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Plant autophagy—more than a starvation response
Diane C Bassham
Autophagy is a conserved mechanism for the degradation of
cellular contents in order to recycle nutrients or break down
damaged or toxic material. This occurs by the uptake of
cytoplasmic constituents into the vacuole, where they are
degraded by vacuolar hydrolases. In plants, autophagy has
been known for some time to be important for nutrient
remobilization during sugar and nitrogen starvation and leaf
senescence, but recent research has uncovered additional
crucial roles for plant autophagy. These roles include the
degradation of oxidized proteins during oxidative stress,
disposal of protein aggregates, and possibly even removal of
damaged proteins and organelles during normal growth
conditions as a housekeeping function. A surprising regulatory
function for autophagy in programmed cell death during the
hypersensitive response to pathogen infection has also been
identified.
Addresses
Department of Genetics, Development and Cell Biology, 253 Bessey
Hall, Iowa State University, Ames, IA 50011, USA
Corresponding author: Bassham, Diane C ([email protected])
Current Opinion in Plant Biology 2007, 10:1–7
This review comes from a themed issue on
Cell Biology
Edited by Ben Scheres and Volker Lipka
1369-5266/$ – see front matter
# 2007 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.pbi.2007.06.006
Introduction
Autophagy (‘self-eating’) is a universal mechanism in
eukaryotic cells for digestion of cell contents to recycle
needed nutrients, degrade damaged or toxic components,
or to reclaim cellular materials as a precursor to cell death.
In plants, two major autophagic pathways have been
described, microautophagy and macroautophagy [1]. In
microautophagy, material is engulfed directly by the
vacuole via invagination of the tonoplast, followed by
pinching off of the membrane to release a vesicle containing the cytoplasmic constituents inside the vacuole lumen.
Microautophagy-like processes have been observed during
the deposition of storage proteins in developing wheat
seeds [2,3] and release of rubber particles into the vacuole
[4], though these processes do not result in immediate
degradation of the material deposited into the vacuole.
Microautophagy also occurs in some species during seed
germination for degradation of starch granules and storage
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protein in vacuoles [5,6]. Macroautophagy, by contrast, is
initiated in the cytoplasm with the formation of cup-shaped
membranes of unknown source that enclose the material to
be degraded. The membranes elongate and eventually
fuse together to produce a double-membrane autophagosome containing cytoplasmic material. The outer autophagosomal membrane fuses with the tonoplast, releasing
an autophagic body consisting of the inner membrane and
contents into the vacuole. Vacuolar acid hydrolases then
degrade the autophagic body, and the degradation products are presumably transported back to the cytosol [1].
The functions of macroautophagy in suspension cultures
and whole plants in response to sucrose and nitrogen
starvation and during senescence have been well described
for a number of plant species (for example, see references
[7–10]). The recent development of markers to rapidly and
easily detect the occurrence of autophagy in plant cells by
the specific labeling of autophagosomes has facilitated the
study of autophagy. These markers include a fusion of
green fluorescent protein (GFP) with the autophagosomelocalized protein ATG8 [11–13] and the acidotrophic
fluorescent dyes monodansylcadaverine (MDC [11]) and
LysoTracker Red [14]. In this review, I will summarize
advances over the past two years in our understanding of
macroautophagy, hereafter simply termed autophagy, and
in particular focus on additional physiological roles for
autophagy in plants that are now coming to light.
Pathways for autophagy in plants
Examination of the morphological characteristics of
autophagy in different plant species has led to some
discrepancies regarding the precise pathway and site of
degradation of cytoplasmic material (Figure 1). For
example, when the cysteine protease inhibitor E-64
was used to block autophagosome degradation in Arabidopsis and barley, cytoplasmic inclusions accumulated as
autophagic bodies within the central vacuole, whereas in
tobacco they accumulated in smaller organelles outside
the central vacuole that are termed autolysosomes [15].
Extensive analyses of the stages of autophagosome formation and delivery to the vacuole has suggested that in
plants, two distinct pathways exist in which autophagosomes can either fuse directly with the vacuole or can first
be engulfed by a smaller lysosome-like or endosome-like
organelle, which begins the degradation of the contents,
before eventual fusion with the vacuole [16,17]. The
species and conditions under investigation may determine which pathway is predominant at any given time.
Arabidopsis proteins required for autophagy
On the basis of sequence similarity to yeast proteins
required for autophagy [18], a number of Arabidopsis
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2 Cell Biology
Figure 1
systems and the ATG9 complex, which may function in
membrane recruitment to the site of autophagosome
formation (Table 1).
