degrading matrix pro-
teins, perhaps LON2 processes or disaggregates matrix pro-
teins, receptor complexes, or receptor-matrix protein complexes
to allow their ubiquitin-dependent retrotranslocation out of the
organelle. In the absence of LON2 function, ubiquitinated matrix
proteins might not efficiently retrotranslocate, resulting in ubiq-
uitination at the surface of the peroxisome, which could trigger
pexophagy. Future studies will be needed to resolve these
Although pexophagy functions in mammals and yeast to de-
grade excess peroxisomes (Iwata et al., 2006; Till et al., 2012),
plant peroxisomes are not known to undergo the dramatic shifts
in abundance that facilitate the study of pexophagy in other
systems. In addition, peroxisomal b-oxidation normally is re-
quired to mobilize carbon from fatty acids released during au-
tophagy of other organelles. For example, autophagy allows
plants to survive periods of carbon starvation, such as that in-
duced by extended darkness (Thompson et al., 2005; Phillips
et al., 2008). Peroxisomes also promote plant survival during
carbon starvation (Dong et al., 2009; Contento and Bassham,
2010), suggesting that membrane lipids freed from extraneous
organelles during autophagy are b-oxidized in peroxisomes.
Gene expression studies suggest that peroxisomes also would
mobilize carbon from fatty acids during senescence (Lopez-
Huertas et al., 2000), another time when autophagy is important
(Doelling et al., 2002; Hanaoka et al., 2002; Thompson et al.,
2005; Xiong et al., 2005). In contrast with these instances in
which the autophagy system collaborates with peroxisomes to
recycle nutrients, our results show that autophagy also can
dispose of damaged or abnormal peroxisomes and demonstrate
a requirement for ATG2, ATG3, and ATG7 in this process. Our data
indicate that pexophagy can dispose of seedling peroxisomes and
provide tools for the future elucidation of pexophagy requirements
and regulation in plant development.
Plant Materials and Growth Conditions
Arabidopsis thaliana accession Columbia-0 (Col-0) or Col-0 transformed
with 35S:PTS2-GFP (Woodward and Bartel, 2005a) were used as the wild
type. All mutants were in the Col-0 accession. lon2-2/SALK_043857
(Lingard and Bartel, 2009), deg15-1/SALK_007184 (Schuhmann et al.,
2008; Lingard and Bartel, 2009), atg2-1/SALK_076727 (Yoshimoto
et al., 2009), atg7-2/GABI_655B06 (Chung et al., 2010), and atg7-3/
SAIL_11_H07 (Lai et al., 2011; Wang et al., 2011) were previously de-
scribed and were from the ABRC. lon2-2 35S:PTS2-GFP (Lingard and
Bartel, 2009) and pex7-2 (Ramón and Bartel, 2010) were previously de-
scribed. lon2-4 is a probable null allele that was isolated from a screen
for Suc dependence and IBA resistance (by A.W. Woodward and M.
Bjornson); it contains a G-to-A transition in the 39 nucleotide of intron 3
that generates a frameshift followed by a premature stop codon. Col-0
transformed with 35S:GFP-PTS1 was described previously (Zolman and
Bartel, 2004), and lon2-2 35S:GFP-PTS1 and atg7-3 35S:GFP-PTS1 lines
were generated by crossing and genotyping the F2 progeny by PCR.
Mutant genotypes were assayed using PCR-based markers (see
Supplemental Table 2 online).
