Feature Review

Autophagosome Biogenesis and the

Endoplasmic Reticulum: A Plant Perspective
Xiaohong Zhuang,1,4,* Kin Pan Chung,1,3,4 Mengqian Luo,1 and Liwen Jiang1,2,*

The autophagosome is a double-membrane compartment formed during Highlights

autophagy that sequesters and delivers cargoes for their degradation or recy- Recent studies have uncovered new
cargoes for selective autophagy in
cling into the vacuole. Analyses of the AuTophaGy-related (ATG) proteins have
plants, including organelles, ribosomes,
unveiled dynamic mechanisms for autophagosome biogenesis. Recent advan- pathogens, proteasomes, and plant-
ces in plant autophagy research highlight a complex interplay between auto- specific proteins.

phagosome biogenesis and the endoplasmic reticulum (ER): on the one hand Recent studies have suggested various
ER serves as a membrane source for autophagosome initiation and a signaling models and mechanisms for autopha-
platform for autophagy regulation; on the other hand ER turnover is connected gosome initiation in yeast, mammals,
and plants. Advanced techniques
to selective autophagy. We provide here an integrated view of ER-based including super-resolution imaging,
autophagosome biogenesis in plants in comparison with the newest findings electron tomography, and cryo-electron
microscopy have been applied in autop-
in yeast and mammals, with an emphasis on the hierarchy of the core ATG
hagy research to reveal the mechanisms
proteins, ATG9 trafficking, and ER-resident regulators in autophagy. of phagophore assembly.

An Update on the Roles of Autophagy in Plants The ER is interconnected with autop-

Macroautophagy, hereafter referred as autophagy (see Glossary), is an evolutionarily con- hagosome biogenesis by serving as
the main membrane/lipid source of
served degradative process for the delivery of harmful or unwanted materials (e.g., damaged autophagosome biogenesis, by pro-
organelles, protein aggregates, and pathogens) into the lytic compartment of the cell: the viding a cradle for the assembly of
lysosome (animals) or vacuole (yeast and plants) [1]. During this process the degradative the core autophagic machinery includ-
ing the ATG1/ULK1 complex, the
cargoes are engulfed into a compartment called the autophagosome, which subsequently
ATG9 complex, and the PI3K complex,
fuses with the lysosome/vacuole (Figure 1), where they are degraded by hydrolases and and by acting as a signaling platform
proteases, and recycled back as building blocks (amino acids and fatty acids) for protein and cargo during ER stress to maintain
synthesis and energy supplies to facilitate survival during stress conditions such as starvation. cellular homeostasis.

ATG9 vesicles from the Golgi–endoso-

A crucial feature of autophagosome biogenesis is that it is a highly dynamic process involving a set mal system have been recently identi-
of ATG proteins (named Atg in yeast and ATG in mammals/plants) [2]. Most of the core ATG fied as a crucial component
proteins are conserved in the plant genome, and these can be divided into five subcomplexes: the contributing to autophagosome bio-
genesis in a plant-specific manner.
ATG1 complex, the class III phosphoinositide 3-kinase (PI3K) complex, the ATG9 complex, and
two ubiquitin-like conjugation systems (ATG5–ATG12 and ATG8). It has long been established
that autophagy is required for plant development at a basal level for nutrient recycling (e.g.,
nitrogen remobilization). Therefore, the direct impact of defective autophagy in plants includes
early leaf senescence as well as reduced yield and seed quality [3,4]. Impairment of autophagy also 1
Center for Cell and Developmental
leads to the accumulation of protein aggregates and dysfunctional organelles (e.g., mitochondria, Biology, State Key Laboratory of
Agrobiotechnology, School of Life
peroxisomes, and chloroplasts), as well as increased sensitivity to oxidative stress accompanied Sciences, The Chinese University of
with enhanced levels of reactive oxygen species [5–13]. In addition, deficiency in autophagy leads Hong Kong, Shatin, New Territories,
to a significantly reduced hypersensitive response and immunity-related programmed cell death Hong Kong, China
2
The Chinese University of Hong Kong
(PCD) upon pathogen infection. Excellent reviews on the physiological functions of plant autoph- Shenzhen Research Institute,
agy have been published recently [3,14–16]. Shenzhen, China
3
Current address: Max Planck
Institute of Molecular Plant
In recent years our understanding of plant autophagy regulation has been greatly expanded at Physiology, Am Mühlenberg 1 14476,
the mechanistic level. For example, signaling transduction kinases responsible for the sensing Potsdam-Golm, Germany

Trends in Plant Science, August 2018, Vol. 23, No. 8 https://doi.org/10.1016/j.tplants.2018.05.002 677
© 2018 Elsevier Ltd. All rights reserved.
 4
of energy (e.g., ATP) and nutrients (e.g., amino acids) have been identified as crucial switches of These authors contributed equally to
this work
autophagy activation in plants. The target of rapamycin (TOR) complex is well established as a
negative regulator in plant autophagy, although a TOR-independent autophagy pathway
induced by oxidative and ER stress has also been recently reported [17,18]. In contrast to *Correspondence:
the mammalian mTORC2 complex which directly senses amino acid levels, the plant TOR xhzhuang@cuhk.edu.hk (X. Zhuang)
and ljiang@cuhk.edu.hk (L. Jiang).
complex can sense the concentration of the amino acid precursors [19]. It has been suggested
that specific adaptation of the TOR signaling network has taken place in plants because two
TOR subcomplexes, mTORC1 and mTORC2, have been identified in mammals while mTORC2
components are absent in plants. On the other hand, another essential sensor kinase: SnRK1
complex (SNF1-related kinase 1), the ortholog of yeast Snf1 and mammalian AMPK, has
recently been characterized in plant autophagy. It was shown that two subunits of the SnRK1
complex, KIN10 and KIN11, positively regulate plant autophagy upstream of TOR [20,21]. In
addition, ubiquitination has been identified as a novel signaling switch in autophagy

(A)
InducƟon ElongaƟon and expansion MaturaƟon and fusion DegradaƟon

Phagophore Autophagosome Vacuole

Autophagic
body

TSPO
AGO1 BES1
Protein
aggreate ER Mitochondrion Chloroplast Peroxisome Proteasome Ribosome Pathogen Selected proteins

