Update
Letters
What about the role of autophagy in PCD?
Wouter G. van Doorn1 and Ernst J. Woltering2
1
Mann Laboratory, Department of Plant Sciences, University of California, Davis, CA 95616, USA
2
Wageningen University and Research Centre, PO Box 17, 6700 AA Wageningen, The Netherlands
Here we discuss to what extent the concept ‘autophagic cell
death’ applies to programmed cell death (PCD) in plants.
When using the classical definition of ‘autophagic cell
death’ the evidence is as yet absent. We examine a possible
redefinition of the term.
An interesting discussion has developed in recent issues
of Trends in Plant Science, on the possible role of autop-
hagy as an executioner of programmed cell death (PCD) in
plants. Andrew Love et al. [1] suggested that autophagy is
responsible for the bulk of cellular dismantling and remo-
bilization of various compounds, during senescence, which
is a type of developmental PCD. Jean-Luc Cacas and Mark
Diamond [2] did not concur. They argued (i) that autopha-
gic cell death has been defined, in animal science, by
autophagic vacuolization of the cytoplasm, and (ii) that a
causal role of autophagy had not been demonstrated in
plant PCD. They claimed that autophagic structures,
unlike other vesicles, possess a double membrane. So
unless a double membrane has been shown in the vesicles
involved in the cellular degradation, they asserted, one
cannot speak of autophagy.
It is useful to make a distinction between developmental
PCD (which can be taken to include stress-induced PCD)
and pathogen-induced PCD. We previously suggested the
following sequence of events at the final stage of many
examples of developmental PCD in plants: the tonoplast
becomes highly permeable (or ruptures); this results in the
release of a massive amount of hydrolases, which rapidly
degrade whatever is by then left of the cytoplasm, and in
some cases also of the cell wall. The action of hydrolytic
enzymes, resulting in self-degradation, we proposed, can
be regarded as a type of autophagy. The cell is after all
‘eating’ itself [3].
We will furthermore distinguish between different
types of autophagy, and between the autophagy found in
yeast, animal and plant cells. Micro-autophagy is the
uptake of materials at the surface of a lytic vacuole. It
occurs by the invagination or protrusion of the vacuolar
membrane. The vesicles produced have only a single mem-
brane. Little, if anything, has been published about the role
of micro-autophagy in developmental plant PCD. Cacas
and Diamond [2] claimed that autophagic structures
should possess a double membrane. This is not correct,
as micro-autophagy is also autophagy and does not involve
a vesicle with a double membrane. The claim [2] that
autophagy has not been shown to be an executioner of
plant PCD, therefore, cannot be based on the argument
that no double-membrane structures have been observed
during plant PCD.
Corresponding author: van Doorn, W.G. (wgvandoorn@ucdavis.edu).
Macro-autophagy starts further away from a lytic
vacuole. In animal cells a portion of the cytoplasm, which
can include various organelles, is surrounded by a growing
double-membrane structure. Upon closure of the double
membrane, the structure with its enclosed cytoplasmic
material is an autophagosome [4]. In animals the outer
membrane of the autophagosome subsequently fuses with
a lysosome, a small vesicle containing hydrolases. This
fusion results in degradation of the material inside the
outer membrane of the autophagosome. Upon fusion with a
lysosome, the autophagosome is called an autolysosome
[4]. Yeasts also have autophagosomes, which fuse with a
large lytic vacuole [5].
Macro-autophagy carried out by autophagosomes and
autolysosomes is ubiquitous in animal cells. ‘Autophagic
cell death’ in animals has been defined by the increase in
the number of autophagosomes, autolysosomes and small
lytic vacuoles produced by autolysosomes (together termed
autophagic vacuolization) in the cytoplasm, independent of
the process being a cause of death or only associated with
death [6]. If macro-autophagy is defined as in animals (by
the presence of autophagosomes and autolysosomes) it has
not yet been demonstrated in plant cells. Nonetheless, in
the root meristem of several plant species, organelles have
been shown that are similar to autophagosomes in animal
cells. A structure with two parallel membranes was formed
around a portion of the cytoplasm, which resulted in
degradation of the inner membrane and the contents of
the enclosed cytoplasm. However, the lumen of these
autophagosome-like structures was acidic and contained
hydrolases from their very inception at the Golgi/endoplas-
mic reticulum interface [7,8]. They therefore cannot be
called autophagosomes because autophagosomes become
acidic and obtain hydrolases only after fusion with a lyso-
some (animals) or after fusion with a lytic vacuole (yeasts).
