Download Protein dynamics and proteolysis in plant vacuoles

Journal of Experimental Botany, Vol. 58, No. 10, pp. 2391–2407, 2007
doi:10.1093/jxb/erm089 Advance Access publication 1 June, 2007
REVIEW ARTICLE
Protein dynamics and proteolysis in plant vacuoles
Klaus Müntz*
Institut für Pflanzengenetik und Kulturpflanzenforschung (IPK), Corrensstr. 3, D-06466 Gatersleben, Germany
Received 1 November 2006; Revised 25 March 2007; Accepted 26 March 2007
Abstract
Plant cells cannot live without their vacuoles. The
tissues and organs of a plant contain a wide variety of
differentiated and specialized vacuoles—even a single
plant cell can possess two or more types of vacuoles.
Vacuolar proteins are encoded by nuclear genes and
synthesized in the cytoplasm. Their transport into the
vacuolar compartment is under cytoplasmic control.
Transcription seems to be a major control level for
differential protein supply to the vacuoles. It is at this
level that vacuole differentiation and functions are
mainly integrated into cellular processes. Recycling
amino acids generated by protein degradation is
a major function of the vacuole. This is most evident
when storage proteins are mobilized in storage tissues
of generative or vegetative organs in order to nourish
the embryo of germinating seeds or sprouting buds.
When specific proteins are transferred to the vacuole
for immediate degradation this compartment contributes to the adaptation of protein complexes in response to changes in developmental or environmental
conditions. Vacuolar proteases are involved in protein
degradation during reversible senescence and programmed cell death, which is also called irreversible
senescence. Vacuoles contribute to defence against
pathogens and herbivores by limited and unlimited
proteolysis. Our present knowledge on functions and
processes of vacuolar protein dynamics in plants is
reviewed. Research perspectives are deduced.
Key words: Complete protein breakdown, differential protein
supply to vacuoles, limited proteolysis, lytic vacuoles, PCD,
protein storage vacuoles, vacuolar protein mobilization,
vacuolar proteins, vacuolar protein transfer, vacuolar
proteolysis in defence.
Introduction
Plant cells cannot survive without their extra-cytoplasmic
vacuolar compartment (Rojo et al., 2001; Surpin and
Raikhel, 2004). Since protein synthesis does not take place
in vacuoles (for reviews on vacuoles and their functions,
see Leigh and Sanders, 1997; De, 2000; Robinson and
Rogers, 2000) all vacuolar proteins have to be imported
from the cytoplasm. One major life-supporting function
of vacuoles is that of protein breakdown. Special sets of
proteins are responsible for the structure and function of
vacuoles. When vacuoles undergo developmental and
environmental changes some of these proteins are
replaced by new imports from the cytoplasm. So far
export of proteins from vacuoles has not been observed.
However, it is unknown whether all substituted proteins
are broken down by vacuolar proteases or whether some,
or at least their fragments, are degraded outside the
vacuole. The vacuolar compartment represents a major
site of amino acid recycling in plant cells. This function is
closely related to plant development and plant response to
environmental effects. In this context various proteins are
imported from the cytoplasm to be degraded in the
vacuole. Breakdown takes place either immediately after
arrival in the vacuole or after short- to long-term storage.
For a long time vacuoles were believed to be the only
site of protein breakdown in the plant cell. Vacuolar
* E-mail: [email protected]
This paper is dedicated with gratitude to Prof. Dr Ulrich Wobus on the occasion of his 65th birthday and his retirement after 15 years as director of IPK,
Gatersleben.
Abbreviations: CCV, clathrin-coated vesicles; CP, carboxypeptidase; DV, dense vesicles; ER, endoplasmic reticulum; ESCRT, endosomal sorting complex
required for transport; HR, hypersensitivity response; H+-V-ATPase, proton-translocating vacuolar ATPase; H+-V-PP, proton-translocating vacuolar
pyrophosphatase; I-CliP, intra-membrane-cleaving protease; LV, lytic vacuole; MVB, multi-vesicular bodies; PAC, precursor-accumulating vesicle; PB,
protein body; PCD, programmed cell death; PI, protease inhibitor; PM, plasma membrane; PSV, protein storage vacuole; PVC, pre-vacuolar compartment;
rER, rough endoplasmic reticulum; SNARE, soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor; TGN, trans-Golgi network; TI,
trypsin inhibitor; TIP, tonoplast intrinsic protein; VPE, vacuolar processing enzyme; VSP, vegetative storage protein.
ª The Author [2007]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.
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2392 Müntz
proteases were the first proteases to be detected, isolated,
and analysed. During the past 20 years, however, our
knowledge of proteolytic systems in other compartments
and of vacuolar proteins and protein import has increased.
It is now known that different vacuoles can exist
simultaneously in a single plant cell. Therefore, the role
of plant vacuoles in protein dynamics and proteolysis has
to be re-evaluated.
Vacuolar proteins
Recent proteomic analysis revealed the presence of 402
proteins in isolated and purified lytic vacuoles (LV) of
Arabidopsis thaliana leaves (Fig. 1), 46.5% of which
could be identified (Carter et al., 2004). Some of these
proteins were typical of other non-vacuolar organelles,
which may reflect contamination or, more probably, an
autophagic uptake into the vacuole.
Vacuolar proteins can be classified, according to their
localization, into luminal and tonoplast proteins, and the
latter further into integral membrane and membrane
peripheral proteins (e.g. cytoskeleton proteins at the outer
and lectins at the inner surface). The proteomic analysis of
vacuolar proteins has been further refined by separating
tonoplast from luminal fractions (Carter et al., 2004;
Shimaoka et al., 2004; Szponarski et al., 2004).
Tonoplast preparations from LV of Arabidopsis leaves
contained 38 transporters belonging to the integral
membrane proteins (Carter et al., 2004). Among them
carrier proteins, ion channels, and ion pumps, which have
several trans-membrane domains, play a role in the
exchange of inorganic and organic compounds between
cytoplasm and vacuole. For example, proton-translocating
vacuolar ATPase (H+-V-ATPase) and the protontranslocating vacuolar pyrophosphatase (H+-V-PP) catalyse energy-dependent proton transfer, and generate the
acidic pH inside vacuoles and the energy gradients needed
for ion transfer. Furthermore, they contribute to maintaining the cytoplasmic pH in narrow neutral limits. Mg2+and Ca2+-ATPases are active transporters of inorganic
cations, whereas ABC-transporters of the tonoplast catalyse the ATP-dependent transfer of organic compounds.
Tonoplast intrinsic proteins (TIPs, also called aquaporins),
which are water channels that facilitate the exchange of
water molecules between cytoplasm and vacuole, are very
abundant. TIPs and transporters are components of a vital
cellular function. By controlling ion concentrations and
the amount of water in the vacuole, the cell can control its
osmotic and turgor pressure and thereby contribute to cell
growth and mechanical stability of the plant itself. Finally
SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) complex proteins have
Fig. 1. Schematic overview of the plant vacuole and functional categorization of the vacuolar proteins identified [Fig. 4 in Carter et al. (2004),
reproduced with permission, which is gratefully acknowledged, of ASPB as copyright holder and the authors]. The proteins identified are grouped
into eight major categories, and the number of proteins belonging to each group is shown in parentheses. Some of the specific activities of each group
and a model of the tonoplast SNARE-pin complex are shown.
Vacuolar protein dynamics 2393
been identified among tonoplast proteins, providing direct
evidence for vesicular protein trafficking into vacuoles.
The luminal fraction of LV is characterized by its high
percentage of hydrolytic enzymes, such as glucosidases,
proteases, chitinases, RNases, glucanases, invertase, and
myrosinase, as well as protease inhibitors (PIs) and lectins.
These proteins indicate the other important functions of
LV, the turnover of biological macromolecules and their
role in defence against pathogens and herbivores. Comprehensive lists of proteins found in the vacuolar lumen were
published by De (2000) and Carter et al. (2004).
Protein storage vacuoles (PSV) in cells of vegetative
(root, stem parenchyma, tubers, pods) and generative
(endosperm, cotyledon mesophyll, embryonic axis) organs
are sites of short- or long-term deposition of protein
reserves. An extensive proteome analysis of PSV has not
yet been conducted but, using immunohistochemistry,
many proteins have been found to be localized there.
Besides large amounts of polymorphic storage proteins
(Staswick, 1994; Müntz, 1998; Shewry and Casey, 1999)
PSV often contain proteases (Müntz et al., 2001) and
other hydrolytic enzymes, even at the time of protein
deposition.
Protein dynamics
How do proteins get into the vacuole?
Little is known about the mechanisms of vacuole
formation. Early during mitosis of root tip cells, clusters
of pro-vacuolar vesicles of similar appearance have been
observed in both daughter cells, suggesting their allocation from the mother cell and a persistence of the vacuolar
system through cell division (Marty, 1999). However,
a very early de novo generation of pro-vacuolar vesicles in
the emerging daughter cells cannot be excluded. De novo
generation of a vacuole was even observed in specializing
cells after cell division ceased (Hoh et al., 1995). It is not
clear whether de novo formation of vacuoles starts from
the trans-Golgi network (TGN) as suggested by Marty
(1999) or from the endoplasmic reticulum (ER). In any
case, vesicular biogenesis of vacuoles takes place (Fig. 2).
During cell differentiation the numerous pro-vacuolar
vesicles merge into a few larger vacuoles. These grow by
material transfer in vesicles that are generated from the
ER or Golgi apparatus. Several different types of vesicles
for vacuolar protein transfer are known (Hara-Nishimura
et al., 2004; Herman and Schmidt, 2004; Vitale and Hinz,
2005; Neuhaus and Paris, 2006).
If pro-vacuolar vesicles persist through cell division as
suggested by Marty (1999) then the primary sets of
vacuolar membrane and lumen proteins are derived from
the mother cell. In the case of de novo formation of
vacuoles, the primary protein sets are provided by the
biosynthetic pathway (Herman, 1994). Biosynthesis of
vacuolar proteins takes place at the rough endoplasmic
reticulum (rER). The nascent precursors of vacuolar
proteins are sequestered co-translationally into the ER
lumen. Concomitantly the N-terminal signal peptide is
cleaved from the precursor and it enters the biosynthetic
pathway (also called the secretion pathway). Large segments of the precursor correspond to the mature vacuolar
protein. Precursors can contain more than one subunit of
the mature protein (e.g. 2S and 11S storage proteins;
Müntz, 1998) or even multiple copies of a protein (e.g.
proteinase inhibitors and cyclotides; see sections below
entitled ‘Proteinase inhibitors’ and ‘Cyclotides’, respectively). Precursor segments corresponding to mature
vacuolar proteins can be flanked by pro-peptides, and the
different segments can be connected by linker peptides.