(B) YFP–ATG8e (C) AnƟ-SH3P2

50 μm 50 μm 100 nm 100 nm
Control BTH+

Figure 1. An Overview of Plant Autophagy. (A) Schematic model showing the autophagosome formation in plants. During autophagy, an isolation membrane
structure called the phagophore is formed. Following its elongation and expansion, the phagophore grows to sequester the cargo. After the phagophore is sealed, it
becomes the double-membrane autophagosome. The autophagosome undergoes further maturation steps and finally fuses with the vacuole to become an
autophagic body with a single limiting membrane inside the vacuole. Ultimately, the autophagic body as well as the cargoes will be degraded. So far, several
cargoes have been identified in both selective and non-selective autophagy pathways, including protein aggregates, ER, mitochondria, chloroplasts, peroxisomes,
proteasomes, ribosomes, pathogens, and specific proteins (e.g., AGO1, TSPO, BES1). (B) Confocal images showing the patterns of an autophagosome marker YFP–
ATG8e expressing in transgenic arabidopsis (Arabidopsis thaliana) root cells before and after BTH (acibenzolar-S-methyl) treatment. Ring-like autophagosome
structures (insets) were observed upon autophagic induction with BTH treatment. (C) TEM images showing autophagosomal structures labeled by anti-SH3P2-
associated gold particles in transgenic arabidopsis root cells after BTH treatment. Abbreviations: ER, endoplasmic reticulum; TEM, transmission electron microscopy.

678 Trends in Plant Science, August 2018, Vol. 23, No. 8

dynamics. It was shown that, under starvation conditions, members of the SINAT E3 ubiquitin Glossary
ligase family trigger the ubiquitination of BES1, a central growth transcriptional regulator in Autophagic body: the outer
brassinosteroid signaling, leading to targeting by the ubiquitin receptor DSK2 and autophagic membrane of the autophagosome
degradation of BES1 [11]. Another study has showed that the SINAT family of E3 ubiquitin fuses with the vacuolar membrane,
releasing the inner membrane and
ligases, together with two tumor necrosis factor receptor-associated factor family proteins associated cargo into the inside of
(TRAF1a and TRAF1b), participate in autophagy by mediating ATG6 degradation upon star- vacuole. This inner membrane-
vation [22]. containing structure inside the
vacuole is called an autophagic
body.
Understanding where the autophagosome forms and how ATG proteins are coordinated in this Autophagic flux: the ratio of
event is a central point of focus in autophagy [23]. In yeast and mammalian cells, recent studies cargoes that are recognized,
have revealed how ATG proteins orchestrate membrane remodeling to create autophago- segregated, and degraded through
the autophagy pathway; this is used
somes in four distinct steps, including nucleation and initiation of a crescent-shaped membrane
as an indicator of autophagic activity
called the phagophore, followed by its expansion and closure [24,25]. Pioneering work has instead of measuring
also characterized the conserved function of the ATG machinery in plants, particularly the two autophagosome numbers.
conjugating systems [26–28], but investigations into the molecular machinery for autophago- Autophagosome: a double-
membrane structure formed to engulf
some initiation in plants still lag considerably behind research in yeast and mammals. Recent
cytoplasmic components which are
evidence in plants suggests that the ER might serve as a membrane source and signaling then delivered to the vacuole for
platform for autophagosome biogenesis, especially under ER stress [29–32]. In this review we degradation.
attempt to provide an integrated view of ER-based autophagosome biogenesis in plants in Autophagy: the sequestration of
cytoplasmic contents (e.g., abnormal
comparison with the newest findings in yeast and mammals. In doing so we have focused on intracellular proteins, excess or
the molecular roles of the core ATG proteins and ER-related regulators in membrane dynamics damaged organelles) and their
for autophagosome initiation. subsequent delivery into the vacuole
for degradation or recycling.
Coat protein complex II (COPII)
The ER as a Membrane Source for Autophagosome Biogenesis vesicles: the vesicles that mediate
Early work on autophagosome biogenesis stems from budding yeast (Saccharomyces cer- protein trafficking from the ER to the
evisiae) and used microscopy to detect autophagosomal structures [25]. In the wake of Golgi.
ER exit sites (ERES): specialized
innovative techniques [e.g., super-resolution imaging, electron tomography (ET), and cryo-
regions of the endoplasmic reticulum
electron microscopy (cryo-EM)] (Box 1), our understanding of autophagosome biogenesis has (ER) for the export of proteins and
been raised to a higher level. Working on different experimental systems, multiple membrane lipids by forming COPII vesicles.
sources, especially the ER and ER-derived compartments, have been shown to be involved in LIR/AIM motif: LC3-interacting
region (LIR) or the Atg8-interacting
autophagosome formation (Figure 2A).
motif (AIM), a conserved motif for
targeting autophagy receptors to
LC3/ATG8 family proteins anchored
Box 1. Application of Super-Resolution Imaging, ET, and Cryo-EM in Autophagy Studies
in the phagophore membrane.
Over recent decades laser scanning confocal microscopy (LSCM) has become a popular tool for imaging autophago- Omegasome: an omega-shaped
some dynamics and autophagic flux using fluorescence-tagged/labeled autophagosome markers [104]. Compared to protrusion derived from a
the max 200 nm resolution of LSCM, advancing super-resolution imaging systems including stimulated emission phosphatidylinositol 3-phosphate
depletion (STED) microscopy, structured illumination microscopy (SIM), and stochastic optical reconstruction micro- (PI3P)-enriched ER subdomain that
scopy (STORM) allow researchers to clearly visualize diverse types of structures involved in autophagy with an improved serves as a platform for
resolution of 20–100 nm. For example, super-resolution microscopy has revealed the assembly of ULK1 complex on autophagosome biogenesis in
the ER and its association with ATG9 and ERGIC membrane compartments [42]. mammalian cells.
Phagophore: also called the
Furthermore, in the history of autophagy research, transmission EM (TEM) has been an indispensable tool in almost all of isolation membrane, a cup-shape
the milestone findings to verify autophagosome structures at the nanoscale [104]. CLEM is an integrated system which double-membrane structure that
enables the sample to be imaged by an electron beam and an optical light path simultaneously, and ultra-high resolution matures into autophagosomes.
structural information of the same targets can be obtained by EM. This method has been used to study the nature of Pre-autophagosomal structure
ATG9 vesicles as well as the relationship between the ULK1 complex and the early autophagic machinery [42,80]. ET is (PAS): also known as the
an extension of traditional TEM in which a tilt series of 2D projections is used to retrieve 3D structural information, such phagophore assembly site, the PAS
as of the phagophore–ER connection [31,41]. Single-particle EM is useful for obtaining high resolution of the con- is a site for ATG protein recruitment
formational complexity in the autophagic complex (e.g., TRAPPIII and PI3K complexes in yeast) [37]. Cryo-EM is a and assembly in yeast.
combination of TEM with cryo-techniques at liquid nitrogen temperatures. It is a powerful emerging structural biology Selective autophagy: a selective
technique that enables the visualization of macromolecular complexes in their functional cellular context by avoiding autophagic process for specific
harmful preparation methods. New mechanistic insights in autophagy research should be achieved with these new cargoes involving specific receptors,
technological breakthroughs in the future. such as mitophagy for mitochondrial

