Autophagy in Development and Stress Responses of Plants

Abstract

The uptake and degradation of cytoplasmic material by vacuolar autophagy in plants has been studied extensively by electron microscopy and shown to be involved in developmental processes such as vacuole formation, deposition of seed storage proteins and senescence, and in the response of plants to nutrient starvation and to pathogens. The isolation of genes required for autophagy in yeast has allowed the identification of many of the corresponding Arabidopsis genes based on sequence similarity. Knockout mutations in some of these Arabidopsis genes have revealed physiological roles for autophagy in nutrient recycling during nitrogen deficiency and in senescence. Recently, markers for monitoring autophagy in whole plants have been developed, opening the way for future studies to decipher the mechanisms and pathways of autophagy, and the function of these pathways in plant development and stress responses.

Full text

©2005 LANDES BIOSCIENCE. DO NOT DISTRIBUTE.
[Autophagy 2:1, 2-11; January/February/March 2006]; ©2006 Landes Bioscience
2Autophagy 2006; Vol. 2 Issue 1
Diane C. Bassham1,*
Marianne Laporte2
Francis Marty3
Yuji Moriyasu4
Yoshinori Ohsumi5
Laura J. Olsen6
Kohki Yoshimoto5
1Department of Genetics, Development and Cell Biology; Iowa State University;
Ames, Iowa USA
2Department of Biology; Eastern Michigan University; Ypsilanti, Michigan USA
3Université de Bourgogne; Dijon, France
4School of Food and Nutritional Sciences; University of Shizuoka; Shizuoka, Japan
5National Institute for Basic Biology; Division of Molecular Cell Biology; Okazaki , Japan
6Department of Molecular, Cellular and Developmental Biology; University of
Michigan; Ann Arbor, Michigan USA
*Correspondence to: Diane C. Bassham; Department of Genetics, Development and
Cell Biology; Iowa State University; Ames, Iowa 50011 USA; Tel.: 515.294.7461;
Fax: 515.294.1337; Email: bassham@iastate.edu
Received 07/20/05; Accepted 08/09/05
Previously published online as an Autophagy E-publication:
http://www.landesbioscience.com/journals/autophagy/abstract.php?id=2092
KEY WORDS
ATG genes, autophagosome, autophagy, nutrient
starvation, senescence, vacuole formation
ACKNOWLEDGEMENTS
F.M. is supported by the Ministère de l'Education
Nationale, de l'Enseignement Supérieur et de la
Recherche. D.C.B. is supported by grants from the
US Department of Agriculture and the National
Science Foundation.
Review
Autophagy in Development and Stress Responses of Plants
ABSTRACT
The uptake and degradation of cytoplasmic material by vacuolar autophagy in plants
has been studied extensively by electron microscopy and shown to be involved in devel-
opmental processes such as vacuole formation, deposition of seed storage proteins and
senescence, and in the response of plants to nutrient starvation and to pathogens. The
isolation of genes required for autophagy in yeast has allowed the identification of many
of the corresponding Arabidopsis genes based on sequence similarity. Knockout mutations
in some of these Arabidopsis genes have revealed physiological roles for autophagy in
nutrient recycling during nitrogen deficiency and in senescence. Recently, markers for
monitoring autophagy in whole plants have been developed, opening the way for future
studies to decipher the mechanisms and pathways of autophagy, and the function of these
pathways in plant development and stress responses.
Plants frequently encounter adverse environmental conditions, and their cellular and
physiological responses to these conditions are critical for survival. Common stresses
imposed upon plants include nitrogen deficiency, due to lack of available nitrogen in the
soil, and carbon deficiency, caused by conditions that limit photosynthetic efficiency and
thus carbon fixation. A key process by which eukaryotic cells respond to and survive such
stresses is vacuolar autophagy. Autophagy, which etymologically means “to eat oneself”, is
a catabolic process by which eukaryotic cells degrade portions of their own cytoplasm.
This process is conserved between yeast, animal, and plant cells, and in plants has also
been known for a number of years to be involved in cellular architectural (re)modeling
that occurs during differentiation and development.
The function of autophagy was revealed by morphogical observations with the electron
microscope. The electron micrographs on which the concept of cellular autophagy rests
show the bulk sequestration of cytoplasmic fragments and their subsequent digestion in
the lytic vacuole. While many plant cells have at least two different types of vacuoles, lytic
and storage, the lytic vacuole is thought to be functionally equivalent to the yeast vacuole
or animal lysosome. Macroautophagy and microautophagy have been defined according
to the size of the cytoplasmic material taken up for destruction (see Fig. 1) and the site of
sequestration. Macroautophagy (hereafter referred to as autophagy) sequesters regions of
cytoplasm, including organelles such as endoplasmic reticulum (ER), Golgi stacks, mito-
chondria, plastids and peroxisomes, into double-membrane bound structures called
autophagosomes. The content of the autophagosome including the inner limiting
membrane, which is defined as the autophagic body once it has been released into the
vacuole lumen, is subsequently degraded. In contrast, in microautophagy the uptake was
originally envisaged as occurring in small “gulps” in which a few cytosolic molecules were
transferred to the vacuole interior within a membrane-delimited vesicle directly formed by
invagination of the vacuolar surface, followed by disintegration of the membrane. The
internalized vesicle formed after septation of the invaginated membrane usually looked
empty. The reader is referred to previous reviews (refs. 1–5) for detailed information on
earlier work on cellular autophagy in plants. In this review, we will attempt to integrate more
recent molecular data on autophagy in plants cells with morphological and physiological
information on the role of autophagy both during plant development and as a stress response.