Pro- and linker peptides act in maintaining a conformational state of the precursor that makes it compatible with
the structural demands of intra-cellular protein traffic
(Müntz, 1998) and/or maintains enzyme precursors in an
inactive state (e.g. precursors of proteases; see Müntz and
Shutov, 2002). In addition, precursors of several proteins
become glycosylated in the ER and their glycosyl side
chains are further trimmed and modified in the Golgi
apparatus (e.g. 7S globulin of Phaseolus vulgaris; Müntz,
1998). Precursors of vacuolar proteins contain sorting and
targeting signals which direct the interaction with sorting
receptors when transfer vesicles are generated at the ER or
Golgi apparatus (Jiang and Rogers, 2003; Robinson et al.,
2005; Vitale and Hinz, 2005). After they have completed
their function, pro- and linker peptides, as well as in some
cases N- or C-terminal peptides containing targeting
signals, are detached by site-specific limited proteolysis in
the vacuole (Müntz, 1998) or even already in the prevacuolar compartment (PVC) (Otegui et al., 2006). This
proteolytic tailoring transforms precursors into mature
vacuolar proteins at their destination. It is, for example, in
the vacuole that storage proteins assume the conformation
which is compatible with their vacuolar deposition, and
inactive enzyme precursors are activated.
From the ER, tonoplast and luminal proteins are transported to the vacuole either directly or after passage
through the Golgi apparatus. In cotyledons of developing
pumpkin seeds, precursor-accumulating vesicles (PAC)
transfer storage proteins from the ER directly to the PSV
(Hara-Nishimura et al., 1998). Direct ER-to-vacuole
transfer has also been observed for the sorting receptor
AtSRC2 (Oufattole et al., 2005). Transfer of proteins from
the ER to the cis-Golgi takes place in coat protein
complex II vesicles (Aniento et al., 2003; Hanton et al.,
2006; Matheson et al., 2006). At the cis-Golgi, dense
vesicles (DV) are generated that contain storage globulins
destined for the PSV (Hillmer et al., 2001), whereas from
the TGN clathrin-coated vesicles (CCV) bud that carry
proteins destined for the LV (Robinson et al., 1998a; Hinz
et al., 1999), for example, acid hydrolases. On their way
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Fig. 2. Scheme of mechanisms and routes of protein transfer to the vacuole. Upper part: vacuole generation during cell division by de novo
generation or apportionment from mother cells (Marty, 1999). Lower part: transfer of proteins into the vacuole. Proteins can arrive by endomembrane
progression when they are transported by vesicular transfer via the Golgi apparatus (Hara-Nishimura et al., 2004; Herman and Schmidt, 2004; Vitale
and Hinz, 2005; Neuhaus and Paris, 2006). Vesicular transfer of proteins from the ER to the Golgi is indicated by white circles. On their way from
the TGN to the vacuole many proteins with sorting determinants are contained in transfer vesicles, such as DV or CCV, which discharge their cargo
to MVB/PVC or might even bypass this endosomal compartment. The ER generates several vesicles (PAC, ER bodies, PB) where proteins can be
transported to the vacuole without progression through the Golgi. A specific variant of this mechanism represents the direct formation of PB from the
ER. Autophagy transfers cytoplasmic proteins or organelles into the vacuole. PM proteins can be transported to the vacuole by endocytosis. CCV,
clathrin-coated vesicles; ctVSD, C-terminal vacuolar sorting determinant; DV, dense vesicles; ER, endoplasmic reticulum; LV, lytic vacuole; MVB,
multi-vesicular bodies; PB, protein bodies; PM, plasma membrane; PSV, protein storage vacuole; PVC, pre-vacuolar compartment; PAC, precursoraccumulating vesicles; ssVSD, sequence-specific vacuolar sorting determinant. Greek letters indicate characteristic isoforms of TIPs of different types
of vacuoles.
to the vacuole both types of vesicles enter multi-vesicular
bodies (MVB) which have been identified as endosomal
pre-vacuolar compartments (PVC) (Robinson et al., 2000;
Jiang et al., 2002, Tse et al., 2006). Storage proteins have
been found in the luminal matrix of MVB (Robinson
et al., 1998a). The presence of sorting receptors for
vacuolar proteins in MVB (Li et al., 2002; Miao et al.,
2006) is additional proof of vesicular transfer of vacuolar
proteins from the Golgi to this compartment. According to
Jiang et al. (2002) the intra-luminal vesicles contain acid
hydrolases known to be transferred in CCV. In the acidic
environment, MVB receptors involved in vesicular protein
transfer to vacuoles dissociate from their cargo protein
before the latter are further transferred into vacuoles. The
recycling of receptors involved in vesicular protein transfer to vacuoles starts from MVB (Lam et al., 2006).
Correct storage protein transfer to vacuoles was shown to
depend on intact receptor recycling to the Golgi. Recent
experimental results indicate that similarly to yeast
(reviewed by Bowers and Stevens, 2005) a retromer
complex must also be involved in this receptor recycling
to the Golgi in plants. In Arabidopsis, mutation of a gene
encoding a protein homologous to a component of the
yeast retromer complex interferes with vacuolar storage
protein transfer (Shimada et al., 2006). In the MVB/PVC
of the same plant species, components of a retromer
complex that interact with a vacuolar sorting receptor
were found (Oliviusson et al., 2006). It is unclear whether
DV and CCV can also bypass MVB/PVC and go directly
to the PSV as hypothesized by Vitale and Hinz (2005).
The mechanism of protein transfer from MVB into
vacuoles has still to be verified experimentally but fusion
with pre-existing vacuoles is assumed to be most probable
(Robinson et al., 2000). In Arabidopsis cells, two different
populations of endosomes have been observed, one of
which seems to act as an early endosome in endocytosis,
Vacuolar protein dynamics 2395
whereas the other seems to act as a PVC in vacuolar protein transfer (Ueda et al., 2004). Possibly, docking of different transfer vesicles and receptor-recycling retromer
vesicles at the MVB leads to modifications, turning the
MVB into a PVC that finally fuses with a vacuole. In this
PVC, precursors of storage proteins are already processed
by limited proteolysis before entering the PSV (Otegui
et al., 2006).
Protein traffic to the vacuole seems to be a one-way
passage. In yeast, receptors and other components of the
vesicular protein traffic machinery undergo a retrograde
transport from the vacuole to the MVB and TGN (Bryant
et al., 1998; Bowers and Stevens, 2005). The docking of
transfer vesicles at the membrane boundary of LV in
Arabidopsis leaves is reflected by the presence of SNARE
proteins in its tonoplast membranes (Carter et al., 2004).
Secretion of vacuolar luminal hydrolases was observed in
suspension-cultured tobacco cells (Kunze et al., 1998), but
the authors could not rule out that the secreted enzymes
came from a PVC, in which at least precursors of storage
globulins can undergo maturation by limited proteolysis
(see above). In conclusion, so far no retrograde traffic of
resident tonoplast or other vacuolar proteins has been
conclusively shown for plants.
Another pathway of protein (and other cargo) supply to
the vacuole is endocytosis (Fig. 2) (Murphy et al., 2005;
Šamaj et al., 2005, 2006). Endocytotic vesicles which are
generated at the plasma membrane (PM) can transfer PM
proteins to the LV (Takano et al., 2005). On their way to
the LV, the PM proteins first pass through MVB or the
TGN (Lam et al., 2006). In yeast, heteromeric protein
complexes have been detected that are required for the
vesicular transport of membrane proteins to MVB. The
Arabidopsis genome contains genes encoding proteins that
are homologous to the components of the yeast endosomal
sorting complex required for transport (ESCRT) to MVB
(Winter and Hauser, 2006). It remains to be shown
whether in the plant the encoded proteins have functions
similar to the yeast ESCRT machinery. The intra-luminal
vesicles within MVB originate, at least in part, from invagination of endocytotic vesicles. The endocytotic pathway from the PM to the LV and the biosynthetic pathway
(secretory pathway) of protein transfer from the Golgi
apparatus to the LV and, partially, the PSV converge in
MVB. It is unclear, however, whether proteins from the
endocytic pathway and those of the biosynthetic pathway
can enter the same MVB.
In yeast, some proteins are known to be transferred
directly from the cytosol into the vacuole (Noda et al.,
2000). The yeast vacuole is often used as a model for
plant vacuoles, but so far no similar mechanism has been
observed in plant cells.
Aside from the targeted transfer so far described, cytoplasmic material, including proteins, is taken up into the
vacuole by autophagic processes (Robinson et al., 1998b;
Herman and Schmidt, 2004; see also section below
entitled ‘Programmed cell death’). According to Marty’s
hypothesis about vacuole generation from the TGN
(Marty, 1997, 1999), cytoplasmic material is trapped
autophagically by the emerging vacuole and is afterwards
degraded by lytic enzymes. Material uptake by autophagy
(Fig. 2) was observed in cells of germinating bean seed
cotyledons (Herman et al., 1981; Toyooka et al., 2001).
As a result of this process remnants of plastids and mitochondria are transiently present in vacuoles. Recently
autophagy has been shown to be a basic process involved
in amino acid recycling that continuously takes place in
young as well as in stressed and senescing plant cells
(Yashimoto et al., 2004; Slávikova et al., 2005).
Changes in vacuolar protein composition
The vacuolar compartment of a plant cell undergoes
development- and environment-dependent structural and
functional changes which are accompanied by alterations
in its protein composition. Best known is the formation of
PSV from LV during seed development and the transformation of PSV into LV during seed germination and
seedling growth. Analogous processes take place during
storage and mobilization of vegetative storage proteins
(VSP) (Staswick, 1994). Different vacuoles can be formed
in leaf cells during senescence (Otegui et al., 2005) and in
salt-stressed ice plants (Epimashko et al., 2004). The
transformation of one type of vacuole into another adapts
the vacuolar compartment to different functions. The
simultaneous presence of different vacuoles generates
a physical separation of different physiological functions
in a cell. A structural separation of different vacuolar
functions has even been attributed to vacuolar subcompartments which are observed in PSV (Jiang and Rogers, 2002;
see section below entitled ‘Intra-vacuolar compartments’).
It is disputed whether already existing vacuoles undergo
structural and functional transformation or whether vacuoles are formed de novo in order to substitute preceding
ones and to form additional new ones. Independent of the
mechanism that underlies vacuole differentiation in a cell,
this process has to be accompanied by proteolytic elimination of specific proteins and the insertion of new ones.
Deposition of proteins in PSV: Two mechanisms have been
described for PSV generation: transformation of LV
during early seed development or substitution of LV by
a newly formed PSV (Herman, 1994; Robinson and Hinz,
1997; Vitale and Hinz, 2005). In both cases the newly
formed PSV contain a-TIP instead of c-TIP, which is
characteristic of the tonoplast of the LV, and the lumen of
the vacuole is progressively filled with storage proteins.
Several tonoplast and luminal proteins have to be substituted or added during PSV formation. There is evidence
that membrane proteins such as TIPs and luminal proteins
2396 Müntz
such as storage proteins use independent trafficking
mechanisms to the PSV. They are probably transported in
different vesicles from their site of biosynthesis at the rER
to the PSV (Gomez and Chrispeels, 1993; Park et al.,
2004) independently of whether the proteins pass or
bypass the Golgi apparatus and MVB/PVC. However, it
is disputed whether a-TIP and storage proteins are transferred separately in different vesicles, since Hinz et al.