Trends in Plant Science, August 2018, Vol. 23, No. 8 679

In yeast, autophagosomal structures are often observed under fluorescent microscopy as a components, pexophagy for
single dot in close proximity to the vacuole [25]. The term pre-autophagosomal structure peroxisomal substrates, and
chlorophagy for chloroplast contents.
(PAS) was initially used in yeast as a location for the assembly of Atg proteins. Atg9 vesicles Transport protein particle
have been shown to be a major source for autophagosome formation in yeast [33] (Figure 2Ai). (TRAPP) III complex: this
During starvation, a few Atg9 vesicles are recruited to the PAS by the Atg1 complex for comprises the TRAPPI complex and
phagophore nucleation [33,34]. Meanwhile, the transport protein particle (TRAPP) III an additional subunit, Trs85–an
autophagy-specific guanine
complex is recruited by the Atg9 vesicles to the PAS to promote autophagosome formation nucleotide exchange factor that is
[35]. Microscopy studies have revealed that the PAS localizes in close proximity to both ER exit recruited to the phagophore
sites (ERES) and Atg9 vesicles, and mutants in functions essential for ERES are defective in assembly site by the Rab GTPase
Ypt1 during autophagy.
autophagy, suggesting that ERES may contribute to phagophore formation [36]. Indeed,
Ubiquitination: a process by which
single-particle EM of the TRAPPIII complex showed that the ERES-derived coat protein ubiquitin is conjugated to a substrate
complex II (COPII) vesicles are recruited to the PAS, which then tether with the Atg9 vesicles protein and directs these proteins for
via the TRAPPIII complex and the Atg1 complex [37]. COPII vesicles may possibly carry their degradation either by the
proteasome or the lysosome/
cargoes from the ER to the PAS to facilitate membrane remodeling and fusion. For example, it is
vacuole.
reported that the ER-resident SNARE protein Ufe1 is exported in specific COPII vesicles for Unfolded protein response (UPR):
autophagosome biogenesis [38], whereas COPII vesicles are reprogrammed during autophagy the UPR is activated to cope with ER
in which several COPII cargo adaptors (e.g., Sec23/Sec24 and Sec13) interact with Atg9 to stress by reducing the levels of
misfolded proteins.
regulate autophagosome number [39].

In contrast to yeast, ER subdomains in mammals appear to play a direct role in the early stages
of autophagosome formation which involve a specialized structure named the omegasome
[40] (Figure 2Aii). Omegasome formation is determined by the levels of PI3P (phosphatidyli-
nositol 3-phosphate), ULK1, and the PI3K complexes [40]. Upon autophagic induction, ATG
proteins are recruited to PI3P-enriched ER subdomains, and the growing phagophore or
isolation membrane becomes sandwiched between two ER cisternae, resulting in the omega-
shaped omegasome [40]. ET has provided direct evidence for a physical connection between
the ER and the phagophore via narrow tubules [41]. Recently, using super-resolution micros-
copy and correlative EM (CLEM) after live imaging, ULK1 complex assembly was shown to
occur on ER tubular-vesicular membranes coalescing with mATG9 vesicles, which are in close
proximity with the phosphatidylinositol synthase (PIS)-enriched ER subdomains [42,43]. In
addition, both ERES and the ERGIC (ER–Golgi intermediate compartment) have been impli-
cated in autophagosome biogenesis [42,44]. ER–organelle contact sites that are not involved in
vesicular trafficking, such as ER–mitochondria contact sites and ER–plasma membrane (PM)
contact sites, have also been reported to serve as a platform for omegasome formation,
probably via the production of phospholipids and recruitment of components of the molecular
machinery [45,46]. For example, the ER-resident SNARE protein syntaxin 17 (STX17) was
shown to translocate to ER–mitochondria contact sites during autophagy, and recruited
ATG14 to initiate phagophore formation [45]. In addition, autophagosome formation is signifi-
cantly suppressed upon knockdown of ER–mitochondrion tethering factors (e.g., mitofusin 2
and PACS-2) or of ER–PM tethering factors (e.g., extended synaptotagmins E-Syts1/2/3)
[45,46].

Although the mechanisms underlying autophagosome initiation in plants remain elusive, a

working model for autophagosome formation has emerged in recent years, pointing to the ER
as an essential membrane source for autophagosome formation [2] (Figure 2Aiii). Several
previous EM studies on plant cells have identified omegasome-like structures that are closely
associated with the ER. Dynamic analysis of the fusion protein ATG5–GFP also showed that
ATG5 is present at the growing phagophore, defining a torus-shaped phagophore structure
overlying the ER [30]. 3D ET analysis further demonstrated that plant autophagosome-related
tubules grow directly out from a PI3P-enriched ER subdomain in a process that requires ATG9

680 Trends in Plant Science, August 2018, Vol. 23, No. 8

(A)
(i) Yeast (ii) Mammals (iii) Plants

Atg9 ULK1/ATG13 ATG1/ATG13

PAS
vesicle complex complex

mATG9
PI3K vesicle
TRAPPIII Phagophore PI3K Phagophore PI3K TRAPPIII? Phagophore
complex Omegasome TRAPPIII complex
complex
Atg1/Atg13
complex ATG9
COPII vesicle
COPII COPII
vesicle PI3P vesicle PI3P vesicle

ER ER ER
ERES ERES ERES

(B) (i) Yeast (ii) Mammals (iii) Plants

ATG11
P
Atg29 Atg18 ATG101
Atg17 P FIP200 P ATG101
Atg31 P P
Atg13 P ATG13
Atg2 ATG13
Atg9 vesicle mATG9 vesicle ATG9 vesicle