AUTOPHAGY PROCESSES DURING PLANT DEVELOPMENT
Autophagy is a key process in the formation of the vegetative
vacuole in plant cells. In nonvacuolated meristematic cells (the plant
version of nondifferentiated stem cells), numerous autophagosomes
resulting from the enwrapping of large portions of cytoplasm by
double-membrane envelopes are made. The origin of the isolation
membranes is still a subject of speculation.3Because the enveloping
structure is a flattened sac possibly formed within minutes, it was
considered likely, as still postulated in mammalian cells, that it arises
directly from preexisting ER, which is also largely made of sacs.
However, the landmark description of a complete high-resolution
3-D autophagic sequence in differentiating cells pointed to a totally
different mechanism.1,6 Provacuoles (similar to yeast preautophago-
somal structures; see below) arise as vesicles at the trans-Golgi network
(TGN). Biochemical markers including phosphatidylinositol
phosphate, GTPases and various phosphatases now support this
structural model in plant and animal cells.7,8 The provacuoles are a
critical junction in post-Golgi trafficking at which the endocytic and
vacuolar pathways converge.9,10 These vesicular structures elongate
into tubules, which make cagelike traps around portions of cytoplasm.
Autophagosomes are completed when the tubules fuse to completely
seal the region in a double-membrane compartment. Tubules and
derived autophagosomes contain acid hydrolases in an acidic environ-
ment. The lumen of the tubules contains microvesicles presumably
derived from invagination of their membrane. It is speculated that
this microautophagy might be responsible for the direct
cytosol-to-provacuole transport of components required for the
maturation of the content. The cytoplasm sequestered in the
autophagosome is finally degraded by the digestive enzymes, which
are released from the surrounding cavity, formed from the tubules,
as the inner membrane of the autophagosome deteriorates.
Autophagic-like pathways are used to deliver materials to the
vacuole at numerous stages of plant development. During seed
development, protein storage vacuoles (PSVs) replace the vegetative
(lytic) vacuoles in the cells of maturing legume cotyledons (seed
leaves). During this time, the vegetative vacuole is entrapped by a
tubulo-cisternal envelope whose origin is not clearly understood.11,12
The vegetative vacuole is progressively degraded as the novel PSV
takes it over and fills up with reserve proteins. Degradative
autophagy pathways are also operating when vegetative (lytic) vacuoles
are substituted for PSVs in germinating legume seedlings. Fragments
of cytoplasm are indeed engulfed by PSVs via local invaginations of
their limiting membrane before they are pinched off and subse-
quently degraded.13-15
Some protein precursors and other substances are deposited in
the vacuole using an ER-to-vacuole targeting (ERvt) pathway, which
is presented as a convergent analogue to the cytoplasm-to-vacuole
targeting (Cvt) pathway described in yeast (see below).16 In wheat
grain, the endosperm storage protein prolamin accumulates tran-
siently in ER-derived protein bodies, which enter the vacuoles by a
process similar to microautophagy. The ER-derived limiting
membrane is then degraded and the naked prolamin core combines
with others to produce large storage protein aggregates that are
resistant to vacuolar hydrolysis.17 Similarly, in transgenic tobacco
seeds expressing seed storage protein genes from maize, ER-derived
protein bodies are engulfed into the vacuoles by autophagy.18 A
comparable process is responsible for the release of rubber particles
into the vacuole. Unlike the ER-bodies with a ribosome-bearing
membrane, rubber particles synthesized at the ER are coated by a
single-unit membrane. After internalization into the vacuole the
rubber membrane is digested and the stripped particles coalesce to
form large aggregates.19
Plant-specific, ER-derived vesicles containing precursors of cys-
teine proteinases (PPVs) have been described in seed-storage tissues
and senescing tissues of vegetative organs.20-25 These ER-derived
vesicles have the potential to accumulate a large repertoire of protease
precursors stored in stable, inactive aggregates. They are delivered to
the vacuole by a microautophagy-like protrusion autophagy process
(see Fig. 1). Because the proteases would be activated only when
autophagy occurs, it has been speculated that this Golgi-bypass traf-
ficking could be useful when sudden and massive changes in prote-
olytic activities are required such as in storage protein mobilization
and programmed cell death16 (PCD). This is also a regulated cellular
suicide strategy activated in the hypersensitive response (HR) to
pathogens in plants.21,26 HR is one strategy for resistance of plants
to pathogens in which the rapid induction of PCD in infected cells
restricts the growth and spread of the pathogen to adjacent cells.
General cellular autolysis resulting from a sharp increase of vacuolar
proteolytic activities followed by the permeabilization of the tono-
plast (vacuolar membrane) is involved in the PCD of several plant
tissues.27
Distinctively, it has been recently demonstrated that autophago-
some-mediated autophagy, i.e., macroautophagy, plays an important
role during the innate immune response in plants. In order to prevent
the HR from playing a pathologic rather than protective role,
autophagy functions to restrict PCD to infection sites.28 It is specu-
lated that autophagy may eliminate death-promoting signals moving
out of the pathogen-infected area or may clean up cellular damage
www.landesbioscience.com Autophagy 3
Autophagy in Development and Stress Responses of Plants
Figure 1. Autophagy pathways in plant cells. Macroautophagy: (1)
autophagosome formation from preautophagosomal structures (PAS or
provacuoles); (2) completed autophagosome containing intact cytoplasm
(class 1 autophagosome as described by Rose et al 2005);58 (3) autophago-
some in which the sequestered cytoplasm is being digested (class 2
autophagosome); (4) autophagosome fusing with the central vacuole, the
digestion of the sequestered cytoplasm is almost complete (class 3
autophagosome); (5) the autophagic body is released into the lumen of the
central vacuole. Microautophagy: (6) a small vesicle is formed directly by
invagination of the vacuole membrane and released in the vacuolar lumen
where it is degraded. Protrusion autophagy: (7) whole organelles (ER-
derived bodies, mitochondria, plastids, peroxisomes, rubber particles, etc.)
and large portions of cytoplasm are delivered into the central vacuole by
protrusion (8) and direct engulfment (9). Further processing is achieved by
vacuolar hydrolases (10).