The best studied proteins involved in plant autophagy are
those of the ubiquitin-like conjugation systems [19]. The
first involves the formation of a covalently linked conjugate of ATG5 and ATG12. The reaction is reminiscent
of the attachment of ubiquitin to proteins to tag them for
degradation, though the ATG5-ATG12 conjugate is
stable and seems to be the functional form of the two
proteins. The conjugation enzymes show similarity to
those of the ubiquitin pathway, consisting of an E1-type
enzyme ATG7 and an E2-like ATG10. While ATG12
does not have any sequence homology with ubiquitin, the
recent crystal structure of Arabidopsis ATG12 reveals that
the protein has a canonical ubiquitin fold [20]. The
second system is more unusual in that the ATG8 protein
is conjugated to a lipid, phosphatidylethanolamine (PE),
rather than a protein. The same ATG7 E1 enzyme performs this reaction, along with an E2, ATG3. A processing
enzyme that cleaves the C-terminus of ATG8 is also
required. Both of these systems are required for autophagosome formation in Arabidopsis [12,13,20], and
knockout mutants in several of the corresponding genes
have been identified and show starvation sensitivity and
early senescence phenotypes as expected for disruption
of autophagy [8,12,13,20].
Pathways for autophagy in plant cells. (a) In many species, including
Arabidopsis, upon induction of autophagy a double-membrane
autophagosome forms around a portion of cytoplasm. The outer
membrane of the autophagosome fuses with the tonoplast and the inner
membrane and contents enter the vacuolar lumen, where they are
degraded (upper panel). Inhibition of vacuolar proteases by E-64 or
concanamycin A (ConA) leads to accumulation of autophagic bodies
inside the vacuole (lower panel). (b) In tobacco, two autophagy
pathways operate. In addition to the pathway described in
(a), autophagosomes can also fuse with small lysosomes or
endosomes, in which the contents can be degraded before fusion
with the vacuole (upper panel). E-64 or ConA leads to accumulation of
these autolysosomes in the cytoplasm as well as accumulation of
material in the central vacuole (lower panel). Vac, vacuole.
proteins can be predicted to function in autophagy,
primarily in autophagosome formation [1,19]. While a
large amount of information is now available on the yeast
proteins, in many cases their precise function in autophagy is still unknown. Most of the autophagy proteins
can be classified into several major complexes or processes,
including protein kinases involved in the initiation or
regulation of autophagosome formation, a phosphatidylinositol 3-kinase complex, two ubiquitin-like conjugation
Components of the ATG9 complex have also been studied in Arabidopsis using genetic approaches. Atg9 is an
integral membrane protein that may be required for the
delivery of membrane to the forming autophagosome
[18], and disruption of the Arabidopsis ATG9 gene results
in typical autophagy phenotypes [9]. In yeast, Atg9 interacts with Atg2, and Atg18 is required for the correct
localization of both of these proteins. Disruption of
ATG2 or ATG18 in Arabidopsis also prevents autophagosome formation [15,21], suggesting conserved functions
for the proteins in plants.
Not all proteins required for autophagy are specific for that
pathway. For example, in yeast, Atg6 is required for
biosynthetic trafficking to the vacuole [22] in addition to
being a component of the phosphatidylinositol 3-kinase
complex required for autophagy [23], and the SNARE Vti1
is probably required for all vesicle transport pathways
terminating at the vacuole [24]. Several homologs of
Vti1 with partially overlapping functions are present in
Arabidopsis [25], and VTI12 is thought to be the homolog
required for autophagy [26]. ATG6 also appears to be a
multi-functional protein in plants, and a knockout mutant
in the Arabidopsis ATG6 gene is defective in pollen tube
germination [27,28], probably because of a vacuolar trafficking defect. In tobacco, an ATG6 homolog is involved in
the regulation of programmed cell death, and this function
is related to a role in autophagy (see below [29]).
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Plant autophagy—more than a starvation response Bassham 3
Table 1
Arabidopsis proteins potentially involved in autophagy
Complex/process
PI-3 kinase complex
Ubiquitin-like conjugation
Ubiquitin-like conjugation
ATG9 complex and localization
Regulation
SNARE
Proteins
Potential function
ATG6, VPS15, VPS34
ATG5, 7, 10, 12
ATG3, 4, 7, 8
ATG9, 2, 18
TOR, ATG1, 13
VTI12
Autophagosome formation
Conjugation of ATG12 and ATG5
Conjugation of ATG8 to phosphatidylethanolamine
Membrane recruitment to autophagosome
Initiation of autophagy
Fusion of autophagosomes with the vacuole
See references [1,19] for a more detailed description.