Atg1 P
ULK1 P ATG1
VPS34 P
P
P P P
P ?
Atg6 BECN1 AMBRA1

(C) (i) Yeast (ii) Mammals TBC1D5 (iii) Plants

AP2

Rab11

Endosome Endosome Recycling endosome Endosome Recycling endosome

TRAPPIII
TBC1D14
TRAPPIII

ATG23/ TRAPPIII Bif1

Ypt1 Rab1
ATG27 Ypt1 AP4

Rab1
Ypt1

Golgi ATG9 vesicles Phagophore Golgi ATG9 vesicles Phagophore

Golgi ATG9 vesicles Phagophore

Figure 2. Current Models for Autophagosome Initiation and Its Relationship to the ER in Yeast, Mammals, and Plants
For a Figure360 author presentation of Figure 2, see the figure legend at https://doi.org/10.1016/j.tplants.2018.05.002
(A) (i) In yeast, the Atg1 complex activates Atg9 vesicles and the PI3K complex such that the Atg9 vesicles nucleate to form a phagophore. COPII vesicles from ERES are
also recruited to the PAS by the TRAPPIII complex to contribute to phagophore initiation. (ii) In mammals, a specific PI3P-enriched ER subdomain called the
omegasome is formed for the assembly of the phagophore. The ATG1 complex activates and recruits the PI3K complex to the omegasome. Both mATG9 vesicles and
COPII vesicles are implicated in phagophore initiation. (iii) In plants, omegasome-like structures are formed during autophagosome initiation and ATG9 vesicles are
required for phagophore outgrowth from the ER subdomain. Whether COPII vesicles and the TRAPPIII complex are involved in autophagosome formation is unclear
(dashed arrow and question mark). (B) Schematic illustration of the role of the ATG1/ULK1 complex for autophagosome initiation in yeast (i), mammals (ii), and plants (iii).
Representative substrates phosphorylated by ATG1/ULK1 are labeled. (C) Proposed models for ATG9 trafficking in yeast (i), mammals (ii), and plants (iii). Whether other
pathways are involved in ATG9 trafficking in plants is unclear (dashed arrow). Abbreviations: ER, endoplasmic reticulum; ERES, ER exit site(s); P, phosphorylation; PAS,
pre-autophagosomal structure; PI3P, phosphatidylinositol 3-phosphate.

vesicles and PI3K activity [31]. It appears that distinct mechanisms and models for autopha-
gosome initiation may exist in different organisms, at least in yeast and mammals/plants
(Figure 2A). ER subdomains or its derived vesicles appear to have a direct role in the initiation
of autophagosome formation in all eukaryotes. Although the roles of ERES, COPII vesicles, and
ER–organelle contact sites in plant autophagy remain to be explored, counterparts of these
protein complexes are also highly conserved in plants [47,48] (Box 2). For example, the protein
complex for ER–PM contact sites in arabidopsis (Arabidopsis thaliana) has been identified, and
includes the VAP27 protein family, SYT1 (counterpart of mammalian E-SYT1), and NET3C

Trends in Plant Science, August 2018, Vol. 23, No. 8 681

 Box 2. ER–Organelle Contact Sites, ERES, and COPII Vesicles
The ER has been long considered to be the central organelle responsible for lipid synthesis, protein synthesis, and
calcium storage. Distinct contacts between the ER and other organelles, known as ER–organelle contact sites, have
been characterized in higher eukaryotes, such as ER–mitochondria contact sites and ER–PM contact sites [105]. In
plants, conserved and plant-unique specific ER contact sites have been characterized, including the ER–chloroplast
contact site and specialized ER–PM contact domains named plasmodesmata [106]. Protein complexes participating in
these contacts have been identified, and recent studies focusing on these protein tethers have revealed their diverse
roles in plant growth and development, including the regulation of intracellular Ca2+ dynamics and signaling as well as
the control of lipid traffic and homeostasis [48,106]. For example, ER–PM contact sites in plants employ a protein
complex that is unique to plants, which is composed of VAP27 protein family, SYT1 (a Ca2+ sensor), and NET3C (a
plant-specific NET family protein interacting with the actin cytoskeleton). ER–PM contact cites are often associated with
cortical microtubules and actin, whereas ER–PM contact cites may function as a hub for cytoskeleton organization [49–
51].

By definition, ERES are specialized ER export sites for the export of proteins and lipids from the ER mediated by COPII
vesicles [47]. COPII, which comprises five cytosolic components (Sar1, Sec23/24, and Sec13/31), is highly conserved
for the formation of COPII vesicles in eukaryotes. Extensive expansion of COPII subunits has been identified in
arabidopsis (21 paralogs), and some have been shown to participate in ER export. Phenotypic discrepancy between
paralogs was observed in arabidopsis, and it is suggested that the COPII subunits in arabidopsis may be evolutionarily
divergent from their yeast counterparts [53]. Moreover, recent studies have suggested that diverse COPII vesicles, with
distinct combinations of COPII subunit paralogs, may also be present in plants [53].

(a plant-specific NET family protein interacting with the actin cytoskeleton) [49–51]. Intriguingly,
VAP27 is found to overlap with NAP1, a component of the SCAR/WAVE complex that regulates
both microtubules and actin filaments [52]. Knockout of NAP1 reduces the number of auto-
phagosomes during starvation and leads to autophagy defects that render these plants less
tolerant to salt and nitrogen starvation [52]. In addition, the COPII subunits have been
characterized in arabidopsis, and these exhibit distinct functions in protein export from the
ER under ER stress via specific pairing or the formation of specific COPII vesicles [53]. Because
COPII vesicles are conversely required for autophagy in yeast and mammals, in future studies it
will be interesting to find out whether specific COPII vesicle(s)/subunit(s) contribute to auto-
phagosome formation in plant cells under particular stress condition(s), and how other ER-
related subdomains are engaged in this process.

Hierarchy of Core ATG Components for Autophagosome Initiation from the

ER
The ATG1/ULK1 Complex
Upon starvation, the first step in phagophore formation requires the assembly of the ATG1
complex for autophagy activation. The major role of the ATG1 complex is to activate the
downstream substrates by phosphorylation, to recruit downstream regulators for assembly
onto the PAS, and to tether membranes/vesicles for the development of phagophore (including
ATG9 and COPII vesicles) [24].