(1999) co-localized both by immuno-cytochemical methods in DV (see also Robinson and Hinz, 1999). Different
mechanisms participate in the sorting of storage proteins
into transfer vesicles, including their reversible binding to
receptors (Robinson et al., 2005). Electron microscopic
observations of developing pea cotyledons indicated an
autophagocytic elimination of the old vacuole whereby
tonoplast and luminal proteins of the original LV have to
be degraded in de novo generated PSV (Hoh et al., 1995;
Robinson et al., 1998a). Proteases of this PSV either
derive from the preceding LV or arrive by vesicle
transport from the rER. In order to accumulate, storage
proteins must either be resistant against or physically
separated from these proteases, otherwise the elimination
of the LV components has to be completed and the
proteases involved eliminated before storage proteins can
accumulate in the PSV. During LV–PSV transformation,
when c-TIP is substituted by a-TIP, the former has to be
eliminated by degradation in the membrane (membrane
proteases?), by degradation in the cytoplasm, or/and in the
vacuolar lumen after detachment from the membrane.
Mature storage proteins accumulate to high levels in cells
of developing storage tissue, which indicates that their
turnover is low or at least is vastly exceeded by their
deposition in the PSV. Half lives of 2 weeks and >5
weeks have been measured for storage proteins in developing soybean and pea seeds, respectively (Madison
et al., 1981). Storage proteins are synthesized as transportcompatible precursors that are transferred by vesicles into
PSV (Fig. 2) where they are processed into mature vacuolar proteins by limited proteolysis (see section above entitled ‘How do proteins get into the vacuole?’). When this
transport pathway includes passage through MVB/PVC
the separate pathways of storage protein precursors and
processing proteases converge there and the first processing steps can take place in this compartment (Otegui
et al., 2006). Excised targeting, pro- and linker peptide
fragments are degraded in the PSV at the latest. It has been
suggested that vacuolar degradation of mis-structured
proteins forms part of the cellular protein qualitycontrol system (Tamura et al., 2004; Vitale and Ceriotti,
2004; Pimpl et al., 2006) but there is still no proof that
this process occurs in vivo in PSV. Highly cleavagespecific proteases catalyse limited proteolysis while proteases with broad cleavage specificity mediate complete
polypeptide breakdown. The intact storage protein molecules have to be protected against degradation by structural
inaccessibility or a differential subcompartmentation. According to Shutov et al. (2003) and Jiang et al. (2001)
both mechanisms act in PSV. Controlled peptide degradation concomitant to storage protein deposition indicates
that even PSV exhibit some lytic functions at that stage.
In the starchy endosperm of developing cereal grains
storage proteins are deposited directly in ER-derived
protein bodies (PB) (e.g. zeins of maize, prolamins of
rice) or in PSV (e.g. some wheat prolamins, oat globulins,
globulin-like rice glutelins). Autophagic processes (Fig. 2)
can be involved in the transfer of wheat prolamins to the
PSV (Levanony et al., 1992). At the end of grain
maturation, the cells of the starchy endosperm undergo
programmed cell death (PCD, see section below entitled
‘Senescence and cell death’) leaving only the cells of the
aleurone layer alive (Young and Gallie, 2000). In aleurone
cells, at least two types of vacuoles have been found
(Swanson et al., 1998): PSV where 7S globulins and
lectins are stored and LV containing various hydrolases.
Plants store protein reserves in vegetative propagation
organs too, such as shoot (e.g. patatin in potato) and root
tubers (e.g. sporamin in sweet potato). In addition,
deciduous trees accumulate VSP in bark parenchyma cells
during autumn for mobilization in spring. Transient
deposition of protein reserves occurs even in leaves, shoot
axes, and pods of various grain legumes, such as soybean
VSP (Staswick, 1990; Herman, 1994), or in roots of
alfalfa (Cunningham and Volonec, 1996). VSP are
a heterogeneous group of polypeptides, unrelated in
amino acid sequences and without evolutionary relationship to seed storage proteins. Nevertheless, they are also
deposited in vacuoles. In soybeans and related legumes,
VSP are deposited in a single large central LV, whereas
tubers contain several vacuoles with VSP aggregations.
In soybean leaves, non-coated transfer vesicles containing VSP have been observed that bud from the Golgi
apparatus. These vesicles seem to discharge their cargo
directly into the vacuole (Klauer and Franceschi, 1997).
This may be interpreted as Golgi-to-vacuole transport of
VSP which bypasses MVB/PVC, although the involvement of the endosomal compartment has not been
excluded by these experiments. The tonoplast of VSPaccumulating leaf vacuoles contains d- but not a-TIP
(Jauh et al., 1998). However, Park et al. (2004) found
a-TIP in the tonoplasts of seed storage proteinaccumulating leaf cell PSV of transformed plants, as well
as in tonoplasts of leaf PSV of untransformed Phaseolus
beans. It is not clear whether PSV with d-TIP and PSV
with a-TIP can both occur concomitantly in the same
leaf cell.
Multiplicity and transformation of vacuoles in vegetative
organs: In the aleurone cells of barley seeds lectin is
localized in PSV, whereas aleurain, a cysteine protease, is
contained in a different vacuole, probably an LV. In root
Vacuolar protein dynamics 2397
tip cells of barley and pea as well as in the plumula of
pea, lectin and aleurain co-localized with a-TIP and
c-TIP, respectively, indicating that, at certain developmental stages, vegetative cells can contain at least two different
types of vacuoles simultaneously, the PSV and LV (Paris
et al., 1996).
Developmental diversification of vacuoles was observed
in young root tip cells (Flückiger et al., 2003). In the
youngest stage, only one type of small vacuole is present.
The tonoplast of early stage vacuoles contains a- and
sometimes a- and d-TIP. Later on, tonoplasts normally
contain a-, c-, or d-TIP (Fig. 2), though the tonoplast of
some vacuoles contains a- and c- or c- and d-TIP (Jauh
et al., 1998, 1999). A hypothetical scheme for vacuole
diversification has been proposed by Jauh et al. (1999). It
remains to be shown whether the different types of
vacuoles are generated de novo or whether vacuole
transformation takes place. It seems conceivable that
vacuoles with two different TIPs in their tonoplast are the
result of fusion of two ancestor vacuoles (Fig. 2).
Double labelling of vacuoles with TIP- and lumen
protein-specific antibodies has seldom been reported (Hoh
et al., 1995). If only TIPs or lumenal proteins are labelled,
their assignment to different types of vacuoles is problematic. Nevertheless, several studies confirm that the plant
cell uses different vacuoles to compartmentalize different
vacuolar functions and that vacuolar diversity varies
depending on the developmental stage of a cell and environmental effects. This was shown in the regeneration
of two different vacuoles in evacuolated tobacco leaf cell
protoplasts (Di Sansebastiano et al., 2001; Tamura et al.,
2003) and senescent leaves of soybean and Arabidopsis
thaliana (Otegui et al. 2005; see section below entitled
‘Vacuolar proteolysis in defence against herbivores and
pathogens’).
Besides the differential distribution of different TIP subfamily members, multiplicity and diversification of vacuoles are reflected in other tonoplast proteins as well. When
maize endosperm cells differentiate into aleurone and
starchy endosperm, an H+-V-PP is only inserted into vacuoles of the aleurone cells but not in starchy endosperm
cells (Wisniewski and Rogowsky, 2004). In petal cells of
opening flower buds of morning glory (Ipomea tricolor
L.), anthocyans in the vacuole change colour from purple
to blue due to a pH shift from weakly acidic (pH 6.6) to
weakly basic (pH 7.7). This process is regulated by a
Na(K)-antiporter protein which by active Na+ and/or K+
transport into the vacuole mediates the pH shift (Yoshida
et al., 2005). Under induction conditions in tomato and
petunia cells, anthocyans accumulate in vacuoles bounded
by d-TIP-containing tonoplasts (Jauh et al., 1998).
Environment-dependent changes in tonoplast proteins
and vacuole functions have been documented for salt
stress. In tomato [Lycopersicum esculentum (¼Solanum
lycopersicum)], isoform A1 of the catalytic subunit of the
+
H -V-ATPase prevails in leaf cell vacuoles, whereas A2 is
characteristic for root cell vacuoles. Under salt stress, A1
transcript and protein concentration doubled in leaves,
whereas in the root cells transcript and protein levels of
A2 did not change (Bageshwar et al., 2005). Differential
vacuole formation also occurs in the ice plant (Mesembryanthemum crystallinum), where two different vacuoles
stabilize the cytoplasmic pH in response to salt stress
(Epimashko et al., 2004).
Intra-vacuolar compartments: The PSV of storage tissue
cells in seeds of pumpkin, castor bean, tobacco, and
several other plants are substructured. Phytate globoids
and protein crystalloids embedded in an amorphous PSV
matrix can be identified under the microscope. For PSV
from pumpkin cotyledons it has been shown that crystalloids and PSV matrix contain identical storage globulins. The phytate globoid cavity seems to be delimited by
a one-unit membrane (Jiang et al., 2001). Interestingly,
the tonoplast components H+-V-PP and c-TIP, which are
characteristic of LV, were detected in the globoid
membrane (Jiang et al., 2001), whereas the PSV tonoplast
only showed d-TIP-specific immunolabelling. Using
immuno-electron microscopy, the same authors localized
DIP (dark intrinsic protein), which is also a member of the
d-TIP subfamily of tonoplast proteins, to purified crystalloids from pumpkin and tobacco PSV (Jiang et al., 2000).
In tobacco crystalloids, a new receptor-like membrane
protein was detected, which shares the receptor domain
for vacuolar protein targeting with BP-80. These and other
results indicate that the crystalloid also has a membrane
boundary (Jiang et al., 2000). To explain the differing
protein compositions of the membrane boundaries of PSV
and intrinsic crystalloids and globoids, Jiang and Rogers
(2002) published a model to show how PSV subcompartments might be generated and how proteins characteristic
of the different PSV subcompartments are transported
from the ER to the PSV on separate pathways that can be
traced back to the MVB/PVC.
Under the electron microscope, PSV of Brassicaceae
seeds appear to contain only globoids and no crystalloids.
However, in PSV studies from Brassica napus seeds the
isolation of a crystalloid-like fraction has been reported
(Gillespie et al., 2005). A new protein, BPEP (Brassicaceae
PSV-embedded protein), was isolated from this globoid
fraction. In immunofluorescence studies, BPEP- and DIPspecific antibodies label the same substructures in B. napus
and A. thaliana PSV. Since peptide fragments responsible
for protein–protein interactions were found in BPEP the
authors suggest that it may act as a scaffolding protein
forming an internal membrane network in globoids and
crystalloid-like structures as well.
There appears to be no subcompartmentation in LV
and in vacuoles that store VSP. In PSV of seeds, however,
subcompartmentation could explain how lytic enzymes
2398 Müntz
can be stored separately from storage proteins. At the time
of germination when amino acids have to be mobilized in
order to nourish the embryo, controlled fusion of subcompartments could give the proteases access to their
substrates.
Proteolysis in vacuoles
Controlled proteolysis of tonoplast proteins?