Autophagy during oxidative stress
In addition to its role in nutrient recycling during
starvation, autophagy may be a more general response
to a variety of abiotic stress conditions. Exposure of
Arabidopsis seedlings to oxidative stress, either by direct
addition of H2O2 or by addition of methyl viologen (MV)
to generate reactive oxygen via the photosynthetic electron transport chain, led to a rapid and strong induction of
autophagy [30] (Figure 2). RNAi-AtATG18a transgenic
plants, which are defective in autophagosome formation
because of decreased expression of an ATG18 homolog,
are hypersensitive to oxidative stress, indicating a physiological role for autophagy in response to this stress. One
potential reason for the hypersensitivity was revealed by
the analysis of oxidized proteins in wild-type and RNAiAtATG18a plants under oxidative stress conditions. Oxidized proteins accumulated in RNAi-AtATG18a plants,
and their rate of degradation was reduced compared with
that of wild-type plants, suggesting that autophagy is
required for the degradation of damaged cellular components. In situ localization of oxidized proteins provided
further evidence for this, as when vacuolar degradation
was blocked using ConA, oxidized proteins accumulated
in the vacuole in wild-type plants, whereas they remained
Figure 2
Autophagy is induced during oxidative stress. In the presence of
methyl viologen (MV) to cause oxidative stress, autophagy is
induced in Arabidopsis roots as revealed by staining with the
autophagosome-specific fluorescent dye monodansylcadaverine (MDC;
lower right). Very few autophagosomes can be seen in the absence of
stress (upper right). Visible light images are shown at left. Scale
bar = 50 mm (modified from reference [30], copyright American Society
for Plant Biologists).
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in the cytoplasm in RNAi-AtATG18a plants (Figure 3).
Together, these data indicate that, during oxidative
stress, oxidized proteins and potentially other cell components are transferred to the vacuole by autophagy for
degradation [30]. Whether autophagy also plays a role in
clearing damaged proteins during other abiotic stresses
remains to be seen.
Autophagy during programmed cell death
While under abiotic stress conditions autophagy is generally considered to be a response to assist in cell survival,
autophagy is also well known to contribute to programmed
cell death (PCD) by degradation of cellular contents before
death [31]. Classical apoptosis as seen in animal cells is
unlikely to occur in most plant systems, as the presence of
the cell wall would preclude engulfment of cellular remains
by surrounding cells. By contrast, many instances of PCD in
plants show typical morphological features of autophagic
cell death, including an increase in vacuole and cell size,
uptake of organelles into the vacuole followed by organelle
degradation, and eventual lysis of the vacuole resulting in
cell death. These characteristics have been described by
electron microscopic observations in such diverse systems
as soybean and foxglove nectaries upon termination of
nectar production [32,33] and death of suspensor cells
during embryogenesis in gymnosperms [34].
Vacuolar proteases with caspase-like activity are regulators of PCD in response to a variety of pathogens [35–37].
A role for a caspase-type protease activity in autophagic
PCD has been indicated recently in barley seed development [38]. While the protein responsible for the
activity has yet to be identified, the protease activity
correlated with PCD and was inhibited by caspasespecific inhibitors. In situ labeling with fluorescent substrate demonstrated the localization of the protease to
vesicles that were also labeled with the autophagosome
marker MDC [38], providing an intriguing connection
between the protease and autophagy activity. Whether
this protease is a regulator of autophagy, is involved in
degradation of cellular components during autophagic
cell death, or has some other role is not yet clear.