The Atg1 complex in yeast consists of the serine/threonine kinase Atg1, the adaptor protein
Atg13, and the scaffolding subcomplex Atg17–Atg29–Atg31 for bulk autophagy, or Atg11 for
selective autophagy [25] (Figure 2Bi and Table 1). Atg13 is highly phosphorylated and loses
its binding affinity for Atg1 under nutrient-rich conditions; however, upon starvation Atg13 is
rapidly dephosphorylated to promote Atg1 kinase activity and the assembly of the Atg1
complex. Atg1 has a serine/threonine kinase domain at its N-terminus as well as the microtu-
bule-interacting and transport (MIT) domain at its C-terminus (Table 1). Using consensus
peptide screening, Atg9 was identified as a direct Atg1 substrate, whereas phosphorylation
of Atg9 is essential for the recruitment of both Atg18 and Atg8 to the PAS for phagophore
expansion [34]. Other substrates include Atg2 and Atg6, but the biological significance of their

682 Trends in Plant Science, August 2018, Vol. 23, No. 8

 Table 1. The Core ATG Machinery in Yeast, Mammals, and Plantsa
Yeast Mammal Plant Function annotation in plants Refs
(Saccharomyces cerevisiae) (Homo sapiens) (Arabidopsis thaliana)

ATG1/ S/T kinase for autophagy: interacts with [62]

ULK complex ATG13 and ATG8e

ATG13a (603 aa) Regulates subunit for autophagosome [62]

ATG13b (625 aa) biogenesis

Scaffold for autophagy: ATG11 interacts with [63]

ATG8e, ATG101 and ATG13

Class III Vacuole development, pollen germination [71]

PI3K complex

Vacuole development, pollen germination [70]

Autophagy, PCD: interacts with BAG6 and [73–76]

Bax inhibitor-1

Not characterized [72]

Trends in Plant Science, August 2018, Vol. 23, No. 8

Not identified

Vacuolar trafficking: interacts with ATG6 [72]

ATG9 Regulates autophagosome progression from [31]

the ER

a
Not all ATG components are listed. Abbreviations: aa, amino acids; AIM/LIR, Atg8-interacting motif/LC3-interacting region; BAG6, Bcl-2-associated athanogene 6; BI-1, Bax inhibitor 1; BARA, beta-alpha
repeated autophagy-specific domain; BATS, Barkor/Atg14L autophagosome targeting sequence; BH3, Bcl-2 homology 3 domain; CC, coiled-coil domain; EAT, early autophagic targeting; HORMA,
Hop1p, Rev7p and MAD2 proteins; HEAT, huntingtin, EF3, A subunit of PP2A, TOR1; MIM, MIT-interacting motif; MIT, microtubule interacting and trafficking motif; TMD, transmembrane domain; PCD,
programmed cell death; WD, also known as the WD40 repeat, a motif of approximately 40 aa in length terminating in a tryptophan–aspartic acid (W–D) dipeptide.
683
phosphorylation remains unknown. On the other hand, upon activation, Atg17, together with its
two adaptors Atg29 and Atg31, which form a unique S-shaped heterodimer, are the earliest
protein to arrive at the PAS and to recruit Atg1/Atg13 and Atg9 vesicles for phagophore
initiation [54–57]. Structure-based analysis revealed that Atg13 serves as a flexible bridge for
Atg1 complex self-assembly and downstream substrate recruitment for PAS organization, in
which the MIM (MIT-interacting motif) domain of Atg13 binds to Atg1, while its intrinsically
disordered region and HOMOR (Hop1p, Rev7p, and Mad2) domain interact with Atg17 and
Atg9, respectively [57,58] (Figure 2Bi).

In contrast to yeast, the ULK1 complex in mammals includes ULK1 (yeast Atg1 homolog),
ATG13, FIP200 (also known as RB1-inducible coiled-coil protein 1), and ATG101 [24]
(Figure 2Bii). A striking difference between yeast and mammals is that the mammalian
ULK1 forms a stable complex with ATG13, FIP200, and ATG101, which seems not to be
regulated by nutrient signaling. The ULK1 kinase transduces signals by phosphorylating several
substrates including both the ULK1 subunits and PI3K components for their activation and
redistribution to the ER to promote autophagy (Figure 2Bii) [24]. Very recently it has been shown
that, in mammals, ULK1 plays a distinct role in phosphorylating mATG9 to enable mATG9
translocation from the PM and juxtanuclear region to the peripheral pool for autophagosome
initiation [59]. FIP200 is suggested to be a hybrid of Atg17 and Atg11 that functions as an S-
shaped scaffold. Using super-resolution imaging, FIP200 was shown to associate with PIS-
enriched ER subdomains at the initiation stage before other ATG proteins [42,43]. Meanwhile,
ATG13 binds to the EAT domain of ULK1 and the HORMA domain of ATG101 for their
recruitment to sites of phagophore initiation [24]. Recently, structural and cellular analysis
revealed that human ATG13 lacks a sequence for capping, thus requiring ATG101 for
stabilization of ‘uncapped’ ATG13, whereas ATG101 is absent from budding yeast that
contains ‘capped’ ATG13 [60,61]. In addition, a newly identified WF finger in ATG101 was
shown to be essential for recruitment of downstream regulators, including PI3P-binding
proteins DFCP1 and WIPI1 (homolog of yeast Atg18), to the isolation membrane [60].

The counterpart of the ATG1/ULK1 complex in arabidopsis is composed of ATG1(a–c and t),
ATG13, ATG11, and ATG101 [62,63] (Figure 2Biii and Table 1). Four isoforms of ATG1 in
arabidopsis have been identified, including ATG1a–c and ATG1t, the latter with only a truncated
kinase domain, likely representing a novel adaptation of the ATG1 kinase family in seed plants
[62]. ATG1a autophosphorylation has been demonstrated to occur in arabidopsis, and this
requires ATG11 activity, but other ATG1 substrates and their roles in plant autophagy remain to
be explored. Hyperphosphorylated forms of ATG13a appear to be regulated by nutrient
conditions, but it is unclear whether the binding affinity between ATG13 and ATG1 is nutri-
ent-dependent. Of note, arabidopsis ATG11 is named for its sequence homology to the Atg11
domain. However, arabidopsis ATG11 is more likely to be an ortholog of FIP200, representing a
hybrid of yeast Atg11 and Atg17, because it also contains Atg17-like coiled-coil motif and
interacts with ATG1, ATG13, as well as with ATG101 for both bulk and selective autophagy
[63]. It is suggested that the ATG1 complex in arabidopsis acts at the step(s) that encloses
autophagosomes because ATG8–phosphatidylethanolamine (PE) conjugation is detectable in
both atg13a/atg13b and atg11 plants [62,63] (Figure 3). However, Atg8/LC3 lipidation also
occurs in various atg mutants in both yeast and mammalian cells, such as in yeast atg1 and
atg17 mutants [64] and in mammalian FIP200 knockout cells [65]. Instead, the levels of
lipidation in these atg mutants are compromised to different degrees but are not completely
inhibited. In fact, both autophagosomal structures (labeled by GFP–ATG8) and autophagic
flux (revealed by a degraded GFP core) are completely or profoundly inhibited in atg13a/
atg13b and atg11 mutants [62,63] (Figure 3). Accordingly, the ATG1 complex in plants may