Transfer vesicles (Fig. 2) fusing with the MVB/PVC or
vacuoles have to be recycled. As mentioned in the section
above entitled ‘How do proteins get into the vacuole?’
indication for MVB/PVC-to-Golgi retrograde protein
transport in plant cells has been obtained by analysing
appropriate mutants of Arabidopsis thaliana. However, so
far no export of a tonoplast ‘resident’ protein from
vacuoles has been found. It is therefore possible that
removal and substitution of these vacuolar proteins may
only occur via protein degradation. During seed germination and seedling growth the PSV is transformed into an
LV (see section below entitled ‘Mobilization of storage
proteins in PSV’). Concurrently with this transformation
a- and d-TIP are removed and substituted by c-TIP. Other
tonoplast proteins may also undergo controlled substitution (see section above entitled ‘Multiplicity and transformation of vacuoles in vegetative organs’). Complete
proteins or fragments generated by limited proteolysis
might be removed. Their subsequent proteolysis could
occur in the cytosol or vacuolar lumen. Intra-membranecleaving proteases (I-CliP) are known from animal and
bacterial cells. I-CliP-encoding genes have also been
found in plants (Weihofen and Martoglio, 2003). I-CliP
are localized in membranes and catalyse the release of
extra-membranous domains and intra-membrane peptides
from membrane proteins. In bacterial and animal cells this
generates regulatory poly- or oligopeptides or results in
the degradation of cleavage products. So far, intramembrane proteases have not been reported for tonoplasts
of plant vacuoles.
Proteolysis in the lumen of vacuoles
Many proteases reside in the lumen of plant vacuoles
(Table 1). Protein degradation (Fig. 3) occurs either
immediately after proteins have entered the lumen of LV
or, in the case of storage proteins, after temporary
deposition in PSV when these are transformed into LV.
Amino acids are recycled for metabolic processes that
take place outside the vacuole. Peptide and amino acid
transporters of the tonoplast catalyse the export of small
protein degradation products (Waterworth et al., 2001).
Immediate protein degradation in LV: The acidic environment of an LV might facilitate conformational changes of
proteins and thus make them more accessible to resident
lumen proteases. After endocytosis (Figs 2, 3) PM
proteins can be transferred to the vacuole and degraded.
In pericycle cells of A. thaliana, boron deficiency leads to
the formation of a specific boron transporter protein
(BOR1) which is inserted into the plasmalemma. This is
followed by increased boron transfer into the xylem for
supply to shoot organs. When normal boron nutrition is
restored the BOR1 protein is removed by endocytosis and
degraded in the vacuole (Takano et al., 2005). Posttranslational control processes act outside the vacuole,
which in this case is just a non-specific site of proteolysis.
Dismantling and degradation of complex cell structures
after autophagic uptake into the vacuole are described in
the section above entitled ‘Changes in vacuolar protein
composition and the section below entitled ‘Senescence
and cell death’.
Mobilization of storage proteins in PSV: At the onset of
mobilization, the mechanisms that protect stored proteins
against degradation by stored proteases have to be
overcome; for example, structural inaccessibility of proteins (Shutov et al., 2003), membrane boundaries separating stored proteins from proteases (Jiang and Rogers,
2002), or pH values that maintain proteases inactive (Fath
et al., 2000). In addition, de novo biosynthesis of
proteases and the activation of protease precursors
Table 1. List of vacuolar proteases known to be involved in storage protein metabolism of seeds (Müntz, 2003)
Protease
Type
Function
Plant
Legumains (syn. VPE):
Asn-specific CPR
Endopeptidase
Precursor processing,
protein breakdown
Papain-like CPR
Endopeptidase
Protein breakdown
Aspartic proteinase
Endopeptidase
Subtilisin-like serine proteinase
Metalloproteinase
Carboxpeptidase
Endopeptidase
Endopeptidase
Exopeptidase
Precursor processing,
protein breakdown
Protein breakdown
Protein breakdown
Protein breakdown
Arabidopsis thaliana, Nicotina tabaccum,
Ricinus communis, Vicia sativa,
Vigna radiata, and others
Glycine max, Hordeum vulgare, Ricinus
communis, Vigna radiata, Vicia sativa,
and many others
Arabidopsis thaliana, Brassica napus
CPR, Cysteine proteinase; VPE, Vacuolar processing enzyme.
Glycine max
Fagopyrum esculentum
Hordeum vulgare, Triticum sativum,
Vigna radiata, and various others
Vacuolar protein dynamics 2399
Fig. 3. Schematic overview of the functions of vacuolar proteolysis in cellular protein breakdown and amino acid recycling. Upper part: precursors
of vacuolar proteases are synthesized at the ER and transferred in vesicles to the vacuole either via endomembrane progression through the Golgi and
MVB (e.g. CCV) (for references see legend to Fig. 2) or bypassing the Golgi (PAC, precursor protease vesicles, ER bodies, riconosomes) (Chrispeels
and Herman, 2000; Gietl and Schmidt, 2001; and references in the legend to Fig. 2). Lower part: in MVB and vacuoles (LV, PSV) precursors of
processing enzymes (e.g. VPE) are activated auto-catalytically by limited proteolysis (precursor processing). Activated processing proteases process
other precursor polypeptides and thereby activate enzymes and generate polypeptides involved in defence processes. Proteins to be degraded get into
vacuoles as shown in Fig. 2. Only vacuolar protein transfer by variants of autophagy is indicated here because of its specific contribution to
senescence and PCD. Limited and complete vacuolar proteolysis is a major process of cellular amino acid recycling after long-term storage of protein
reserves, when selected proteins are broken down immediately after getting into the vacuole or during senescence. Mega-autophagy represents the
final step in PCD when the release of proteases leads to cell corpse degradation.
(Okamoto and Minamikawa, 1998) contribute to the
initiation of protein reserve mobilization. Protease action
and protein mobilization are spatially and temporally
highly regulated (Müntz et al., 2001).
When globulin precursors arrive in PSV at the time of
protein deposition, exposed Asn-flanked processing sites
are cleaved by Asn-specific vacuolar processing enzymes
(VPEs or legumains) yet the majority of Asn-flanked
peptide bonds remain uncleaved in these globulins
(Shutov et al., 2003). This shows that certain protective
conformations exist in the stored proteins that have to
change before breakdown can start. As described in the
section above entitled ‘Intra-vacuolar compartments’,
fusion of vacuolar subcompartments could give the
proteases access to their substrates. Activation of stored
proteinase precursors in PSV has not been described so
far. In seedlings of cereals, as well as those of dicotyledonous plants, de novo formation of proteinases is
under transcriptional control. This is reflected by the
coincidence of histo-patterns of immuno-localization of
proteinases and in situ hybridization of corresponding
mRNAs in cotyledons of legume seedlings (Tiedemann
et al., 2001). Precursors of new proteinases are synthesized at the rER and transferred via the PVC into the
vacuole where they are activated by proteolytic processing. The mobilization of proteins does not take place
simultaneously in all storage tissue cells but proceeds in
a layer, a few cells wide, moving through the storage
tissue, leaving behind cells with LV free of storage
proteins (Müntz et al., 2001; Tiedemann et al., 2001).
Protein mobilization in storage tissue is under the control
of the growing embryo which, in seedlings, acts as the
major amino acid sink. In cereal aleurone cells, control is
exhibited by the antagonistically acting phytohormones
gibberellic acid and abscisic acid, and mediated mainly by
transcription regulation (Bethke et al., 2006). So far no
conclusive evidence has been presented that a similar
hormonal control acts in seedlings of dicotyledonous
plants. It cannot be excluded that there is a feedback
control of protease activities mediated by concentration
gradients of amino acids between embryo and storage
tissue. This would allow a fine tuning of amino acid
provision from the source according to the demand in the
sink represented by the growing embryo.
2400 Müntz
PSV contain stored proteinases that start protein mobilization within a few hours of seed imbibition (Müntz et al.,
2001; Schlereth et al., 2001). During germination of dicotyledonous seeds, protein degradation begins in radicle
tips, prevascular strands, and in subepidermal cell layers
where growth and differentiation are initiated. Vacuoles
dispose of families of proteases (Table 1) which undergo
compositional changes during germination and seedling
growth. At the end of germination the protein reserves of
the embryonic axis are nearly exhausted and functional
vascular strands have been established between axis and
storage cotyledons. In the cotyledons of seedlings, the
major mobilization of storage proteins is catalysed by de
novo formed proteases. Storage protein mobilization proceeds in a species-specific histological pattern through the
storage cotyledons. The emptying PSV merge and transform into a few or a single central LV. In Phaseolus beans
it has been found that, concomitantly with changes in the
luminal protein composition during this fusion process, aTIP is substituted by c-TIP in the tonoplast.
In cereal grains, 7S globulins are stored mainly in PSV of
the embryo and aleurone layers. The mobilization of these
storage proteins is initiated by an aspartic protease, called
phytepsin, stored in PSV (Bethke et al., 1996). Later
during germination, phytepsin is supplemented by newly
formed cysteine proteases, which were found in PSV as
well as in LV, that have been called secondary lytic vacuoles by Swanson et al. (1998). It is unclear whether these
LV are similar to another type of LV where the cysteine
protease called aleurain was detected in aleurone cells of
barley (Holwerda and Rogers, 1993). Prior to germination
the stored phytepsin is inactive due to the near basic pH of
PSV in mature grains. Acidification of the vacuole which
is controlled by gibberellic acid (Swanson and Jones,
1996) activates phytepsin and leads to the activation of
cysteine proteinases which then mediate protein breakdown. Phytepsin was shown to process the barley lectin
precursor by limited proteolysis and it is highly probable
that it similarly activates cysteine proteases (Fath et al.,
2000). The amino acids generated in this way are used for
de novo biosynthesis of hydrolases, including cysteine
proteinases, which are secreted into the dead starchy
endosperm in order to mobilize the proteins stored there.
Vacuoles of the aleurone cells undergo developmental
changes related to storage protein mobilization (Bethke
et al., 1998). PSV of aleurone layer cells are transformed
into LV that finally contribute to PCD (Jones, 2001; see
section below entitled ‘Programmed cell death’).
Despite many experimental attempts, no conclusive
evidence has so far been presented that in germinating
dicotyledonous seeds a pH-dependent control of protease
activation contributes to the initiation of protein mobilization in a way similar to cereal grains.
Mobilization of vegetative protein reserves follows
similar patterns to those of generative organs, although
a detailed analysis of these processes and the contributing
proteolytic enzymes is still to be done.
Vacuolar proteolysis in defence against herbivores
and pathogens
Although conclusive evidence is rare there is no doubt
that vacuolar proteases are involved in the defence of
plants against pathogens and herbivores (Mosolov et al.,
2001; van der Hoorn and Jones, 2004). The first line of
proteolytic defence against pathogens occurs in the
apoplast. Herbivorous attack destroys cells mechanically
(wounding) and thus confronts insects or nematodes
directly with the proteases of the vacuole. Since pathogen
attack frequently induces local and systemic hypersensitivity response (HR), vacuolar proteolysis forms part of the
defence against pathogens. An additional major defence
mechanism is the inhibition of proteases of pathogens and
herbivores. Precursors of the proteases and PIs are synthesized at membrane-bound polysomes, co-translationally
imported into the ER lumen, and transferred to the vacuole.