Seeds were surface-sterilized, resuspended in sterile water or 0.1%
agar, and stratifiedat4°C for1to3d. Unlessotherwise noted,plants were
grown at 22°C on plant nutrient medium (Haughn and Somerville, 1986)
supplemented with 0.5% (w/v) Suc (PNS) and solidified with 0.6 or 1.0%
agar. After stratification, seeds were placed under continuous light. To
quantify IBA sensitivity, seedlings were grown on PNS plates under light
filtered with yellow long-pass filters (to slow photochemical breakdown of
indolic compounds; Stasinopoulos and Hangarter, 1990) for 4 to 5 d,
transferred to new PNS plates with or without 10 mM IBA, and grown
vertically under yellow light for an additional 3 to 4 d. Lateral roots
emerged from the primary root were counted using a dissecting micro-
scope; primary root lengths were measured with a ruler or using Image J
3.0 (http://rsbweb.nih.gov/ij/) after imaging seedlings using a Gel Doc
Mutant Isolation and Recombination Mapping
Seeds of lon2-2 (Lingard and Bartel, 2009) carrying the 35S:PTS2-GFP
transgene (Woodward and Bartel, 2005a) were mutagenized with ethyl
methanesulfonate (EMS) (Normanly et al., 1997) and grown in 16 pools.
Approximately 5000 M2 seeds from each pool were surface sterilized,
stratified for 2 d at 4°C, and plated on PNS supplemented with 3 mM IBA.
Seedlings were grown vertically under yellow-filtered light for 10 d, and
seedlings with more than three to four lateral roots were moved to soil for
seed production. M3 progeny were retested for lateral root production on
10 µM IBA, and seedlings from the 61 strongest suppressor lines were
tested by immunoblot analysis with anti-PMDH antibodies (as described
below) for rescue of the lon2 PTS2-processing defect. Lines exhibiting
IBA sensitivity and lacking PTS2-processing defects were retained as
lon2 suppressors. Suppressor lines were backcrossed to lon2-2 once
prior to phenotypic analyses shown in Figures 2 and 4. Homozygous
backcrossed suppressor lines were selected from the F2 generation for
IBA-induced lateral root formation and were subsequently genotyped for
lon2-2 homozygosity as described (Lingard and Bartel, 2009).
For recombination mapping, suppressor mutants isolated in the Col-0
background carrying the lon2-2 T-DNA insertion were outcrossed to
a lon2-2 line that had been introgressed into Landsberg erecta by three
successive outcrosses to Landsberg erecta. F2 seedlings from each
4096The Plant Cell
outcross were selected on 10 µM IBA for suppression of lon2-2 IBA
resistance. DNA was isolated from F2 plants exhibiting wild-type lateral
root numbers by homogenizing leaf tissue in extraction buffer (200 mM
Tris, pH 7.5, 250 mM NaCl, 25 mM EDTA, and 0.5% SDS) followed by
chloroform extraction and isopropanol precipitation of the aqueous
(see Supplemental Table 3 online) as previously described (Konieczny and
Ausubel, 1993; Bell and Ecker, 1994). ATG2, ATG3, and ATG7 were PCR
amplified from genomic DNA prepared from mutants with the primer pairs
listed in Supplemental Table 1 online. Amplicons were sequenced directly
(Lone Star Labs) with the primers used for amplification.
Genomic DNA Isolation and Whole-Genome Sequencing
For each suppressor mutant, F3 seedlings from three backcrossed lines
(homozygous for both lon2-2 and the suppressing lesion) were pooled for
DNA extraction to reduce the number of noncausal homozygous muta-
tions identified. Approximately 2000 surface-sterilized seeds were grown
under continuous white light on PNS overlaid with sterile filter paper for
7 or 9 d. Seedlings were ground in liquid nitrogen and homogenized in
7 mL of prewarmed (65°C) Buffer S (110 mM Tris, pH 8.0, 55 mM EDTA,
1.54 M NaCl, and 1.1% hexadecyltrimethylammonium bromide). Ho-
end-over-end, and incubated at 65°C for 2 h with occasional inversion.
Samples were allowed to cool at room temperature for 5 min, after which
4 mL of 24:1 chloroform:isoamylalcohol was added. Samples were mixed
by inversion for 15 min and then centrifuged at 3000 rpm for 20 min. The
aqueous phase was extracted with 4 mL of 24:1 chloroform:isoamyl al-
cohol and DNA was precipitated by adding 0.6 volumes of isopropanol.