684 Trends in Plant Science, August 2018, Vol. 23, No. 8

 WT atg5, atg7 atg11, atg13 atg9
DetecƟon
method
(A) Autophagosome
morphology

EM
FM
ER ER ER ER
(B) GFP–ATG8
cleavage

GFP GFP GFP

FM GFP
GFP GFP GFP
GFP GFP GFP
IB GFP

GFP
Vacuole Vacuole Vacuole Vacuole
lipidaƟon
(C) ATG8

IB Yes No Yes Yes

Key: EM Electron microscopy FM Fluorescence microscopy IB Immunobloƫng

ATG9 PI3P ATG18 ATG8 ATG8–PE ATG5 complex

Figure 3. Schematic Representation of the Methods Used in Detecting Autophagy in Arabidopsis thaliana atg Mutants. (A) Detection of autopha-
gosomal structures or numbers by electron microscopy (EM) or fluorescence microscopy (FM) [29–31,74]. (B) Measurement of the autophagic flux by observing the
number of autophagosomes/autophagic bodies labeled with GFP–ATG8 (e.g., ATG8a, ATG8e, ATG8f) via FM, or by detecting the cleavage ratio of GFP generated by
autophagic bodies inside the vacuole by immunoblotting (IB) with a GFP antibody [29–32,62–63]. (C) In the ATG8 lipidation assay, the conversion of ATG8 to ATG8–PE
before/after autophagic induction is detected by IB with an ATG8 antibody [31,62,63]. Abbreviations: PE, phosphatidylethanolamine; PI3P, phosphatidylinositol 3-
phosphate; WT, wild type.

function independently of the lipidation system in the early steps of autophagosome initiation,
as in yeast and mammals. However, it is not necessary to exclude their additional roles in
autophagosome maturation because both ATG1 and ATG11 contain the AIM/LC3
(Atg8-interacting motif/LC3-interacting region) motif to traffic together with the autophagoso-
mal membrane into the vacuole [62,63] (Table 1 and Box 3). Nevertheless, further biochemical
and structural analysis in parallel with cellular studies will be necessary to clarify the role of the
plant ATG1 complex in autophagosome formation.

The PI3K Complex

The formation of PI3P is another essential early event in autophagy initiation, and is driven by the
class III PI3K complex [25]. In both yeast and mammalian cells there are at least two distinct
PI3K complexes (I and II), both of which contain Vps34/VPS34, Vps15/VPS15, and
Atg6/Beclin-1 (BECN1), together with the autophagy-specific factor Atg14/ATG14L. In recent
years a fifth subunit of the PI3K complex I, termed Atg38 in yeast, and NRBF2 (nuclear receptor
binding factor 2) in mammals, has been identified [66,67] (Table 1). The complete structure of
the human PI3K complex I has been solved, in which the NRBF2 dimer binds to two PI3K I
complexes via interactions with the N-terminus of ATG14L and BECN1, generating a V-shaped

Trends in Plant Science, August 2018, Vol. 23, No. 8 685

 Box 3. ATG8 and AIM/LC3 Motifs
Yeast contains a single gene encoding ATG8, whereas six and nine isoforms of ATG8 have been identified in Homo
sapiens (including LC3 and GABARAP subfamilies) and arabidopsis (ATG8a–8i) respectively [107]. ATG8s are widely
used markers in studying autophagy in various model organisms because ATG8s are associated with autophagic
membranes during different stages of autophagosome formation. ATG8 proteins are attached to membrane by the
conjugation systems and bind to different cargo receptors via a consensus short peptide motif called the
Atg8-interacting motif/LC3-interacting region (AIM/LIR) [96].

In plants, it has been found that family-specific ATG8 clades have evolved, and ATG8 expansion has been suggested to
be driven by functional diversification [107]. Some isoforms of ATG8 have been shown to play a specific role in
autophagy. For instance, ATG8CL but not ATG8IL in Nicotiana benthamiana was shown to specifically interact with the
effector from the Irish potato famine pathogen, Phytophthora infestans PexRD54, and to trigger autophagosome
formation [107]. At the molecular level, various arabidopsis ATG8 isoforms have been shown to be cleaved differentially
by ATG4b, suggesting the potential for non-redundant functional roles of ATG8 isoforms in plants [108].

architecture [66,67]. By contrast, the PI3K complex I displays a different organization in yeast,
where the Atg38 dimer interacts with only one PI3K complex I [66]. The PI3K complex II
displayed a similar structure, but involves another subunit Vps38/UVRAG and may also function
in autophagosome maturation [68]. These detailed structural studies reveal the flexibility of the
PI3K complex membrane-binding and protein-binding interfaces during autophagy. In mam-
mals, the unique ATG14L subunit contains an N-terminal cysteine-rich domain for ER-targeting
and a coiled-coil domain for membrane curvature sensing [69]. In addition, there is a C-terminal
amphipathic helix, termed the BATS domain (Barkor/ATG14L autophagosome targeting
sequence) in ATG14L, which enables ATG14L to function as a membrane sensor to bind
to high-curvature lipids [69]. Although Atg14 in yeast displays a similar structure, whether it has
a direct connection with the ER membrane remains unclear.

So far, four PI3K components including VPS15, VPS34, VPS38, and ATG6 have been studied
in arabidopsis [70–76]. In addition, two possible isoforms of ATG14L/Atg14 in arabidopsis are
predicted; these also contain N-terminus conserved cysteine repeats but their exact roles in
plant autophagy have not yet been characterized [72]. Nevertheless, an essential role for the
PI3K complex in autophagosome initiation seems to be highly conserved in arabidopsis
because autophagy is significantly disturbed by PI3K inhibitor treatment and knockdown of
ATG6 [29,30,73]. In addition, impairment of autophagy has been observed in the vps38 mutant
upon starvation [72]. In arabidopsis, ATG6 has been shown to associate with two novel
autophagic regulators, SH3P2 and FREE1, which are involved in autophagosome expan-
sion/maturation [29,74]. In addition, ATG6 was recently shown to interact with Bcl-2-associ-
ated athanogene 6 (BAG6) and Bax inhibitor-1 (BI-1) to function in plant immunity and PCD
[75,76]. However, the detailed mechanisms underlying how the PI3K components coordinate
during autophagy in plants remain elusive.

ATG9
ATG9 is a six-transmembrane protein which was first identified in yeast to play a key role in
delivering membrane/lipid for autophagosome formation. Although the intracellular behavior of
ATG9 trafficking remains controversial, the Golgi–endosomal system has been suggested as a
key route for the production of ATG9 vesicles in yeast, mammalian, and plant cells [31,77]. It
has been suggested that vesicles containing ATG9 proteins interconnect with autophagosomal
membrane upon membrane fusion, and this process certainly requires a subset of membrane
trafficking regulators (Figure 2C).