caused during the defense response. A role for autophagy in host
defense by limiting the growth of intracellular pathogens is also
suggested. How this negative regulatory role for autophagy in PCD
will be reconciled with the potential positive role described above
remains to be seen.
Autophagy driven by direct invagination of the tonoplast into the
central vacuole (i.e., a form of microautophagy) in cells from senesc-
ing tissues has been reported.29-32 The invagination is pinched off
and the isolated body, containing cytoplasmic materials and
membranes, is finally degraded in the vacuole. Interestingly, during
the senescence of photosynthetic leaf cells from Arabidopsis and
soybean, small specific senescence-associated vacuoles (SAVs) with
intense proteolytic activity are formed in the peripheral cytoplasm.33
They often contain dense aggregates, which may consist of partially
degraded cellular material similar to the contents of late autophagic
vacuoles. However, these SAVs are not derived from classical
autophagosomes and their relationship to more typical autophagy
processes remains to be determined.
SELECTIVE AUTOPHAGY PATHWAYS
The term pexophagy was introduced to describe the selective
elimination of peroxisomes in different yeasts.34 Similarly, it has
been suggested that plants might also use selective sequestration to
remove glyoxysomes when they are no longer needed in oil
seeds.35,36 Occasionally, vacuoles have been seen to engulf mitochon-
dria, plastids or starch grains.29,37-41 However, it is now known that
autophagy does not play a major role in the degradation of chloroplast
proteins,2but catabolites released from the senescing chloroplast are
further metabolized in the vacuole.42 Subregions of the tonoplast
invaginate in round-shaped structures described as “bulbs”, inside
the vacuole of cotyledonary cells and drought-acclimated cells.43,44
They are highly dynamic structures, resistant to lysis and are thought
to be reservoirs for tonoplast proteins. Whatever the mechanisms
involved in the uptake of organelles and tonoplast, one may wonder
whether these intriguing processes are essentially blind or selective.
Several additional autophagy-related pathways have been shown
to operate in other organisms. The Cvt pathway is biosynthetic and
operates under nutrient-rich conditions. It involves sequestration by
a double-membrane envelope, which appears morphologically to be
a small version of the autophagosome. The existence of the Cvt
pathway has only been demonstrated in the yeast S. cerevisiae.45
Another degradative pathway, which operates in animal cells but is
not found in yeast, is chaperone-mediated autophagy46 (CMA), in
which particular cytosolic proteins are delivered to lysosomes in a
molecule-by-molecule fashion with the help of a chaperone complex
in the cytosol and on the lumenal side of the lysosomal membrane.
Finally, the vacuolar import and degradation (Vid) pathway is
involved in the degradation of the gluconeogenic enzyme fructose-
1,6-bisphosphate in yeast.47 None of these alternative autophagy
pathways have been shown as yet to be present in plants.
It is now clear that the vacuolar apparatus forms an intracellular
digestive system and has evolved different autophagy pathways for
recycling intracellular constituents. Because several types of vacuoles
interplay in plant cells,48-52 the question arises as to whether
autophagy is a common function.
MORPHOLOGICAL OBSERVATION OF AUTOPHAGY INDUCED
BY NUTRIENT STARVATION
The degradative autophagy pathway is induced in vacuolated cells
to adapt to various environmental stresses, such as nutrient starva-
tion.37,53-60 In tobacco-cultured BY-2 cells, cytoplasmic degradation
has been suggested to be confined to small lytic compartments,
rather than the central vacuole,57,61 although this remains controversial
as other researchers report no differences between BY-2 cells and
vacuolated cells from other species (Bonneau L, Marty F, unpub-
lished). In sycamore and Arabidopsis-cultured cells autophagosomes
are formed in the cytoplasm shortly after the cells are deprived of
nutrient.53,58 Unlike the situation in yeast cells, the encircled portion
of cytoplasm is usually degraded before the outer membrane of the
autophagosome fuses with the tonoplast. Upon fusion, the internal
vesicle is released into the vacuole (the vesicle is now called an
autophagic body) where the degradation of its membrane and internal
remnants is completed. The degradation of the trapped cell compo-
nents recycles intracellular substrates and makes resources sufficient
for the cell to meet its vital needs and survive. Cellular autophagy
thus plays an essential role in nutrient recycling.55,56,60,62
Sucrose starvation in suspension cultured cells has proven to be
an excellent model system for the analysis of plant
autophagy.37,53,57,58,63 When cells are transferred to sucrose-free
medium, the transvacuolar strands, strands of cytoplasm that connect
peripheral and perinuclear regions of the cytoplasm, degenerate, and
30 to 50% of the total protein is degraded over a two-day period,61
concomitant with an increase in protease activity. Treatment with
cysteine protease inhibitors such as E-64c rescues almost all polypep-
tides from degradation, showing that the decrease in the total protein
derives from nonselective degradation of intracellular proteins and
not from the degradation of specific proteins.57
In BY-2 cells, cysteine protease inhibitors cause the accumulation
of membrane vesicles around the nucleus, which contain
electron-dense particles.57 Among these particles, partially degraded
mitochondria are occasionally seen (Moriyasu Y, Robinson DG,
unpublished results), suggesting that the particles represent portions
of the cytoplasm undergoing degradation. The vesicles are acidic
compartments, containing cysteine protease activity and acid phos-
phatase, a marker enzyme of lysosomes.57 Thus the membrane vesicles
are thought to represent newly formed class 2 autophagosomes
(autolysosomes; see Fig. 1). They are morphologically distinct from
the central vacuole, in that they are smaller (1 to 6 µm in diameter)
and the central vacuole accumulates very few electron- dense parti-
cles upon E-64c treatment.