A role for autophagy in the response to pathogen infection
is clear in animals [39], but recent research indicates an
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Figure 3
Oxidized proteins are delivered to the vacuole for degradation during oxidative stress. Wild-type (WT) and autophagy-defective RNAi-AtATG18a
seedlings were incubated with methyl viologen (MV) and concanamycin A (ConA) for 12 h. The oxidized proteins were derivatized with 2,4dinitrophenylhydrazine (DNPH) and detected using DNP antibodies, followed by confocal microscopy. Oxidized proteins are seen in the vacuole in
wild-type plants but accumulate in the cytoplasm in the RNAi plants. Scale bar = 20 mm (modified from reference [30], copyright American Society for
Plant Biologists).
unexpected function in the innate immune response in
tobacco. Upon infection by tobacco mosaic virus (TMV),
infected cells undergo PCD in a process termed the
hypersensitive response, as a means of limiting pathogen spread. Using a high-throughput virus-induced gene
silencing (VIGS) screen, Liu et al. discovered that a tobacco
homolog of the autophagy protein Atg6 (also called
Beclin1) is involved in TMV-induced PCD [29]. The
tobacco BECLIN1 gene appears to function in autophagy,
as it was able to complement the yeast atg6 mutant phenotype, and silenced plants showed an accelerated senescence phenotype, typical of autophagy mutants. Whereas
the cell death lesions during the TMV-induced hypersensitive response are normally restricted to the infected cells,
VIGS of BECLIN1 (or other autophagy gene homologs)
caused expanded lesions that extended beyond the infection site and were even present in uninfected upper
leaves. These results suggested that BECLIN1 is required
for the restriction of PCD to the TMV infection site.
Arabidopsis Atg6 homologs have autophagy-independent
functions [27,28], possibly in trafficking to the vacuole as
in yeast. The effect of BECLIN1 on cell death, however,
does appear to involve the autophagy pathway. In nonsilenced infected plants, autolysosomes accumulated at
the infection site and were also seen in non-infected
leaves, suggesting that autophagy may be induced systemically during TMV infection [29]. Silencing of
BECLIN1 prevented this induction of autophagy. The
same phenotype was observed upon infection with bacterial and fungal pathogens, indicating that autophagy is
involved in the hypersensitive response in general, rather
than a specific response to TMV. The mechanism by
which autophagy prevents the spread of cell death is still
unknown. One possibility is that autophagy degrades a
death induction signal, preventing it from spreading to
uninfected cells. Alternatively, autophagy may degrade
components damaged by reactive oxygen species in adjacent cells, thereby preventing death.
A role for autophagy in the absence of
stress conditions
It has been speculated that autophagy is involved in
the disposal and degradation of abnormal or damaged
organelles and proteins, even under normal growth conditions without the imposition of environmental stresses.
This has come into question because of the lack of an
obvious growth phenotype of Arabidopsis autophagy
mutants throughout most of their life cycle [8,9,12,
13,21]. With the use of the protease inhibitor E-64d, it
has now been demonstrated that autophagy does occur
under nutrient-rich conditions, at least in certain cell
types [14,15]. When Arabidopsis or barley root tips were
incubated in the presence of nutrients and E-64d, cytoplasmic inclusions were seen to accumulate inside
vacuoles in the root meristem and in the elongation zone,
suggesting that autophagy was active in these cells.
These barley autophagic vacuoles were unusual in containing the tonoplast marker a-TIP, and not d-TIP or gTIP, suggesting functional specialization of the vacuoles
[14].
The mechanism of uptake of cytoplasm under normal
conditions was addressed by incubation of root tips in the
presence of the autophagy inhibitor 3-methyladenine
(3-MA) [40]. 3-MA partially inhibited the accumulation
of cytoplasm inside the vacuole [15], providing evidence
that the accumulation is due to uptake by the classical
autophagy pathway. Arabidopsis knockout mutants with
disruptions in the autophagy genes ATG2, ATG5 and
ATG9 showed reduced accumulation of cytoplasm
within the vacuole, indicating a block in vacuolar uptake
[15]. Further insight was gained using transgenic plants
expressing the autophagosome marker ATG8f fused
with GFP. The fusion protein was taken up into the
vacuole in root tips even under normal conditions and
was found in autophagic bodies when ConA was used to
block vacuolar degradation [41]. Together, these results
demonstrate that the same machinery is required for
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Plant autophagy—more than a starvation response Bassham 5
constitutive autophagy in root tips as for stress-induced
autophagy.
A potential role for constitutive autophagy in degrading
damaged proteins was suggested by the examination of
oxidized proteins and lipids in Arabidopsis plants defective
in autophagy. In plants, reactive oxygen species are
commonly generated via the photosynthetic electron transport chain, and some oxidative damage can occur even
under standard growth conditions. In RNAi-AtATG18a
plants, which are defective in autophagy, an increased
level of oxidized proteins and lipids was observed when
compared with wild-type plants [42], indicating that autophagy may participate in the degradation of these oxidized
molecules. In addition, autophagy-defective plants contain
increased levels of reactive oxygen species and enhanced
expression of reactive oxygen scavenging enzymes
[30,42], suggesting that they are constitutively under a
degree of oxidative stress. One possible explanation is that
these plants are unable to dispose of damaged organelles,
which therefore continue to produce reactive oxygen
species.