686 Trends in Plant Science, August 2018, Vol. 23, No. 8

Yeast Atg9 vesicles are derived from the Golgi apparatus in a process involving Atg23 and
Atg27 [33] (Figure 2Ci). In addition, Atg9 may shuttle between the Golgi and endosomes
dependent on the TRAPPIII complex [78]. In response to autophagic induction under nutrient-
rich conditions, Atg9 vesicles (30–60 nm in diameter) nucleate on the PAS, whereas the
number of mobile cytoplasmic Atg9 vesicles significantly decreases [33]. However, only a
few Atg9 vesicles are recruited to the PAS at the early stage rather than at other steps, and
these are sufficient to accomplish a single round of autophagosome formation. A reconstituted
in vitro system using purified components demonstrates that Atg9 is specifically recognized by
the scaffolding protein Atg17 to promote the tethering of two Atg9 vesicles [79]. After nucleation
on the PAS, Atg9 is embedded in the autophagosomal outer membrane, and it may be recycled
back into the cytoplasm as new Atg9 vesicles for cargo recruitment before autophagosome
fusion with the vacuole [33].

In mammals, mATG9 compartments display a dynamic ability to cycle from the Golgi apparatus
to endosomes (Figure 2Cii) [80]. Intriguingly, mATG9 involves trafficking to the PM and recycling
endosomes [81–83]. Several trafficking regulators have been implicated in mATG9 trafficking
steps, including RAB11 and TBC1D14 [81], TRAPPIII [82], AP2 (adaptor related protein
complex 2), AP4 [83,84], BIF1 and dynamin [85]. Using CLEM and super-resolution imaging,
it has been possible to visualize mATG9-positive tubular-vesicular clusters on the perinuclear
RAB11-positive recycling endosomes or adjacent to the ATG13-positive ER subdomains upon
starvation [42]. However, mATG9 itself was not integrated into the autophagosome membrane
as occurs in yeast [80].

In arabidopsis, ATG9 vesicles also traffic through the early secretory pathway and closely
associate with post-Golgi endosomes because inhibition of ER to Golgi trafficking significantly
relocates ATG9 back to the ER [31] (Figure 2Ciii). Interestingly, ATG9 vesicles are sensitive to
both brefeldin A (which prevents the recycling through endosomes) and wortmannin (which
inhibits transport out of endosomes) treatments by forming aggregates and enlarged ring-like
structures, respectively. These observations suggest that ATG9 may reside on motile compart-
ments; however, it remains unknown whether ATG9 traffics to the PM or to the recycling
endosomes, as occurs in mammals. Upon autophagy, ATG9 vesicles display transient inter-
actions with the autophagosome membrane, suggesting that arabidopsis ATG9 vesicles might
not be integrated into the autophagosome membrane [31]. Of note, in comparison to other
autophagy-deficient mutants (e.g., atg5, atg7, atg11, and atg13) [30,62,63], impairment of
ATG9 function impacts on autophagy flux at a relatively moderate level and autophagosomal
structures are still detectable [31] (Figure 3). Furthermore, unlike other plant atg mutants or
yeast/mammalian ATG9 mutants, lack of ATG9 leads to the accumulation of tubular compart-
ments that are positive for autophagosomal marker ATG8e coalescing with ER subdomains
[31]. It should be pointed out that this defect is particularly evident under ER stress, and
arabidopsis ATG9 possesses only weak sequence homology to the yeast and mammalian
proteins. In comparison with the diverse distributions of ATG9 in yeast and mammals
(Figure 2C), it is tempting to speculate that ATG9 may function in plant autophagosome
formation in a plant-specific manner that acquires different regulators in plants. Efforts will
be required in the future to characterize the identity of ATG9 vesicles and to identify the specific
regulators for ATG9 trafficking in plants.

Role of ER-Resident Proteins in Autophagy Regulation

In addition to a direct contribution of membrane/lipid to autophagosome biogenesis, the ER
may also serve as a central site for stress sensing to regulate autophagy. Signaling sensors
resident on the ER detect changes within the lumen (ER stress) and activate downstream

Trends in Plant Science, August 2018, Vol. 23, No. 8 687

Table 2. List of ER-Resident Proteins in Autophagy Regulationa
Organism Proteins Notes Refs

Yeast (Sc) Atg39 Autophagy receptor for the ER and nucleus [97]

Atg40 Localizes at cortical and cytoplasmic ER; autophagy [97]

receptor for ER

Ufe1 SNARE protein; is exported from the ER in specific COPII [38]

vesicles to autophagosomes upon starvation

Mammal (Hs) FAM134b Autophagy receptor for the ER tubule fragmentation [98]
turnover

RTN3 Autophagy receptor for ER sheet degradation [98]

SEC62 Involved in RecovER-phagy after stress [99]

ITPR Transports calcium ions from the ER to mitochondria; [87,88]

interacts with Beclin1

IRE1 Involved in the ER-triggered UPR; activates the c-Jun N- [86]

terminal kinase pathway to recruit Beclin1

PERK Involved in the ER-triggered UPR; required at ER– [86]

mitochondria contact sites to initiate apoptosis

ATF6 Involved in ER-triggered UPR; activates the transcription [86]

of ER molecular chaperones

VMP1 Recruits Beclin1 to phagophores; regulates ER– [90,91]

microdomain and ER–IM (isolation membrane) contacts

SERCA2 Interacts with VMP1 to regulate calcium levels [91]

E-Syts 1/2/3 ER–PM tethering factor; essential for autophagy- [46]

associated PI3P synthesis at the cortical ER via
recruitment of VMP1

VAP-A Involved in ER–endosome contact sites; interacts with the [89]

cholesterol-sensing Rab7 effector ORP1L to regulate
autophagosome transport and fusion with endosomes/
lysosomes

PIS Phospholipid biosynthetic enzymes; supply [43]

phosphatidylinositol and other phospholipids to isolation
membranes

Mitofusin 2 ER–mitochondria tethering factor [45]

PACS-2 ER–mitochondria tethering factor [45]

STX17 ER-resident SNARE protein; relocalizes to ER– [45]

mitochondria contact sites and binds to ATG14L

Plant (At) ATI1/2 Interact with ATG8f and is involved in ER degradation by [100]
forming ATI bodies

IRE1b Involved in ER turnover during ER stress [32]

a
Abbreviations: At, Arabidopsis thaliana; Hs, Homo sapiens; Sc, Saccharomyces cerevisiae.