Without the addition of cysteine protease inhibitors, the accu-
mulation of autophagosomes in tobacco cells is difficult to detect by
light microscopy,57 suggesting that the digestion of cytoplasm pro-
ceeds very rapidly during normal autophagy. In cells treated with
E-64c, fusion profiles between autophagosomes and central vacuoles
are observed. Thus, it is likely that some of the partially degraded
cytoplasm in autophagosomes is released into the lumen of the central
vacuole for further degradation during a typical autophagic process.
On the other hand, it can be observed by electron microscopy that
the number of empty vesicles increases significantly under sucrose
starvation conditions in the absence of E-64c.61 This suggests that,
unlike in yeast, a significant proportion of autophagosomes finish
the digestion of enclosed materials before their fusion with the central
vacuole.
Bafilomycin A1and concanamycin A are inhibitors of the vac-
uole-type H+-ATPase64-66 and therefore prevent vacuolar hydrolase
activity. Starvation-induced autophagy in BY-2 cells, as in
Autophagy in Development and Stress Responses of Plants
4 Autophagy 2006; Vol. 2 Issue 1

Arabidopsis60 (see section on monitoring of plant autophagy, below),
is perturbed by bafilomycin A1and concanamycin A. In these cells,
autophagic bodies cannot be degraded but are still expelled into the
central vacuole where they accumulate, resembling autophagic bodies
in yeast vacuoles.60
The pathway for maturation of autophagosomes is not fully
understood in plant cells. In mammalian cells, newly formed
autophagosomes fuse with preexisting lysosomes and/or endosomes,
becoming autolysosomes. In yeast cells, autophagosomes fuse with
the vacuole, resulting in the vacuole containing autophagic bodies.67
In plant cells, acid hydrolases have been localized to the enwrapping
autophagic cavities as well as to the precursor structures (provacuoles)
from which they derive.6Some time after the sequestered portion of
cytoplasm has been closed off, the hydrolases are released from the
sequestering cavity into the autophagic body, which is subsequently
degraded. It is therefore suggested that autophagosomes on their
own are functionally self-sufficient to achieve the breakdown of the
sequestered materials.58 As a consequence, autophagosomes can
finish the digestion of trapped components before their fusion with
the central vacuole (see above section). When autophagosomes
mature in BY-2 cells, there seem to be several pathways for transport
of membrane vesicles to autophagosomes. The styryl dye FM 4-64
on the plasma membrane flows into autophagosomes, probably
through an endocytic pathway.10 Thus, it is probable that as in
mammalian cells, the fusion of autophagosomes with endosomes
contributes to the maturation of autophagosomes in tobacco BY-2
cells.9,10 On the other hand, FM 4-64 residing on the membrane of
central vacuoles, along with proteins existing in the membrane and
lumen of central vacuoles, moves to autophagosomes under sucrose
starvation conditions, suggesting that, upon fusion of autophagosomes
with the central vacuole, there may be a flow of proteins and
membrane lipids from the central vacuole to autophagosomes.10
Thus it is possible that hydrolytic enzymes in the central vacuole also
move back to autophagosomes and contribute to the maturation of
autophagosomes. In contrast, in mammalian cells and yeast there is
no evidence for the movement of membrane or proteins from the
lysosome/vacuole to the autophagosome.
IDENTIFICATION OF ARABIDOPSIS HOMOLOGUES
OF YEAST AUTOPHAGY GENES
More than 30 genes in yeast have been identified that are
involved in various steps of autophagy (Table 1). Most of these genes
also have mammalian homologues, and many have apparent homo-
logues in plants.56 Though the functions of some ATG genes in
Arabidopsis have been studied, their exact roles in autophagy are not
known. It has been recently shown that the ATG genes linked to
autophagosome formation are expressed in a coordinated manner at
the onset of starvation, suggesting a role during starvation-induced
autophagy.58 An overview of the putative ATG homologues in
Arabidopsis yields several interesting observations (summarized in
Table 1).
The induction of autophagy by starvation conditions or other
stress signals is accomplished by the Tor kinase signaling cascade.68
There is likely a single TOR gene in Arabidopsis. Tor (‘Target of
rapamycin’)69 causes Atg13 to be phosphorylated, thereby decreasing
its interaction with the kinase Atg1.70 Atg13 is dephosphorylated
under starvation conditions, concomitant with the induction of
autophagy. Although two putative ATG13 genes are found in
Arabidopsis, their similarity is restricted to a relatively small region
of the protein and it is currently unclear if they are true Atg13
homologues. There are, however, multiple genes for ATG1 in
Arabidopsis56—nothing is known about their function, protein-
protein interactions or predicted localization.