Vacuolar processing proteinases, which catalyse sitespecific limited proteolysis, can transform precursors into
active enzymes and enzyme inhibitors. Other vacuolar
proteins involved in defence (reviewed by Shewry and
Lucas, 1997), for example lectins (Peumans et al., 1999)
and cyclotides (Craik et al., 2004), are also synthesized
as precursors and activated by limited proteolysis.
Proteinase inhibitors: Plants contain inhibitors against all
classes of proteases known (Mosolov et al., 2001; Birk,
2003). Endogenous plant proteases usually remain unaffected by these PIs which mainly act on proteases of
pathogens and/or animals. Herbivorous insects have several proteases in their gut against which vacuolar PIs act.
This can retard the development of or even kill the attacking insect (Ryan, 1990; Koiwa et al., 1997). The
constitutive and induced expression of PI genes therefore
forms an important tool in the defence of plants against
pathogens and herbivores.
As an effective mechanism preventing PIs from affecting endogenous cellular processes, most known plant PIs
are synthesized as inactive precursor polypeptides and become active only after arrival in the vacuole where they
are converted into mature active inhibitors by limited
proteolysis. Precursors of serine proteinase inhibitors with
one and multiple inhibitor domains have been found
in Lycopersicum esculentum (¼Solanum lycopersicum)
(Graham et al., 1985), Nicotiana alata (Atkinson et al.,
1993), Capsicum annuum (Moura and Ryan, 2001), and
Nicotiana attenuata (Horn et al., 2005), respectively. In
N. attenuata the multi-domain precursor of trypsin
inhibitors (TIs) comprises six homologous inhibitors of
;6 kDa in tandem (Horn et al., 2005). A seventh is
formed by the fusion of the precursor fragment that
Vacuolar protein dynamics 2401
precedes the first inhibitor domain with the linker peptide
that follows the sixth domain. These inhibitor domains are
excised by a legumain (VPE) and a subtilisin-like serine
proteinase. The formation of the seventh TPI includes
a transpeptidation reaction similar to that which contributes to the formation of mature concanavalin A in PSV of
developing Canavalia ensiformis seeds (Sheldon et al.,
1996). After mechanical wounding by herbivores, methyl
jasmonate acts in the signalling pathway to induce
proteinase inhibitor production for defence (Bergey et al.,
1996). In N. attenuata, methyl jasmonate induces an
increased production of the complex of TI and alters the
quantitative ratios between some of the seven components
by differential effects on the expression of the two
processing proteinases. Whereas an increased inhibitor
production is controlled transcriptionally, the compositional change results from a post-translational control of
inhibitor formation (Horn et al., 2005).
Transpeptidation is also involved in the formation of
some members of the TI families of sunflower and squash,
where head-to-tail peptide bond formation generates
macrocyclic inhibitory peptides; for example, Helianthus
annuus trypsin inhibitor 1 (SFTI 1) and Momordica
cochinenesis TI (MCoTI) I and II. In case of SFTI 1 Asn
flanks one of the processing sites in the P1 position,
indicating that legumain is involved in the excision
from its precursor and its subsequent macrocyclization
(Korsinczky et al., 2004; Mulvenna et al., 2005a, b).
McoTI I and II have an even more complicated macrocyclic (knottin) structure than SFTI1 but are similarly
generated from a precursor by limited proteolysis combined with transpeptidation which results in macrcocycle
formation (Chiche et al., 2004).
Cyclotides: Recently, toxins called cyclotides have been
discovered in plants (Craik et al., 2004; Simonsen et al.,
2005) which also have a macrocyclic ‘knottin’ structure
but differ from MCoTI I and II by the absence of the TI
active loop. Cyclotides affect cellular membranes and are
believed to be involved in defence. The macrocyclic
polypeptides (28–35 amino acids long) are generated from
gene-encoded precursors containing one to three cyclotide
domains separated by linker regions (Dutton et al., 2004;
Mulvenna et al., 2005b). Since one processing site of the
domains is flanked by Asn in the P1 position, legumaincatalysed limited proteolysis may contribute to the
maturation and cyclization of cyclotide in vacuoles.
Proteases: A defence-related rise in the general activity of
vacuolar proteolysis, as well as that of specific proteases,
has been observed frequently (reviewed by Mosolov
et al., 2001; van der Horn and Jones, 2004). Unfortunately, only a few detailed studies on the function of
specific proteases in response to infection and wounding
have been conducted.
VPE (legumains) play a key role in the mechanism
of HR after infection by fungal and viral pathogens
(Hatsugai et al., 2004; Rojo et al., 2004). Since PCD
contributes to HR, this will be discussed in more detail in
the section below entitled ‘Senescence and cell death’. In
addition, VPE contribute to defence by processing proenzymes (Rojo et al., 2003) or precursors of other
defence-related proteins such as TI (see above). In
Lycopersicum esculentum (¼Solanum lycopersicum),
wounding of leaves induces a local formation of a vacuolar
serine carboxypeptidase (CP) (Moura et al., 2001). Two
mature CP are generated from a CP precursor by excision
of a peptide linker. This involves the cleavage of an Asnflanked peptide bond which may be catalysed by a VPE.
Whereas legumains (VPE) contribute directly to storage
protein degradation in germinating seeds (Müntz and
Shutov, 2002; Müntz et al., 2002) it is still unknown
whether they also participate in the digestion of cellular
proteins as part of a defence reaction.
Senescence and cell death
Life ends with death. The order in structures and functions
needed for life breaks down and the respective cell or
organism dies. With the exception of necrosis, death is
preceded by genetically programmed processes called
senescence or PCD. Whereas some authors use the terms
PCD and senescence synonymously (van Doorn and
Woltering, 2004) others see fundamental differences
between both processes (Thomas et al., 2003). Senescence
can be reversed experimentally during early stages, which
can sometimes happen in nature. Only after reaching a
point of no return does death become an unavoidable consequence of progressing senescence, which at the cellular
level corresponds to PCD (Thomas et al., 2003; van
Doorn, 2005). The reversible and irreversible stages of
senescence show differences in the proteolytic processes
taking place in the vacuoles and the proteases involved.
Reversible stage of senescence: During the reversible stage
of senescence the compartmentation of the cell remains
intact and vacuolar proteolysis does not seem to be an
important process (Matile, 1997; Buchanan-Wollaston
et al., 2003). Nevertheless, at this stage mobilization of
amino acids becomes a major metabolic activity. A reduction in protein biosynthesis shifts the balance towards
protein breakdown. Still, transcription and translation continue and the formation of specific proteins might increase
and new senescence-related proteins, including proteases,
are synthesized (Buchanan-Wollaston et al., 2003; Lin
and Wu, 2004). Correlations between senescence and an
increase in the transcription of genes, the activity, and/or
concentration of proteases, in particular vacuolar proteases, have been reported frequently. Vacuoles containing the senescence-associated cysteine proteinase SAG12
2402 Müntz
are formed specifically in leaf cells of A. thaliana and
soybean (Otegui et al., 2005). In mutants lacking this
enzyme the progress in senescence is not inhibited, which
may be due to a functional substitution of SAG12 by
other proteinases. A senescence-correlated increase in
legumains (a- and c-VPE) was observed in Arabidopsis
leaves (Kinoshita et al., 1999). c-VPE not only processes
the precursor of a CP activating this vacuolar enzyme, it is
also involved in the senescence-correlated degradation of
a vacuolar invertase, presumably by activating another
proteinase. Initially c-VPE, the CP precursor, and the
invertase are contained in cytoplasmic vesicles, called ER
bodies. The pH range in these vesicles keeps the legumain
inactive. Once legumain is released into the acidic
environment of the LV it becomes functional and can
trigger the proteolytic activation of CP and the protease
that mediates invertase degradation (Rojo et al., 2003). A
similar process has been proposed to regulate the
maturation of cysteine proteinase RD21 observed in
vacuoles of senescing A. thaliana leaf cells (Yamada
et al., 2001). Senescence-correlated increases in cysteine
and other vacuolar proteinases were also observed in other
plants (Chen et al., 2002; Parrott et al., 2005). During
after-harvest senescence in broccoli florets, antisense
inhibition of the formation of the respective protease
delays the senescence process (Coupe et al., 2003; Eason
et al., 2005). Generally it is still unknown on which
substrates the senescence-related proteases act in vivo. Not
all proteases expressed during reversible senescence have
a real physiological function during this stage but rather
may only accumulate in preparation of vacuolar functions
in the irreversible stage (PCD).
Programmed cell death: PCD starts outside the vacuole
but its completion depends on vacuolar proteolysis. In
plants, vacuolar breakdown processes are prominent
features that all variants of PCD have in common (Jones,
2001). In order to be degraded, cytoplasmic proteins
arrive in the vacuole by autophagic processes. Although
micro- and macro-autophagy have not been conclusively
demonstrated during PCD of plant cells (Thomas et al.,
2003) there is no doubt that these processes are involved
in the recycling of cytoplasmic compounds during
senescence (see section above entitled ‘How do proteins
get into the vacuole?’). Towards the end of PCD the
vacuoles accumulate high levels of hydrolases. The exact
composition of these enzyme mixtures varies according to
cell type and the type of PCD. This reflects the different
genetic programmes of PCD. Finally the vacuole ruptures
and releases the hydrolases into the cytoplasm. This process, called mega-autophagy (van Doorn and Woltering,
2004), represents a post-mortal event that eliminates the
cell corpse and recycles cell material (Jones, 2001).
Whereas most cellular proteins are recycled during developmental PCD, for example in leaves of deciduous
trees, only a minor part is recycled during HR-related
PCD. No recycling occurs during necrotic cell death. Very
specific degradation processes are often involved in
developmental PCD; for example, during endosperm
development in cereals grains, tracheary differentiation,
sieve tube differentiation, or aerenchyma formation.
The role of different plant proteinases in the network of
PCD-related proteolysis is largely unknown. Among the
various proteinases that have been implicated so far to
participate in plant PCD (Coffeen and Wolpert, 2004;
Woltering, 2004; Bozhkov et al., 2005; Rotari et al.,
2005; Sanmartin et al., 2005) the only vacuolar enzymes
are the legumains (VPE) (Yamada et al., 2005) with the
exception of d-VPE which in A. thaliana is localized in
the extracellular space (Nakaune et al., 2005). Some
evidence suggests that legumains are involved in tonoplast
destabilization which is a prerequisite for mega-autophagy
in HR-related PCD (Hatsugai et al., 2004). In A. thaliana
leaves the vacuolar c-VPE is specifically involved in HRrelated PCD (Rojo et al., 2004). Somehow conflicting
with these results is that neither developmental PCDrelated nor senescence-related defects were observed in
legumain knock-out mutants of Arabidopsis, including
mutants in which all members of the legumain family
were inactivated (Gruis et al., 2004).