signaling pathways to restore homeostasis, including the unfolded protein response (UPR)
and autophagy for ER turnover (also termed ER-phagy) [86]. Several ER-resident proteins have
been identified to play a direct role in this process for autophagy regulation (Table 2). For
example, the ER-resident inositol-1,4,5 trisphosphate receptor (ITPR) is known to mediate ER
calcium release and was shown to participate in autophagy regulation by associating with
BECN1 via Bcl-2 [87,88]. In addition, multiple ER–organelle contact sites may bridge auto-
phagosomes with other elements such as endosomes and cytoskeletons for their expansion or
maturation. Recently, a novel type of membrane contact site, that depends on the ER-resident

688 Trends in Plant Science, August 2018, Vol. 23, No. 8

VAP-A and the cholesterol sensor ORP1L, was shown to control autophagosome transport
and fusion with the endosome/lysosome in mammals [89]. A possible role of the ER–PM
contact site in the linkage between autophagosome and cytoskeleton has also been suggested
in plant cells. In arabidopsis, under pressure-induced stress, a cytoskeleton regulatory protein
NAP1 is relocated to ER–PM contact sites labeled by VAP27 (homolog of VAP-A), likely to
regulate autophagosome formation by triggering actin polymerization [52].

Another essential ER-localized protein in autophagy activation is called VMP1 (vacuole mem-
brane protein 1), which is absent in yeast. The mammalian VMP1 interacts with and recruits
BECN1 to the phagophore, and overexpression of VMP1 itself may trigger autophagy by
promoting PI3P generation [90]. VMP1 interacts with the sarco(endo)plasmic reticulum calcium
ATPase 2 (SERCA2), a calcium pump on the ER [90,91]. In plants, two isoforms of the VMP1
homolog (named KMS1 and KMS2) have been characterized in arabidopsis [92]. Although
KMS1 does not interact with ATG6, KMS1 expression is elevated in senescing leaves,
suggesting a possible role in leaf senescence, as found for other arabidopsis atg mutants.
Of note, VMP1 deficiency in green algae Chlamydomonas leads to compromised autophagy
and the accumulation of autolysosome-like structures [93].

Accumulation of misfolded proteins in the ER will trigger the UPR, which also regulates
autophagy in various eukaryotes [86]. In mammalian cells, three branches of the UPR, including
the ER-localized IRE1, PERK, and ATF6, are activated in response to misfolded proteins via
different downstream pathways, and these impact on autophagy in different ways [86]. Among
them, IRE1 activates the c-Jun N-terminal kinase pathway, leading to the activation of BECN1
for autophagic induction [86]. So far, only the IRE1-mediated pathway has been studied in
plants [32,94]. Strikingly, only IRE1b in arabidopsis was specifically required for ER stress-
induced autophagy, although arabidopsis contains two IRE1 isoforms [32]. However, the
autophagic response to starvation is normal in the ire1b mutant, implying a unique role for
IRE1b in ER stress-induced autophagy [32]. In addition, both overexpression of the ER
chaperone BiP and treatment with chemical chaperones can reduce ER stress-induced
autophagy but not the basal level of autophagy, implying a role of autophagy as the UPR
effector to cope with ER stress in plants [95].

Moreover, several ER-resident proteins containing the LIR/AIM motifs as cargo receptors for
ER homeostasis have been identified [96]. In yeast, a perinuclear ER-localized protein Atg39
and another reticulon-related protein, Atg40, which is enriched in the cortical and cytoplasmic
ER, are responsible for ER-phagy [97]. In mammals, two reticulon ER-resident proteins
FAM134b and RTN3 were instead identified as receptors for ER-phagy, but they function
independently and mediate the degradation of ER tubules and ER sheets, respectively [98].
Interestingly, an ER-resident translocon subunit, Sec62, also contains a conserved AIM/LIR
motif involved in the turnover of ER membranes during ER stress [99]. Previously, two potential
arabidopsis receptors with a predicted transmembrane domain, termed autophagy interacting
proteins 1 and 2 (ATI1 and ATI2), were shown to interact with ATG8f and to localize to the ER
[100]. Upon carbon starvation, a non-classical autophagosome containing ATI1 (ATI-body)
forms for the degradation of ER components [100]. Because ER-phagy also operates in plants
[32], the identification of plant-specific ER-resident receptors should open up investigations into
the interconnection between autophagy and ER homeostasis in the future.

Concluding Remarks and Future Perspectives

The diversity and complexity of autophagy have been extensively unveiled in higher eukaryotic
cells, and a canonical role for the core ATG machinery in autophagosome biogenesis has been

Trends in Plant Science, August 2018, Vol. 23, No. 8 689

shown to be highly conserved. Regarding the discrepancies between autophagosome initiation Outstanding Questions
and the autophagic machinery in yeast and mammals, a plant-specific mechanism for auto- Do plants employ plant-specific ER
phagosome initiation and ATG machinery assembly with novel regulators may operate to subdomains or ER–organelle contact
sites as a membrane source or signal-
control the plant-unique development and growth process. An in-depth knowledge of the ing platform to adapt to different types
diverse modes of autophagosome biogenesis at both the cellular and structural levels will be of stresses and cargoes?
essential for our understanding of the mechanism, as well as the significance, of autophagy
under specific physiological or pathological conditions in plants (see Outstanding Questions). What is the lipid composition of PAS,
autophagosomal structure, and how is
So far, only the crystal structures of ATG3, ATG7, and ATG12 in arabidopsis have been studied
lipid biosynthesis regulated and
[101–103]. By combining advanced techniques including super-resolution imaging, ET, and involved in autophagosome biogene-
cryo-EM in multidisciplinary collaborative research efforts, we can anticipate new insights into sis in plants?
plant autophagy and autophagosome biogenesis in the coming years.
How does the core ATG machinery
assemble and orchestrate the initiation
Acknowledgments
of autophagosome biogenesis in
We apologize to researchers whose work could not be included in this manuscript owing to space limitations. This work
plants, in particular, at the structural
was supported by grants from the Research Grants Council of Hong Kong (G-CUHK402/15, G-CUHK403/17, level?
CUHK14130716, CUHK14102417, C4011-14R, C4012-16E, C4002-17G and AoE/M-05/12), the National Natural
Science Foundation of China (31470294 and 31670179), and the Shenzhen Peacock Project (KQTD201101) to L.J. What is the nature of ATG9 vesicles in
plants, and what is the mechanism that
Supplemental Information regulates ATG9 trafficking during
Supplemental information associated with this article can be found, in the online version, at https://doi.org/10.1016/j. autophagy in plants?

tplants.2018.05.002.
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