Many of the components required for the yeast-specific Cvt path-
way are also required for autophagy.67,71-73 Two genes, ATG11 and
ATG19, are specifically required for cargo selection in the Cvt path-
way. Arabidopsis does not have a homologue for either of these
hydrolases. Not surprisingly therefore, Arabidopsis lacks an ATG19
homologue; Atg19 is the cargo receptor for the Cvt pathway. Based
solely on computational analysis, it is not clear whether there are any
ATG11 genes in Arabidopsis. In addition to the Cvt pathway, Atg11
is also required for pexophagy in Pichia pastoris.74 Pexophagy has not
yet been documented in plants, but it is possible that a putative
AtATG11 may play a role in pexophagy or a pexophagy-related
process in plants. Alternatively, it is possible that AtATG11, if it
exists, functions in a unique way in plant autophagy.
Most of the proteins required for autophagy function in formation
of the autophagosome. This process, including vesicle nucleation,
expansion and completion and targeting to the vacuolar membrane,
is complex and poorly understood.75 The regulation of vesicle
induction and nucleation in yeast involves a class III phosphatidyli-
nositol 3-kinase (PI3K), whose catalytic subunit is Vps34. There appears
to be a single homologue in Arabidopsis of VPS34 and of VPS15,
which is required for the kinase activity. Another subunit of the
complex, ATG14, is apparently not found in Arabidopsis. Atg14 acts
as a bridge between Atg6 and Vps15/Vps34. Thus it is likely that
another protein, probably unrelated to Atg14, performs this function
in Arabidopsis. Although a single gene for ATG6 is found in
Arabidopsis, there appears to be 2 splice variants. Whether both
variants are expressed at the same time, in the same place, and/or
produce functional proteins is not known.
There are two ubiquitin-like conjugation pathways that function
during autophagosome membrane expansion and cargo engulf-
ment.35,75 The E1-like enzyme Atg7 activates Atg12 in one complex
and Atg8 in the other complex. Atg12 forms a complex with Atg5
and Atg16. Atg10, an E2-like enzyme, is required for this complex
formation in yeast, and is present as a single gene.76 ATG5 and
ATG7 are present as single genes, and there may be an Arabidopsis
homologue of ATG16. There are two ATG12 genes in Arabidopsis.77
The E2-like protein required for the formation of the Atg8 complex
is Atg3. This protein is coded for by a single Arabidopsis gene. Two
genes for ATG4 (both with distinct splice variants) are also found in
Arabidopsis; Atg4 is a cysteine protease that removes residues from
Atg8 after a conserved C-terminal glycine, and cleaves Atg8-phos-
phatidylethanolamine to remove the lipid moiety, and there are nine
ATG8 genes in Arabidopsis.
Many proteins are involved in regulating autophagy, but their
function is currently defined mostly by their interacting partners.75
Atg17 interacts with Atg1, Atg20, and Atg24 to regulate early steps
in autophagosome formation. There are no apparent Arabidopsis
homologues for ATG17, but the other three proteins are each
produced by multigene families. The exact number and members of
the gene families for ATG20 and ATG24 are difficult to distinguish
using only sequence comparison and motif analysis to assign identity.
Similarly, AtATG18 and AtATG21 gene families resemble each other
in structure, making it hard to assign a given gene to a particular
family.
Atg18 is required for the localization of Atg2 to the preautophago-
somal structure (PAS) in yeast.78-80 Atg9 is an integral membrane
protein that also localizes to the PAS, as well as to a non-PAS structure,
but is not present in the mature autophagosome.81 Thus, Atg9 is
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6 Autophagy 2006; Vol. 2 Issue 1
Autophagy in Development and Stress Responses of Plants
Table 1 Genes involved in autophagy in Arabidopsis and yeast
Yeast Predicted Number of Homologues Functions/Characteristics of Yeast Proteinsa
Protein in Arabidopsis Thaliana
Induction of Autophagy, Signal Transduction, Cargo Selection
TOR Single gene Protein kinase; negative regulator of autophagy
ATG11 None Peripheral membrane protein; interacts with ATG1; required for Cvt pathway, but not for
macroautophagy in S. cerevisiae
ATG19 None Cargo receptor; required for Cvt pathway, but not for macroautophagy in S. cerevisiae
Autophagosome Formation
Phosphatidylinositol-3-kinase complex:
ATG6 Single gene;two predicted splice variants Interacts with ATG14, VPS34, VPS15
ATG14 None Subunit of PI3K complex
VPS15 Single gene Protein kinase required for Vps34
VPS34 Single gene PI-3-K catalytic subunit
Ubiquitin-like conjugation pathways:
Pathway 1:
ATG5 Single gene Forms complex with ATG12
ATG7 Single gene E1-like enzyme; activates ATG12 and ATG8
ATG10 Single gene; two predicted splice variants E2-like enzyme
ATG12 Two genes Interacts with ATG5
ATG16 Single gene with restricted similarity Interacts with ATG5
Pathway 2:
ATG3 Single gene E2 protein for ATG8 complex
ATG4 Two genes; both with predicted splice variants Cysteine protease; processes ATG8 and ATG8-PE;
ATG7 Single gene E1-like enzyme; activates ATG12 and ATG8