Developmental PCD ends with the partial or complete
elimination of all cell components. Partial elimination
occurs in the starchy endosperm cells of cereals during
grain maturation when the membranous boundaries of
PSV, PB, and other cytoplasmic components selectively
disappear, while stored carbohydrates, proteins, and lipids
remain unaffected (Young and Gallie, 2000). These
storage compounds are mobilized during germination as
described in the section above entitled ‘Mobilization of
storage proteins in PSV’. During germination, the cells of
the aleurone layer in the endosperm fulfil a major role in
this mobilization process and also finally undergo PCD
after this function has been completed (Fath et al., 2000).
The aleurone cell PSVs fuse and transform into large
central LVs which contain a set of at least two aspartic
and three cysteine proteinases, for example a-, b- and caleurains, some of which were already present in the PSV.
As long as the central vacuole is still intact it might
execute lytic functions by degrading proteins of autophagically ingested organelles and cytoplasmic fragments. In
the final stages of aleurone cell PCD the tonoplast is
destabilized and proteases are released to eliminate the
cell corpse (Bethke et al., 1998, 1999, 2001). Differences
in the types of PCD during endosperm maturation and
aleurone autolysis are reflected in the sets of proteases.
Whereas mainly genes encoding components of the
cytoplasmic ubiquitin–proteasome system and only a few
vacuolar legumains (b- and c-VPE), presumably acting as
processing enzymes, are expressed during partial PCD in
maturing barley starchy endosperm (Sreenivasulu et al.,
Vacuolar protein dynamics 2403
2006), vacuolar proteases predominate during complete
PCD of aleurone cells during barley germination (Fath
et al., 2000). Similarly, when the pericarp tissues undergo
complete PCD during grain development, genes of
vacuolar proteases are mainly expressed in this tissue
(Sreenivasulu et al., 2006).
In contrast to the starchy endosperm of cereals, most
dicotyledonous plants store proteins in tissues that remain
alive during seed maturation, such as the endosperm of
castor bean or cotyledons of Leguminosae. However, similarly to cereal aleurone PCD, after the storage proteins
have been completely mobilized during seedling growth,
the PSV in these dicotyledonous tissues fuse and are
transformed into LV when the cells undergo PCD. PCDrelated proteases have been studied in more detail in
castor bean. During castor bean seed maturation nucellar
cells degenerate by PCD (Greenwood et al., 2005). Cells
of the major storage tissue, the endosperm, remain alive
but undergo PCD during seedling growth after storage
proteins have been mobilized from PSV (Gietl et al.,
2000; Gietl and Schmid, 2001). In both tissues, membrane-bound and ribosome-studded vesicles, called riconosomes (Fig. 3), bud from the ER during PCD (Schmid
et al., 2001). The major protein component of these
riconosomes is a precursor of a C-terminally KDEL-tailed
cysteine proteinase (Schmid et al., 1999) which is
probably kept inactive by an N-terminal pro-peptide.
According to the authors, riconosomes act as ‘suicide
bombs’ in the senescing endosperm cells. First the rupture
of the PSV tonoplast causes an acidification of the
cytoplasm which, in turn, destabilizes the riconosome
boundary membrane, and finally these release their
content into the cytoplasm. Autocatalytic processing in
the acidic environment may then activate the cysteine
proteinase which is supposed to be the major catalyst of
post-mortal cell corpse degradation during castor bean
endosperm cell PCD. It remains to be elucidated whether
other proteases released from the collapsing PSV also
contribute to cell corpse elimination.
Conclusions
Intact vacuoles are indispensable for protein metabolism
in plant cells. Initially all vacuoles originate with similar
basic functions. Their subsequent differentiation depends
on changes of their protein content which varies in
a tissue- and organ-specific manner and according to
developmental processes and environmental effects. Isoenzymes with different dynamic characters catalyse
similar reactions in vacuoles of different tissues and
organs. Individual plants thus contain a wide variety of
differentiated and specialized vacuoles, and even a single
cell can contain two or more different types of vacuoles.
Differential intra-vacuolar proteolytic processing and de-
gradation may contribute to the specific protein equipment
of vacuoles but, since proteins are supplied by the cytoplasm, vacuole differentiation remains under cytoplasmic
control. Transcription is a major control level for differential protein supply to the vacuoles. At this level the
basic integration of vacuole differentiation and functions
into cellular processes is likely to take place. Nevertheless, control and regulation downstream of transcription
can also contribute to the diversification of vacuolar
protein furnishing. Even the timing of protease release
from transfer vesicles into vacuoles and the activation of
pro-proteases could constitute elements of the control
system. Major tasks of future research will be: (i) to analyse the controlling gene expression network that integrates
vacuole differentiation and function into the complex of
other cellular processes; (ii) to investigate mechanisms
that underlie differential protein transfer in the endomembrane system from the site of protein biosynthesis at the
rER to the vacuole(s); and (iii) to study the substitution
mechanisms for resident vacuolar proteins.
Vacuoles are major sites of cellular proteolysis and
contribute strongly to amino acid recycling in the cell.
This specific function predominates when protein reserves
are mobilized in PSV of generative and vegetative storage
tissues, during senescence, and at the end of developmental and HR-related PCD when proteins of the cell corpse
are degraded. When proteins are transferred from cytoplasmic compartments into vacuoles to be degraded by the
resident proteases, the vacuole not only recycles the
amino acids but also contributes to protein differentiation
in the cytoplasm. New examples of these processes will
be detected in the future and the underlying mechanisms
will be further elucidated. It will also be necessary to
investigate the enzymes and mechanisms of proteolysis
that play a role in the substitution of resident vacuolar
proteins. It is still not known which proteases degrade
storage proteins in vivo, a process which is not only
controlled at the level of protease formation, but which is
also regulated by the interaction of tissues and by tuning
the activity of individual enzymes. Differential furnishing
of vacuoles with proteases has to be studied in relation to
different forms of developmental PCD. Finally it has to be
investigated how much and by which mechanisms vacuolar proteolysis contributes to amino acid recycling during
the reversible stage of senescence and how megaautophagic protein breakdown is controlled and works as
the terminal step of PCD.
Acknowledgements
The present paper was written when the author was a guest of IPK
after retirement. He greatly appreciates the generous support given
by the Council of Directors of IPK and several other colleagues in
the institute. The author wants to express his special gratitude to
Dr R Jung from Pioneer Hi-Bred International Inc., Johnston, IA,
USA for critically reading the manuscript and suggesting many
2404 Müntz
improvements. The author is especially indebted to Dr Twan Rutten
for language editing and to Mrs K Lipfert and U Tiemann for
carefully designing the figures.
References
Aniento F, Helms JB, Memon AR. 2003. How to make a vesicle:
coat protein–membrane interactions. In: Robinson DG, ed. The
Golgi apparatus and the plant secretory pathway. Annual Plant
Reviews, Vol. 9. Oxford: Blackwell Publishing, 36–60.
Atkinson AH, Heat RL, Simpson RJ, Clarke AE,
Anderson MA. 1993. Proteinase inhibitors in Nicotiana alata
stigmas are derived from a precursor protein which is processed
into five homologous inhibitors. The Plant Cell 5, 203–213.
Bageshwar UK, Taneja-Bageshwar S, Moharram HM,
Binzel ML. 2005. Two isoforms of the A subunit of the vacuolar
H+-ATPase in Lycopersicon esculentum: highly similar proteins
but divergent patterns of tissue localization. Planta 220, 632–643.
Bergey DR, Howe GA, Ryan CA. 1996. Polypeptide signalling for
plant defensive genes exhibits analogies to defense signaling in
animals. Proceedings of the National Academy of Sciences, USA
93, 12053–12058.
Bethke PC, Fath A, Jones RL. 2001. Regulation of viability and
death by hormones in cereal aleurone. Journal of Plant
Physiology 158, 429–438.
Bethke PC, Hillmer S, Jones RL. 1996. Isolation of intact protein
storage vacuoles from barley aleurone – identification of aspartic
and cysteine proteinases. Plant Physiology 110, 521–529.
Bethke PC, Hwang Y-S, Zhu T, Jones RL. 2006. Global patterns
of gene expression in the aleurone of wild type and dwarf1
mutant rice. Plant Physiology 140, 484–498.
Bethke PC, Lonsdale JE, Fath A, Jones RL. 1999. Hormonally
regulated programmed cell death in barley aleurone cells. The
Plant Cell 11, 1033–1045.
Bethke PC, Swanson SJ, Hillmer S, Jones RL. 1998. From
storage compartment to lytic organelle: the metamorphosis of the
aleurone protein storage vacuole. Annals of Botany 82, 399–412.
Birk Y. 2003. Plant protease inhibitors. Berlin: Springer Verlag.
Bowers K, Stevens TH. 2005. Protein transport from the late
Golgi to the vacuole in the yeast Saccharomyces cerevisiae.
Biochimica et Biophysica Acta – Molecular Cell Research 1744,
438–454.
Bozhkov PV, Suarez MF, Filonova LH, Daniel G,
Zamyatnin Jr
AA, Rodriguez-Nieto S, Zhivotovsky B,
Smertenko A. 2005. Cysteine protease mcII-Pa executes programmed cell death during plant embryogenesis. Proceedings of the
National Academy of Sciences, USA 102, 14463–14468.
Bryant NJ, Piper RC, Weisman LS, Stevens TH. 1998.
Retrograde traffic out of the yeast vacuole to the TGN occurs via
the prevacuolar/endosomal compartment. Journal of Cell Biology
142, 651–663.
Buchanan-Wollaston V, Earl S, Harrison E, Mathas E,
Navabpour S, Page T, Pink D. 2003. The molecular analysis of
leaf senescence – a genomic approach. Plant Biotechnology
Journal 1, 3–22.
Carter C, Pan S, Zouhar J, Avila EL, Girke T, Raikhel NV.
2004. The vegetative vacuole proteome of Arabidopsis thaliana
reveals predicted and unexpected proteins. The Plant Cell 16,
3285–3303.
Chen G-H, Huang L-T, Yap M-N, Lee R-H, Huang Y-J,
Cheng M-C, Chen S-CG. 2002. Molecular characterization of
a senescence-associated gene encoding cysteine proteinase and its
gene expression during leaf senescence in sweet potato. Plant and
Cell Physiology 43, 984–991.
Chiche L, Heitz A, Gelly JC. 2004. Squash inhibitors: from
structural motifs to macrocyclic knottins. Current Protein &
Peptide Science 5, 341–349.
Chrispeels MJ, Herman EM. 2000. Endoplasmic reticulumderived compartments function in storage and as mediators of
vacuolar remodelling via a new type of organelle, precursor
protease vesicles. Plant Physiology 123, 1227–1233.
Coffeen WC, Wolpert TJ. 2004. Purification and characterization of
serine proteases that exhibit caspase-like activity are associated with
programmed cell death in Avena sativa. The Plant Cell 16, 857–873.
Coupe SA, Sinclair BK, Watson LM, Heyes JA, Eason JR.
2003. Identification of dehydration-responsive cysteine proteinases during post-harvest senescence of broccoli florets. Journal
of Experimental Botany 54, 1045–1056.
Craik DJ, Daly NL, Mulvenna J, Plan MR, Trabi M. 2004.