DNA was collected by centrifugation and dissolved in 4mL of 10 mM Tris-
HCl, 1mMEDTA, pH8.0.Samples weretreatedwith 10µg/mLofRNaseA
(Sigma-Aldrich; R-4875) at 37°C for 1 h. After chloroform extraction, DNA
in the aqueous phase was precipitated by adding 0.1 volumes of 3 M
sodium acetate, pH 5.2, and 2 volumes of ice-cold 95% ethanol. After
centrifugation for 20 min at 2000 rpm, the DNA pellet was washed with
3 mL 70% ethanol. Sample tubes were inverted and allowed to drain for
20 min, dried at 37°C for 20 min, and dissolved in 200 to 500 mL 10 mM
Tris-HCl, 1 mM EDTA, pH 8.0.
Genomic DNA (;20 µg/mutant) was submitted to the Genome
Technology Access Center at Washington University in St. Louis for
sequencing with an Illumina HiSequation 2000 sequencer and compar-
ison to The Arabidopsis Information Resource 10 build of the Arabidopsis
Col-0 genome. Because EMS causes G/C-to-A/T transitions in Arabi-
dopsis (Greene et al., 2003), we disregarded insertions and deletions and
retained single nucleotide polymorphisms consistent with EMS muta-
genesis and located in splice sites or coding sequences. We also dis-
carded heterozygous lesions, those that resulted in synonymous codon
changes, those that were present in our lab stock of Col-0, and two
lesions found in all sequenced suppressors that likely represented
noncausal, fixed lesions present in the starting strain.
Extracts were prepared from seedlings at indicated ages by homoge-
nizing frozen seedlings in 2 volumes of 23 sample buffer (Invitrogen)
containing 0.05 M DTT and heated at 100°C for 5 min. Samples were
electrophoresed on NuPAGE 10% or 12% Bis-Tris gels (Invitrogen) using
13 MES running buffer (50 mM MES, 50 mM Tris base, 0.1% SDS, and
1mM EDTA)or 13 MOPSrunning buffer(50 mMMOPS, 50mM Tris base,
0.1% SDS, and 1 mM EDTA) and transferred to Hybond Nitrocellulose
membrane (Amersham Pharmacia Biotech) using NuPAGE transfer buffer
(Invitrogen). Membranes were blocked in 8% milk solution (or 5% BSA for
the anti-ATG3 antibody) in 20 mM Tris, pH 7.5, 150 mM NaCl, and 0.1%
Tween 20 and subsequently incubated overnight at 4°C with primary
antibodies in blocking solution. Rabbit antibodies against ATG3
(1:10,000; Phillips et al., 2008), ATG7 (1:1000; Doelling et al., 2002), ICL
(1:1000; Maeshima et al., 1988), HPR (1:10,000; Agrisera AS11 1797),
MLS (1:25,000; Olsen et al., 1993), the PED1 isoform of thiolase (1:2500;
Lingard et al., 2009), PEX5 (1:100; Zolman and Bartel, 2004), PEX7 (1:800;
Ramón and Bartel, 2010), PEX14 (1:10,000; Agrisera AS08 372), and
PMDH2 (1:2000; Pracharoenwattana et al., 2007) were diluted as
indicated. Mouse antibodies against HSC70 (1:20,000 or 1:50,000;
StressGen Bioreagents SPA-817) were used. Primary antibodies were
visualized with horseradish peroxidase–conjugated goat anti-rabbit or
anti-mouse IgG secondary antibodies (1:5000 dilution in blocking buffer;
Santa Cruz Biotechnology, SC2030 or SC2031). Horseradish peroxidase
was visualized by incubation with WesternBright ECL reagent (Advansta),
and signal was detected using autoradiography film. Membranes were
reblocked and sequentially probed with the indicated antibodies without
stripping the membrane between incubations.
For quantification of immunoblot images, films were photographed
on a light box, and the resulting TIFF images were analyzed using
ImageJ Gel Analysis software. The areas under the density curves from
selected bands were normalized by dividing by the area of the ap-
propriate HSC70 bands (after subtraction of background density).
These ratios were further normalized by dividing by the wild-type band
for the protein of interest.