ATG8 Small gene family; two loci with predicted Ubiquitin-like modifier; regulates autophagosome size
splice variants
Regulatory machinery:
ATG1 Small gene family Serine/threonine kinase; interacts with ATG13
ATG13 Two possible genes with restricted similarity Phosphoprotein; dephosphorylated under starvation conditions; not required for Cvt
pathway
ATG17 None Interacts with ATG1, ATG20, ATG24; not required for Cvt pathway
ATG18 Small gene family with multiple splice variants Needed for ATG2 localization and ATG9 recycling from PAS
ATG20 Small gene family Interacts with ATG24 and ATG17; required for Cvt pathway, but not for macroautophagy
in S. cerevisiae
ATG21 Difficult to distinguish from ATG18 Required for recruitment of ATG8 to PAS; required for Cvt pathway, but not for
macroautophagy in S. cerevisiae
ATG24 Difficult to distinguish from ATG20 Interacts with ATG1, ATG17, and ATG20; required for Cvt pathway, but not for
macroautophagy in S. cerevisiae
ATG27 None PtdIns3P-binding protein; membrane protein; required for Cvt pathway, but not for
macroautophagy in S. cerevisiae
VAC8 N.D.bArmadillo repeat protein; interacts with ATG13; required for Cvt pathway, but not essential
for macroautophagy in S. cerevisiae
ATG9 COMPLEX
ATG2 Single gene Interacts with ATG9
ATG9 Single gene Interacts with ATG2 and ATG23; integral membrane protein
ATG23 None Recycles ATG9 from PAS; required for Cvt pathway, but not essential for macroautophagy
in S. cerevisiae
Breakdown of Autophagic and Cvt Vesicles in Vacuole
ATG15 None Putative lipase; integral membrane protein
ATG22 Single gene Putative permease homologue; integral membrane protein; not required for Cvt pathway
PEP4 Multigene family Vacuolar proteinase A
PRB1 Small gene family Vacuolar proteinase B
VPE N.D. Plant specific; see Thompson and Vierstra, 2005
Homology was predicted based upon database annotation, sequence similarity, and the presence of conserved domains and residues. aPartially based on Farré and Subramani, 2004; bN.D., not determined.

retrieved from the PAS prior to autophagosome maturation. Atg23
localization in yeast is similar to Atg9,82 but its retrieval from the
PAS is different.80 The retrieval of Atg9 and/or Atg23 from the PAS
requires Atg18, Atg2, and Atg1. It is not yet known whether plants
possess a PAS in the same way as yeast, but there is a single homologue
in Arabidopsis of ATG9.56 Atg23 is not required for autophagy in
yeast—it might be Cvt specific;82 there is no apparent Arabidopsis
homologue of ATG23.
Ultimately, autophagosomes (and Cvt vesicles) fuse with the
vacuole, releasing single-membrane vesicles, which are degraded by
vacuolar enzymes.35,75 Vacuolar proteinase A (Pep4), vacuolar pro-
teinase B (Prb1), and the acidic lumen of the vacuole may act directly
or indirectly to break down the autophagic vesicles.83,84 Arabidopsis
has many homologues of these vacuolar proteases. In addition, the
putative lipase Atg15 and a potential permease, Atg22, also may be
involved in this step.85,86,87 Although there seems to be a single gene
for ATG22 in Arabidopsis, there is not an obvious ATG15 homologue.
In summary, of the 31 genes shown in Table 1 to be involved in
autophagy in yeast, at least 24 have homologues in Arabidopsis.
Several are present as multigene families—e.g., ATG8, ATG18,
VAC8. Some of the genes are predicted to express splice variants
(e.g., ATG4, ATG6, ATG8), although the significance of this is
unknown. Finally, some genes that appear to be homologues of yeast
genes, based on sequence comparisons, may ultimately be shown to
play a different role in plant autophagy or to be involved in different
processes altogether.
REVERSE GENETIC APPROACH FOR STUDYING
PLANT AUTOPHAGY
Sequence homologies between Arabidopsis ATG genes and those
of other species are not particularly high, and only AtATG4,
AtATG6 and AtATG8s have been shown to complement the corre-
sponding yeast atg mutant.28,56,60 Most of the essential residues in
the yeast Atg proteins are conserved in these AtATG proteins, sug-
gesting that most of the autophagic machinery is well conserved in
plants. This prediction has been further supported by experiments
using Arabidopsis T-DNA insertion mutants of ATG genes. For
example, in a T-DNA mutant of AtATG7 (Atatg7-1), wild-type
AtATG7 could complement the mutant phenotype, but
AtATG7C/S, which contains a substitution in a catalytic active
cysteine residue, could not, suggesting that AtATG7 also functions
as an E1 enzyme in two Atg conjugation reactions.55 Recently, a
double knockout mutant (Atatg4a4b-1) of the functionally-redundant
proteins AtATG4a and AtATG4b was obtained, corresponding to
the genes encoding the processing and deconjugation enzyme of
AtATG8. It has been clearly shown that the putative phos-
phatidylethanolamine (PE)-conjugated AtATG8s and AtATG8-
AtATG3 intermediates were detected in wild-type plants but not in
the Atatg4a4b-1 double mutant.60 In addition, the C-terminus of
the nascent form of AtATG8s was cleaved in an AtATG4-dependent
manner, and Ala substitution of the C-terminal Gly of AtATG8s
results in a defect in the normal behavior of AtATG8s. These results
indicated that in plant autophagy an ubiquitination-like Atg8
lipidation system functions as it does in yeast.