The plant autophagosome-like organelles in root meristem
cells were present at a very early stage of development,
prior to the formation of large vacuoles, thus not at the time
of cell death (see [7,8] and a series of excellent papers
discussed therein). Additionally, the same but much larger
structures have been found in association with PCD during
laticifer formation, in two plant species. Although the
observed vesicles were involved in clearing a large part
of the cytoplasm, their causal role in cell death has not been
established [9,10].
Others have claimed evidence for the presence of autop-
hagosomes in intact plants. These claims were based on the
presence of vesicles tagged with green fluorescent protein
(GFP) and/or the autophagy protein 8 (ATG8). Small
vesicles were observed using fluorescence microscopy
[11,12]. The nature of these vesicles has as yet not been
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adequately elucidated at the electron microscope and bio-
chemical level, so we do not know whether the observed
vesicles engulf a portion of the cytoplasm, and where they
obtain their hydrolases from. It cannot be stated at this
time, therefore, that the observed fluorescing vesicles are
true autophagosomes or autolysosomes. Some structures
that are reminiscent, at the electron microscope level, to
autophagosomes/autolysosomes have also been shown in
starving suspension-cultured plant cells [13,14]. However,
it is not yet clear how representative this is for intact
plants, where they get their hydrolases from (if they have
them), and whether the structures are associated with
PCD. So taken together, no autophagosomes – or even
autophagosome-like structures – have as yet been shown
to be causal in plant developmental PCD.
In ‘pathogen-induced’ PCD the situation is different
from developmental PCD, as there is now one example
for the role autophagy genes ATG7 and ATG9 in PCD. The
presence of ATG7 in this system was correlated with the
occurrence of vesicles that are similar to small vacuoles
[15]. However, as the biochemistry and development of
these vesicles are not yet known, one cannot claim that the
PCD process answers to the definition of ‘autophagic cell
death’ in animals.
Thus, when adhering to the definition of ‘autophagic cell
death’ that is used in animal science, it is not yet possible to
categorize a type of plant PCD as such. Nonetheless, this is,
in our opinion, counterintuitive. We know that in numer-
ous examples of developmental PCD the final event – the
actual death of the cell – is a lytic process which occurs after
the rupture of the vacuolar membrane. In at least many
types of cells rupture of the tonoplast is the last committed
physiological step leading to developmental cell death.
Tonoplast rupture, however, is not adequate for cell death.
It occurs without release of a large amount of lytic enzymes
from the vacuole in phloem sieve cells, as we previously
discussed [3], which stay very alive for a considerable time
after tonoplast collapse. To cause cell death, tonoplast
rupture must be followed by adequate activity of lytic
enzymes released from the vacuole. This self-eating lytic
activity, we argued, can be regarded to be a type of autop-
hagy. Autophagy, in that view, is the cause of death of
most, if not all, examples of developmental PCD in plants
[3]. The idea of ‘autophagic cell death’ in plants was there-
fore extended (or stretched if you will) by us to include the
release of hydrolases from the vacuole, which usually is the
final cause of developmental cell death [3].
Whether or not autophagy is a predominant feature of
developmental PCD in plants thus hinges on the definition
of ‘autophagic cell death’. If it turns out that true autop-
hagosomes do not occur in plants, but plant autophago-
some-like structures are shown to cause a type of PCD, we
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will need a re-definition of the concept ‘autophagic cell
death’ if we want to call any plant PCD by that name.
Furthermore, if ‘autophagic cell death’ is taken to include
the large-scale hydrolysis of the cell, after permeabiliza-
tion/rupture of the vacuolar membrane, most or all devel-
opmental PCD belongs to the category. We need not
necessarily adhere to the definitions as currently in use
in animal science, and we might need a new definition of
‘autophagic cell death’ that accommodates both what is
known about animals and plants.
To move forward in this field we need a better under-
standing of the genesis of autophagosome-like structures
in plants. Future work on the role of autophagy in the
induction of PCD should tie together both biochemical data
and detailed electron microscope data on the vesicles
involved.
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1360-1385/$ – see front matter ß 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tplants.2010.04.009 Trends in Plant Science 15 (2010) 361–362