Discovery, structure and biological activities of the cyclotides.
Current Protein & Peptide Science 5, 297–315.
Cunningham SM, Volonec JJ. 1996. Purification and characterization of vegetative storage proteins from alfalfa (Medicago sativa
L.) taproots. Journal of Plant Physiology 147, 625–632.
De DN. 2000. Plant cell vacuoles. Collingwood, VIC, Australia:
CSIRO Publishing.
Di Sansebastiano GP, Paris N, Marc-Martin S, Neuhaus J-M.
2001. Regeneration of a lytic central vacuole and of neutral
peripheral vacuoles can be visualized by green fluorescent protein
targeted to either type of vacuoles. Plant Physiology 126, 78–86.
Dutton JL, Renda RF, Waine C, Clark RJ, Daly NL,
Jennings CV, Anderson MA, Craik DJ. 2004. Conserved
structural and sequence elements implicated in the processing of
gene-encoded circular proteins. Journal of Biological Chemistry
279, 46858–46867.
Eason JR, Ryan DJ, Watson LM, Hedderley D, Christey MC,
Braun RH, Coupe SA. 2005. Suppression of the cysteine
protease, aleuraine, delays floret senescence in Brassica oleracea.
Plant Molecular Biology 57, 645–657.
Epimashko S, Meckel T, Fischer-Schliebs E, Lüttge U, Thiel G.
2004. Two functionally different vacuoles for static and dynamic
purposes in one plant leaf mesophyll cell. The Plant Journal 34,
294–300.
Fath A, Bethke P, Lonsdale J, Meza-Romero R, Jones R. 2000.
Programmed cell death in cereal aleurone. Plant Molecular
Biology 44, 255–266.
Flückiger R, DeCaroli M, Piro G, Dalessandro G,
Neuhaus J-M, Di Sansebastiano GP. 2003. Vacuolar system
distribution in Arabidopsis tissues, visualized using GFP fusion
proteins. Journal of Experimental Botany 54, 1577–1584.
Gietl C, Schmid M. 2001. Riconosomes: an organelle for developmentally programmed cell death in senescing plant tissue.
Naturwissenschaften 88, 49–58.
Gietl C, Schmid M, Simpson D. 2000. Riconosomes and aleuraine containing vacuoles (ACVs): protease-storing organelles. In: Robinson
DG, Rogers JC, eds. Vacuolar compartments. Annual Plant
Reviews, Sheffield: Sheffield Academic Press, 5, 90–111.
Gillespie J, Rogers SW, Deery M, Dupree P, Rogers JC.
2005. A unique family of proteins associated with internalised
membranes in protein storage vacuoles of the Brassicacea. The
Plant Journal 41, 429–441.
Gomez L, Chrispeels MJ. 1993. Tonoplast and soluble vacuolar
proteins are targeted by different mechanisms. The Plant Cell 5,
1113–1124.
Graham JS, Pearce G, Merryweather J, Titani K, Ericsson L,
Ryan CA. 1985. Wound-induced proteinase inhibitors from
tomato leaves. I. The cDNA-deduced primary structure of the
pre-inhibitor I and its post-translational processing. Journal of
Biological Chemistry 260, 6555–6560.
Vacuolar protein dynamics 2405
Greenwood JS, Helm M, Gietl C. 2005. Riconosomes and
endosperm transfer cell structure in programmed cell death of the
nucellus during Ricinus seed development. Proceedings of the
National Academy of Sciences, USA 102, 2238–2243.
Gruis D, Schulze J, Jung R. 2004. Storage protein accumulation
in the absence of vacuolar processing enzyme family of cysteine
proteinases. The Plant Cell 15, 270–290.
Hanton SL, Matheson LA, Brandizzi F. 2006. Seeking a way out:
export of proteins from the plant endoplasmic reticulum. Trends
in Plant Sciences 11, 335–343.
Hara-Nishimura I, Kinoshita T, Hiraiwa N, Nishimura M.
1998. Vacuolar processing enzymes in protein-storage vacuoles
and lytic vacuoles. Journal of Plant Physiology 152, 668–674.
Hara-Nishimura I, Matsushima R, Shimada T, Nishimura M.
2004. Diversity and formation of endoplasmic reticulum-derived
compartments in plants: are these compartments specific to
plants? Plant Physiology 136, 3435–3439.
Hatsugai N, Kuroyanagi M, Yamada K, Meshi T, Tsuda S,
Kondo M, Nishimura M, Hara-Nishimura I. 2004. A plant
vacuolar protease, VPE, mediates virus-induced hypersensitive
cell death. Science 305, 855–858.
Herman EM. 1994. Multiple origins of intravacuolar protein accumulation in plants. Advances in Structural Biology 3, 243–283.
Herman EM, Baumgartner B, Chrispeels MJ. 1981. Uptake and
apparent digestion of cytoplasmic organelles by protein bodies.
European Journal of Cell Biology 24, 226–235.
Herman EM, Schmidt M. 2004. Endoplasmic reticulum to
vacuole trafficking of endoplasmic reticulum bodies provides an
alternate pathway of protein transfer to the vacuole. Plant
Physiology 136, 3440–3446.
Hillmer S, Movafeghi A, Robinson DG, Hinz G. 2001. Vacuolar
storage proteins are sorted in the cis-cisternae of the pea Golgi
apparatus. Journal of Cell Biology 152, 41–50.
Hinz G, Hillmer S, Bäumer M, Hohl I. 1999. Vacuolar storage
proteins and the putative vacuolar sorting receptor BP-80 exit the
Golgi apparatus of developing pea cotyledons in different transport vesicles. The Plant Cell 11, 1509–1524.
Hoh B, Hinz G, Jeaong B-K, Robinson DG. 1995. Protein storage
vacuoles form de novo during pea cotyledon development.
Journal of Cell Science 108, 299–310.
Holwerda BC, Rogers JC. 1993. Structure, functional properties
and vacuolar targeting of the barley protease, aleurain. Journal of
Experimental Botany 44, (Suppl.), 321–330.
Horn M, Pantakar AG, Zavala JA, Wu J, DolečkováMarešová L, Vůjtěchová M, Mares M, Baldwin IT. 2005.
Differential elicitation of two processing proteases controls the
processing pattern of the trypsin proteinase inhibitor precursor in
Nicotiana attenuata. Plant Physiology 139, 375–388.
Jauh G-Y, Fischer AM, Grimes HD, Ryan CA, Rogers JC.
1998. d-Tonoplast intrinsic protein defines unique plant vacuole
functions. Proceedings of the National Academy of Sciences, USA
95, 12995–12999.
Jauh G-Y, Phillips TE, Rogers JC. 1999. Tonoplast intrinsic
protein isoforms as markers for vacuolar functions. The Plant
Cell 11, 1867–1882.
Jiang L, Erickson AH, Rogers JC. 2002. Multivesicular bodies:
a mechanism to package lytic and storage functions in one
organelle? Trends in Cell Biology 12, 362–367.
Jiang L, Phillips TE, Hamm CA, Drozdowicz YM, Rea PA,
Maeshima M, Rogers SW, Rogers JC. 2001. The protein
storage vacuole: a unique compound organelle. Journal of Cell
Biology 155, 991–1002.
Jiang L, Phillips TE, Rogers SW, Rogers JC. 2000. Biogenesis
of the protein storage vacuole crystalloid. Journal of Cell Biology
150, 755–770.
Jiang L, Rogers JC. 2002. Compartmentation of proteins in the
protein storage vacuole: a compound organelle in plant cells.
Advances in Botanical Research 35, 140–170.
Jiang L, Rogers JC. 2003. Sorting of lytic enzymes in the plant
Golgi apparatus. In: Robinson DG, ed. The Golgi apparatus and
the plant secretory pathway. Annual Plant Reviews, Vol. 9.
Oxford: Blackwell Publishing, 114–140.
Jones AM. 2001. Programmed cell death in development and
defence. Plant Physiology 125, 94–97.
Kinoshita T, Yamada K, Hiraiwa N, Kondo M, Nishimura M,
Hara-Nishimura I. 1999. Vacuolar processing enzyme is upregulated in the lytic vacuoles of vegetative tissues during
senescence and under various stress conditions. The Plant
Journal 19, 43–53.
Klauer SF, Franceschi VR. 1997. Mechanism of transport of
vegetative storage proteins to the vacuole of paraveinal mesophyll
in soybean leaf. Protoplasma 200, 174–185.
Koiwa H, Bressan RA, Hasegawa PM. 1997. Regulation of
proteinase inhibitors and plant defence. Trends in Plant Sciences
2, 379–384.
Korsinczky MLJ, Schirra HJ, Craik DJ. 2004. Sunflower
trypsin inhibitor 1. Current Protein & Peptide Science 5, 351–364.
Kunze I, Kunze G, Bröker M, Manteuffel R, Meins Jr F,
Müntz K. 1998. Evidence for secretion of vacuolar amannosidase, class I chitinase, and class I b-1,3-glucanase in
suspension cultures of tobacco cells. Planta 205, 92–99.
Lam SK, Tse YC, Jiang L, Oliviusson P, Heinzerling O,
Robinson DG. 2006. Plant prevacuolar compartments and
endocytosis. In: Šamaj J, Baluska F, Menzel D, eds. Plant
endocytosis. Berlin: Springer Verlag, 37–62.
Leigh RA, Sanders D, eds. 1997. The plant vacuole. Advances in
Botanical Research, Vol. 25. San Diego, CA: Academic Press.
Levanony H, Rubin R, Altschuler Y, Galili G. 1992. Evidence
for a novel route of wheat storage proteins to vacuoles. Journal of
Cell Biology 119, 1117–1128.
Li YB, Rogers SW, Tse YC, Lo SW, Sun SS, Jauh GY, Jiang L.
2002. BP-80 and homologs are concentrated on post-Golgi
probable lytic prevacuolar compartments. Plant Cell Physiology
43, 726–742.
Lin J-F, Wu S-H. 2004. Molecular events in senescing Arabidopsis
leaves. The Plant Journal 39, 612–628.
Madison JT, Thompson JF, Muenster AE. 1981. Turnover of
storage proteins in seeds of soya bean and pea. Annals of Botany
47, 65–74.
Marty F. 1997. The biogenesis of vacuoles: insights from
microscopy. Advances in Botanical Reasearch 25, 1–42.
Marty F. 1999. Plant vacuoles. The Plant Cell 11, 587–599.
Matheson LA, Hanton SL, Brandizzi F. 2006. Traffic between
the plant endoplasmic reticulum and Golgi apparatus: to the Golgi
and beyond. Current Opinion in Plant Biology 9, 601–609.
Matile P. 1997. The vacuole and cell senescence. Advances in
Botanical Research 25, 87–112.
Miao Y, Yan PK, Kim H, Hwang J, Jiang L. 2006. Localization
of green fluorescent protein fusions with the seven Arabidopsis
vacuolar sorting receptors to prevacuolar compartments in
tobacco BY-2 cells. Plant Physiology 142, 945–962.
Mosolov VV, Grigor’eva LI, Valueva TA. 2001. Involvement of
proteolytic enzymes and their inhibitors in plant protection.