MONITORING OF PLANT AUTOPHAGY
The lack of a convenient assay for autophagy in plants has ham-
pered previous efforts to study this process. It was predicted that an
ubiquitin-like protein, Atg8, would be suitable as a marker for
monitoring plant autophagy,60,63 as it has proven useful in other
organisms. Atg8 is modified with a lipid molecule PE by ubiquitina-
tion-like reactions; consequently, Atg8-PE behaves like an intrinsic
membrane protein, and the PE-conjugated form is involved in
autophagosome formation. The Atg8 conjugated to PE is localized
to autophagosomes and their intermediates, and finally a portion of
it is transferred to the vacuole during autophagy. Therefore, in yeast,
Atg8 is a useful molecular marker for monitoring the autophagic
process.88 In mammalian cells, an Atg8 homologue, LC3, is used as
a molecular marker for monitoring the autophagic process.89
In order to address this need for a marker protein, transgenic
plants expressing N-terminally GFP-fused AtATG8 protein
(GFP-AtATG8) were generated, and were observed by confocal laser
scanning microscopy.60 In wild-type roots expressing GFP-AtATG8,
under nutrient-rich conditions, GFP-AtATG8 is observed as many
ring-shaped and punctate structures, which correspond to
autophagosomes and their intermediates, respectively (Fig. 2A). In
contrast, these structures are not observed in roots of Atatg4a4b-1
plants expressing GFP-AtATG8, although some small dot structures,
possibly aggregates, are observed (Fig. 2C). Under nitrogen-starved
conditions, GFP-AtATG8 is delivered to vacuolar lumens in
wild-type roots (Fig. 2B). On the other hand, in the Atatg4a4b-1
mutant, none of the GFP-AtATG8s, even GFP-AtATG8i, which has
no C-terminal extension after the Gly residue, are delivered to the
www.landesbioscience.com Autophagy 7
Autophagy in Development and Stress Responses of Plants
Figure 2. Monitoring of the autophagic process in plant roots. Localization
of GFP-AtATG8a in wild-type and Atatg4a4b-1 roots. Under nutrient-rich
conditions (MS), many ring-shaped structures, which are thought to be
autophagosomes, are observed in wild-type roots (A, inset), but not in
Atatg4a4b-1 roots (C). When the plants are transferred to nitrogen-starved
conditions (MS-N), GFP-AtATG8a is delivered to the vacuolar lumen in
wild-type roots (B), but not in Atatg4a4b-1roots (D). Larger GFP-labeled
structures are nuclei, into which most likely the GFP fusion can diffuse due to
its small size. Bar=1 µm (A) and 20 µm (D). [modified from Yoshimoto K, et al.
Plant Cell 2004; 16:2967-83].
A B
C D

vacuole (Fig. 2D and data not shown), suggesting that AtATG4s are
essential for autophagosome formation not only as processing
enzymes but presumably also deconjugation enzymes of putative
PE-conjugated AtATG8s. These results suggest that AtATG8s are
suitable for monitoring the autophagic process in plants. The general
utility of this approach has been demonstrated by transient transfec-
tion of protoplasts with GFP-AtATG8. Whereas starved protoplasts
from wild-type plants contained many GFP-labeled autophagosomes,63
those derived from transgenic lines with reduced expression of the
autophagy gene AtATG18a lack these structures,90 indicating that
transient transfection provides a rapid assay for identifying
autophagy defects.
When roots are treated with concanamycin A, a V-ATPase
inhibitor, many spherical bodies are observed in vacuolar lumens of
wild-type roots, but not in those of Atatg4a4b-1 roots using conven-
tional light microscopy (data not shown). Electron microscopy
revealed that these spherical bodies contain cytoplasmic structures,
such as mitochondria, endoplasmic reticulum, and Golgi bodies
(Fig. 3A and B), suggesting that these structures correspond to
autophagic bodies in plants. Concanamycin A is known to raise the
pH of the vacuolar lumen by inhibiting the V-type ATPase when
exogenously added to plant cells. Consequently, under such high pH
conditions, vacuolar hydrolases cannot act, resulting in accumula-
tion of autophagic bodies in the vacuole. Similarly, when roots are
treated with the cysteine protease inhibitor E-64d, aggregates, pre-
sumably consisting of cytoplasmic degradation products, are
observed in the vacuolar lumen in wild-type roots, but not
Atatg4a4b-1 roots (data not shown). Treatment with Concanamycin
A or E-64d are therefore very easy ways to detect whether autophagy
occurs in plants. Using these monitoring systems, it is now clear that
AtATG proteins are responsible for autophagy.
Recently, it has been proven that AtATG12-AtATG5 conjugates
are also essential for plant autophagy by using mutants in the respon-
sible genes.76,77 Using anti-AtATG12b antibodies that recognize
both AtATG12a and AtATG12b, AtATG12-AtATG5 conjugates
can be detected in wild-type and AtATG10/Atatg10-1 (heterozygous)
plants. As expected, in Atatg4a4b-1, whose mutated genes are
known not to be involved in Atg12-Atg5 conjugation,
AtATG12-AtATG5 conjugates are also detected. In contrast, in
Atatg5-1 and Atatg10-1plants, which are deficient mutants of the
target and E2-like enzymes of AtATG12, respectively,
AtATG12-AtATG5 conjugates are not detected. Instead, unconju-
gated AtATG12s increase in these mutants compared with plants
that can form AtATG12-AtATG5
conjugates. It was further examined
whether these mutants have a defect
in autophagy by concanamycin A
treatment. In wild-type and
AtATG10/Atatg10-1 (heterozygous)
roots treated with concanamycin A,
many autophagic bodies are detected
in the vacuoles, but not in those of
Atatg5-1 and Atatg10-1 roots. The
result shows that Atatg5-1 and
Atatg10-1 plants are defective in
autophagy, suggesting that
AtATG12-AtATG5 conjugates are
essential for plant autophagy. In
mammalian cells, it is known that
the Atg12-Atg5 conjugate dissociates
from autophagosomes when their formation is complete; therefore
this is a good marker for monitoring autophagosome formation.91
As in mammalian cells, it is expected that AtATG5 will provide a
useful marker for monitoring the process of plant autophagosome
formation.