Morphologically, the formation of vacuoles in meristematic
cells appears to involve an autophagy process, in which
vesicles and tubules fuse around portions of cytoplasm,
eventually forming an autophagosome-like structure in
which the cytoplasmic contents are degraded [1,43]. Surprisingly, Arabidopsis knockout mutants defective in
autophagy do not show developmental phenotypes, other
than early senescence, even though vacuole formation is
essential for embryo development in Arabidopsis [44].
These disparate observations have been at least partially
reconciled in recent work by Yano et al. [45], who used a
mini-protoplast system to study vacuole biogenesis. Miniprotoplasts lacking large central vacuoles were prepared
from tobacco BY-2 protoplasts and incubated for one to two
days, during which time large vacuoles reformed. In the
presence of cysteine protease inhibitors the newly formed
vacuoles contained cytoplasmic material, suggesting an
autophagy process is also operational in vacuole formation
in these cells. The autophagy inhibitors 3-MA and wortmannin, which block starvation-induced autophagy in BY2 cells [40] and also constitutive autophagy in Arabidopsis
root tips [15], had no effect on vacuole formation in the
mini-protoplasts [45]. Together, these results suggest that
autophagy is involved in vacuole biogenesis, but that the
mechanism of autophagy is distinct from the conserved
pathway of stress-induced or constitutive autophagy
involving the ATG genes.
Evidence for selective autophagy
Autophagy is in general considered to be non-selective,
with autophagosomes enclosing random portions of cytoplasm for delivery to the vacuole. However, some intriguing recent data have led to the suggestion that in some
cases the autophagic machinery may selectively transport
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specific types of cargo at a faster rate than the bulk
cytoplasm. A fusion between cytochrome b5 and red
fluorescent protein (Cytb5-RFP) was observed to form
punctate aggregates in the cytoplasm when expressed in
tobacco cells under normal growth conditions [16]. By
contrast, during nitrogen limitation, the fusion protein
was found in the central vacuole and processed to a lower
molecular weight form. Cytb5-RFP fluorescence in the
cytoplasm co-localized with the autophagosome marker
YFP-ATG8, suggesting that Cytb5-RFP was taken up
into autophagosomes, and transport to the vacuole was
blocked by 3-MA. Together these data indicate that the
transport occurs via autophagy.
Two lines of evidence suggest that there may be a degree
of selectivity in the transport of Cytb5-RFP. First, the
degradation rate of Cytb5-RFP was compared with that
of other proteins, and Cytb5-RFP was found to be processed by vacuolar enzymes faster than other proteins.
Second, the degree of co-localization with YFP-ATG8
was higher for Cytb5-RFP than for mitochondria,
suggesting Cytb5-RFP is incorporated into autophagosomes at a higher frequency. The authors conclude that
autophagy may be selective for protein aggregates, like
those formed by Cytb5-RFP [16]. The mechanism
behind the selectivity is not known and could be due
to direct recognition of aggregated protein or, more
generally, the shape or size of the aggregates formed.
While some questions remain regarding the results
reported (for example, the stability of the Cytb5-RFP
fusion in the vacuole is not known nor is its rate of
synthesis, both of which could affect the interpretation
of the results), this study provides the first indication that
autophagy may be capable of selectively removing certain proteins or structures in plant cells.
Conclusions
In addition to known functions in nutrient recycling,
autophagy in plants has now been shown to play diverse
roles in responses to abiotic and biotic stresses, programmed cell death and vacuole formation. Many important questions remain: Are there additional examples of
selective autophagy in plant cells, and how are substrates
selected for vacuolar delivery? What is the relationship
between autophagy and vacuole formation, and which
proteins are required for the formation of vacuoles during
development? What determines the switch from autophagy as a stress survival response to autophagy as a
programmed cell death pathway? How is autophagy
induced and regulated in these different circumstances?
Many exciting advances can be anticipated in the future as
these and other questions are addressed and our understanding of the roles of autophagy in plants is enhanced.
Acknowledgement
Work in the author’s laboratory is supported by grant no. IOB-0515998 from
the National Science Foundation.
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6 Cell Biology
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