Confocal Fluorescence Microscopy
Cotyledons of light-grown seedlings were mounted in water, and fluores-
cence was visualized using a Carl Zeiss LSM 710 laser scanning confocal
microscope equipped with a meta detector. lon2-2 suppressor seedlings
were from self-fertilized progeny of the original isolates. Samples were
imaged using a 363 oil immersion objective. GFP and chlorophyll were
excited with a 488-nm argon laser, GFP emission was collected be-
tween 493 and 572 nm, and chlorophyll autofluorescence was detected
between 620 and 719 nm. Each image is an average of four different
exposures using a 47.1-mm pinhole, corresponding to a 0.8-mm optical
the ClustalW default settings with the Gonnet series protein weight matrix.
Sequence data from this article can be found in the Arabidopsis Genome
Initiative or GenBank/EMBL databases under the following accession
numbers: ATG2 (At3g19190), ATG3 (At5g61500), ATG7 (At5g45900),
DEG15 (At1g28320), LON2 (At5g47040), and PEX7 (At1g29260).
The following materials are available in the online version of this article.
Supplemental Figure 1. ATG7 Protein Alignment Depicting atg7
Supplemental Table 1. Primers Used for Candidate Gene Sequencing.
Supplemental Table 2. PCR-Based Markers Used for Mutant
Supplemental Table 3. PCR-Based Markers Used for Recombination
Supplemental Data Set 1. Mutations Identified in lon2-2 Suppressors
by Whole-Genome Sequencing.
Peroxisomes Are Regulated by Autophagy4097
We thank Richard Vierstra (University of Wisconsin–Madison) for the anti-
ATG3 and anti-ATG7 antibodies, John Harada (University of California,
Davis) for the anti-MLS antibody, Steven Smith (University of Western
Australia) for the anti-PMDH2 antibody, Masayoshi Maeshima (Nagoya
University) for the anti-ICL antibody, and the ABRC for seeds from
T-DNA insertion lines. We thank Andrew Woodward (University of Mary
Hardin-Baylor) and Marta Bjornson (Univeristy of California, Davis) for
sharing lon2-4 seeds prior to publication, Matthew Lingard (St. Louis) for
crossing lon2-2 and atg7-3 to 35S:GFP-PTS1, Wendell Fleming (Rice
University) for assistance with bioinformatics, and Lucia Strader (Wash-
ington University) for advice on genome sequencing. We thank Wendell
Fleming, Kim Gonzalez (Rice University), Yun-Ting Kao (Rice University),
and Andrew Woodward for critical comments on the article. We thank the
Genome Technology Access Center in the Department of Genetics at
Washington University School of Medicine for help with genomic
analysis. This center is supported by National Cancer Institute Cancer
Center Support (P30 CA91842), the National Institutes of Health (NIH)
National Center for Research Resources (UL1RR024992), and NIH
Roadmap for Medical Research. Confocal microscopy was performed
on equipment obtained through a Shared Instrumentation Grant from the
NIH (S10RR026399-01). This research was supported by the NIH
(R01GM079177), the National Science Foundation (MCB-0745122),
and the Robert A. Welch Foundation (C-1309).
L.M.F. and B.B. conceived and designed the experiments. L.M.F.
executed most of the experiments. M.A.R. conducted the confocal
microscopy and assisted with protein stability experiments. P.G.Y.
assisted with genotyping and atg3 characterization. C.H.D. assisted
with suppressor mapping and genotyping. S.E.B. assisted with protein
stability experiments and genotyping. L.M.F. and B.B. wrote the article
with input from the other authors. All authors approved the final version
of the article.
Received May 8, 2013; revised September 9, 2013; accepted October 8,
2013; published October 31, 2013.
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4100 The Plant Cell
; originally published online October 31, 2013; 2013;25;4085-4100
Lisa M. Farmer, Mauro A. Rinaldi, Pierce G. Young, Charles H. Danan, Sarah E. Burkhart and Bonnie
Role for the LON2 Protease in Peroxisomal Matrix Protein Degradation
Mutant and Reveals a
Disrupting Autophagy Restores Peroxisome Function to an
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