PHYSIOLOGICAL ROLES OF AUTOPHAGY IN WHOLE PLANTS
The Atatg mutant plants that have been described can achieve
normal embryogenesis, germination, cotyledon development, shoot
and root elongation, flowering, and seed production under normal
nutrient-rich conditions. However, careful phenotypic analyses
revealed some differences between wild-type and Atatg
mutants.55,56,60,77,90 In Atatg mutants, leaf senescence is accelerated
even in nutrient-rich conditions (Fig. 4A and B). In addition, bolting,
the production of inflorescence (floral) stems, of Atatg9-1 is acceler-
ated under normal conditions.56 These results suggest that
autophagy plays some roles in normal developmental processes even
in nutrient-rich conditions.
When Atatg mutants are grown under nitrogen depleted or limiting
conditions, they exhibit drastic acceleration of starvation-induced
yellowing of the leaves. When leaves are artificially starved by
detachment and dark-treatment, the mRNA levels of senescence
marker genes such as SEN1 increase at earlier times in the mutants
than wild-type, resulting from acceleration of artificially induced
senescence.55,56,90 An Arabidopsis mutant of the v-SNARE VTI12,
whose yeast homologue has been shown to be involved in docking
and fusion of autophagosomes with the vacuole, also exhibited a
similar acceleration of senescence in detached leaves, suggesting a
function in autophagy.62 It is currently unclear why Atatg mutants
show an early senescence phenotype. In Atatg mutants, chlorophyll
degradation is accelerated compared with wild-type plants; there-
fore, it seems that autophagy is not necessary for degradation of
chloroplast proteins. This result is consistent with the idea that the
degradation of chloroplast proteins during the initial stages of leaf
senescence occurs within the chloroplast itself.2,42,92 Thus, the accel-
erated senescence seen in Atatg mutants may reflect the need for
vacuolar degradation of components that reside outside of the
chloroplast.
Atatg mutants also show some subtle growth defects. They develop
fewer inflorescence branches because of early senescence, resulting in
lower yield of seeds. In addition, root elongation of Atatg mutants is
inhibited under nutrient-depleted conditions compared with those
8 Autophagy 2006; Vol. 2 Issue 1
Autophagy in Development and Stress Responses of Plants
Figure 3. Plant autophagic bodies. In wild-type Arabidopsis roots treated with concanamycin A, a V-ATPase
inhibitor, many autophagic bodies accumulating in the vacuolar lumens of root cells are observed (A), but not
in Atatg4a4b-1 mutant roots (C). The autophagic bodies contain cytoplasmic structures, such as mitochondria,
endoplasmic reticulum, and Golgi bodies (B). Bar=10 µm (A and C) and 500 nm (B). [modified from Yoshimoto
K, et al. Plant Cell 2004; 16:2967-83].
ABC

of wild-type (Fig. 4C). It is thought that when the nutrient supply
from the environment is limited, autophagy is required for efficient
recycling of protein in the cells, and so its defect may cause a less
efficient supply of nutrients; consequently cell growth is inhibited.
Further phenotypic analysis of Atatg mutants also provides novel
information on potential differences in autophagy in plants com-
pared with other organisms. The phenotypes seen in Atatg4a4b-1 are
more severe than those of Atatg9-1. In Atatg9-1 roots, although
autophagic bodies accumulate more slowly compared with
wild-type, they finally accumulate in the vacuolar lumen in the
presence of concanamycin A, despite the presence of a null mutation.
This is also seen in another mutant allele, Atatg9-2. Atatg9 mutants
are leaky for unknown reasons, even though autophagy is completely
abolished in the yeast atg9 mutant. These results suggest that higher
plants are in some ways distinct from yeast and may have further
plant-specific autophagy pathways.
PERSPECTIVES
Research on plant autophagy is still in its infancy, and many
important questions remain to be answered. One issue that is still
unresolved is the possible variation in the degradative compartment.
It has been proposed that the main lytic compartments of autophagy
in plant cells are autophagosomes and not the central vacuole.53,57,58
However, in vacuolated plant cells, autophagosomes appear to fuse
with the central lytic vacuole as they do in yeast cells.53,93 These
observations suggest that plant cells may use two types of lytic
compartments in autophagy and that the contributions of these
organelles are controlled differently in different cells. It is also possible
that plant species may differ in the extent of degradation of cyto-
plasmic components within autophagosomes, before fusion with the
vacuole, rather than in the mechanism of autophagy itself. How the
contribution of these two pathways to autophagy is regulated will be
one of the future issues to be addressed in autophagy of plant cells.
Autophagy monitoring systems established recently in whole
plants allow the determination of when and where autophagy
occurs, and under which conditions autophagy is induced. These
systems will contribute greatly to the further understanding of the
physiological roles of plant autophagy. In plants, the autophagy-
defective phenotype is rather weak compared with that of slime
mold, nematode, fruit fly, and mammals.94,95,96,97 It is striking that
while autophagy pathways have been shown by electron microscopy
to function in numerous developmental processes, these functions
are not always reflected in the phenotypes of autophagy-defective
mutants. There is no doubt that bulk protein degradation is impor-
tant in various aspects of plant life, and other vacuolar degradation
pathways probably exist in plants. Atatg mutants are likely to provide
powerful tools for the investigation of such pathways in the future.
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