Applied Biochemistry and Microbiology 37, 115–123.
Moura DS, Bergey DR, Ryan CA. 2001. Characterization and
localization of a wound-inducible type I serine carboxypeptidase
from leaves of tomato plants (Lycopersicon esculentum Mill.).
Planta 212, 222–230.
2406 Müntz
Moura DS, Ryan CA. 2001. Wound-inducible proteinase inhibitors in pepper: differential regulation upon wounding, systemin
and methyl jasmonate. Plant Physiology 126, 289–298.
Müntz K. 1998. Deposition of seed proteins. Plant Molecular
Biology 38, 77–99.
Müntz K. 2003. The role of seed proteases in deposition and
mobilization of storage proteins. Recent Research Development in
Plant Biology 3, 95–114.
Müntz K, Belozersky MA, Dunaevsky YE, Schlereth A,
Tiedemann J. 2001. Stored proteinases and the initiation of
storage protein mobilization in seeds during germination and
seedling growth. Journal of Experimental Botany 52, 1741–1752.
Müntz K, Blattner FR, Shutov AD. 2002. Legumains – a family
of asparagine-specific cysteine endopeptidases involved in propolypeptide processing and protein breakdown in plants. Journal of
Plant Physiology 159, 1281–1293.
Müntz K, Shutov AD. 2002. Legumains and their functions in
plants. Trends in Plant Science 7, 340–344.
Mulvenna JP, Foley FM, Craik DJ. 2005a. Discovery, structural
determination, and putative processing of the precursor that
produces cyclic trypsin inhibitor sunflower trypsin inhibitor 1.
Journal of Biological Chemistry 280, 32245–32253.
Mulvenna JP, Sando L, Craik DJ. 2005b. Processing of a 22 kDa
precursor protein to produce the circular protein Tricyclon A.
Structure 13, 691–701.
Murphy AS, Bandyo Padhyay A, Holstein SE, Peer WA. 2005.
Endocytotic cycling of PM proteins. Annual Review of Plant
Biology 56, 221–252.
Nakaune S, Yamada K, Kondo M, Kato T, Tabata S,
Nishimura M, Hara-Nishimura I. 2005. A vacuolar processing
enzyme, dVPE, is involved in seed coat formation at the early
stage of seed development. The Plant Cell 17, 876–887.
Neuhaus J-M, Paris N. 2006. Plant vacuoles: from biogenesis to
function. In: Šamaj J, Baluska F, Menzel D, eds. Plant
endocytosis. Berlin: Springer Verlag, 63–82.
Noda T, Ohsumi Y, Klionsky DJ. 2000. The yeast vacuole:
a paradigm for plant cell biologists? In: Robinson DG, Rogers JC,
eds. Vacuolar compartments. Annual Plant Reviews, Vol. 5.
Sheffield: Sheffield Academic Press, 1–19.
Okamoto T, Minamikawa T. 1998. A vacuolar cysteine endopeptidase (SH-EP) that digests seed storage globulin: characterization, regulation of gene expression, and posttranslational
processing. Journal of Plant Physiology 152, 675–682.
Oliviusson P, Heinzerling O, Hillmer S, Hinz G, Tse YC,
Jiang L, Robinson DG. 2006. Plant retromer, localized to the
prevacuolar compartment and microvesicles in Arabidopsis,
may interact with vacuolor sorting receptors. The Plant Cell 18,
1239–1252.
Otegui MS, Herder R, Schultze J, Jung R, Staehlin A. 2006.
The proteolytic processing of seed storage proteins in Arabidopsis
thaliana embryo cells starts in the multivesicular bodies. The
Plant Cell 18, 2567–2581.
Otegui MS, Noh Y-S, Martinez DE, Vila Petroff MG,
Staehlin LA, Amasino RM, Guiamet JJ. 2005. Senescenceassociated vacuoles with intense proteolytic activity develop in
leaves of Arabidopsis and soybean. The Plant Journal 41, 831–344.
Oufattole M, Park JH, Poxleitner M, Jiang L, Rogers JC. 2005.
Selective membrane protein internalisation accompanies movement from the endoplasmic reticulum to the protein storage
vacuole pathway in Arabidopsis. The Plant Cell 17, 3066–3080.
Paris N, Stanley CM, Jones RL, Rogers JC. 1996. Plant cells
contain two functionally distinct vacuolar compartments. Cell 85,
563–572.
Park M, Kim SJ, Vitale A, Hwang I. 2004. Identification of
the protein storage vacuole and protein targeting to the vacuole
in leaf cells of three plant species. Plant Physiology 134,
625–639.
Parrott D, Yang L, Shama L, Fischer AM. 2005. Senescence is
accelerated and several proteases are induced by carbon ‘feast’
conditions on barley (Hordeum vulgare L.) leaves. Planta 222,
989–1000.
Peumans WJ, Van Damme EJM. 1999. Seed lectins. In: Shewry
PR, Casey R, eds. Seed proteins. Dordrecht: Kluwer Academic
Publisher, 657–684.
Pimpl P, Taylor JP, Snowden C, Hillmer S, Robinson DG,
Denecke J. 2006. Golgi-mediated vacuolar sorting of the
endoplasmic reticulum chaperon BIP may play an active role in
quality control within the secretory pathway. The Plant Cell 18,
198–211.
Robinson DG, Bäumer M, Hinz G, Hohl. 1998a. Vesicle transfer
of storage proteins to the vacuole: the role of Golgi apparatus and
multivesicular bodies. Journal of Plant Physiology 152, 659–667.
Robinson DG, Galili G, Herman EM, Hillmer S. 1998b. Topical
aspects of vacuolar protein transport: autophagy and prevacuolar
compartments. Journal of Experimental Botany 49, 1263–1270.
Robinson DG, Hinz G. 1997. Vacuole biogenesis and protein
transport to the plant vacuole: a comparison with the yeast
vacuole and the mammalian lysosome. Protoplasma 197, 1–25.
Robinson DG, Hinz G. 1999. Golgi-mediated transport of seed
storage proteins. Seed Science Research 9, 267–284.
Robinson DG, Oliviusson P, Hinz G. 2005. Protein sorting to the
storage vacuoles of plants: a critical appraisal. Traffic 6, 615–625.
Robinson DG, Rogers JC, eds. 2000. Vacuolar compartments.
Annual Plant Reviews, Vol. 5. Sheffield: Sheffield Academic Press.
Robinson DG, Rogers JC, Hinz G. 2000. Post-Golgi, prevacuolar
compartment. In: Robinson DG, Rogers JC, eds. Vacuolar
compartments. Annual Plant Reviews, Vol. 5. Sheffield: Sheffield
Academic Press, 270–298.
Rojo E, Gillmor CS, Kovaleva V, Somerville CR, Raikhel NV.
2001. VACUOLELESS 1 is an essential gene required for
vacuole formation and morphogenesis in Arabidopsis. Developmental Cell 1, 303–310.
Rojo E, Martin R, Carter C, et al. 2004. VPEc exhibits
a caspase-like activity that contributes to defence against
pathogens. Current Biology 14, 1897–1906.
Rojo E, Zouhar J, Carter C, Kovaleva V, Raikhel NV. 2003. A
unique mechanism for protein processing and degradation in
Arabidopsis thaliana. Proceedings of the National Academy of
Sciences, USA 100, 7389–7394.
Rotari VI, He R, Gallois P. 2005. Death proteases in plants:
whodunit? Physiologia Plantarum 123, 376–385.
Ryan CA. 1990. Protease inhibitors in plants: genes for improving
defence against insects and pathogens. Annual Reviews of
Phytopathology 28, 425–449.
Šamaj J, Baluška F, Menzel D, eds. 2006. Plant endocytosis. In:
Robinson DG, ed. Plant cell monographs, Vol. 1. Berlin:
Springer Verlag.
Šamaj J, Read ND, Volkmann D, Menzel D, Baluška F. 2005. The
endocytic network in plants. Trends in Cell Biology 15, 425–433.
Sanmartin M, Jaroszewski L, Raikhel NV, Rojo E. 2005.
Caspases: regulating death since the origin of life. Plant
Physiology 137, 841–847.
Schlereth A, Standhardt D, Mock H-P, Müntz K. 2001. Stored
proteinases start globulin mobilization in protein bodies of
embryonic axes and cotyledons during vetch (Vicia sativa L.)
seed germination. Planta 212, 718–727.
Schmid M, Simpson D, Gietl C. 1999. Programmed cell death in
castor bean endosperm is associated with the accumulation and
release of a cysteine endopeptidase from riconosomes. Proceedings
of the National Academy of Sciences, USA 96, 14159–14164.
Vacuolar protein dynamics 2407
Schmid M, Simpson D, Sarioglu H, Lottspeich F, Gietl C. 2001.
The riconosomes of senescing plant tissue bud from the
endoplasmic reticulum. Proceedings of the National Academy of
Sciences, USA 98, 5353–5358.
Sheldon PS, Keen JN, Bowles DJ. 1996. Post-translational peptide
bond formation during concanavalin A processing in vitro.
Biochemical Journal 320, 865–870.
Shewry PR, Casey R, eds. 1999. Seed proteins. London: Chapman
& Hall.
Shewry PR, Lucas JA. 1997. Plant proteins that confer resistance
to pests and pathogens. Advances in Botanical Research 26,
135–192.
Shimada T, Koumoto Y, Li L, Yamazaki M, Kondo M,
Nishimura M, Hara-Nishimura I. 2006. AtVPS29, a putative
component of a retromer complex, is required for the efficient
sorting of seed storage proteins. Plant Cell Physiology 47,
1187–1194.
Shimaoka T, Ohnishi M, Sazuka T, Mitsuhashi N, HaraNishimura I, Shimazaki KI, Maeshima M, Yokota A,
Tomizawa KI, Mimura T. 2004. Isolation of intact vacuoles
and proteomic analysis of tonoplast from suspension-cultured
cells of Arabidopsis thaliana. Plant Cell Physiology 45, 672–683.
Shutov AD, Bäumlein H, Blattner FR, Müntz K. 2003. Storage
and mobilization as antagonistic functional constraints on seed
storage globulin evolution. Journal of Experimental Botany 54,
1645–1654.
Simonsen SM, Sando L, Ireland DC, Colgrave ML, Bharathi R,
Göransson U, Craik DJ. 2005. A continent of plant defence
peptide diversity: cyclotides in Australian Hybanthus (Violaceae).
The Plant Cell 17, 3176–3189.
Sláviková S, Shy G, Yao Y, Glozman R, Levanony H,
Pietrokovski S, Elazar Z, Galili G. 2005. The autophagyassociated Atg8 gene family operates both under favourable
growth conditions and under starvation stress in Arabidopsis
thaliana. Journal of Experimental Botany 56, 2839–2849.
Sreenivasulu N, Radchuk V, Strickert M, Weschke W,
Wobus U. 2006. Gene expression patterns reveal tissue-specific
signalling networks controlling programmed cell death and ABAregulated maturation in developing barley seeds. The Plant
Journal 47, 310–327.
Staswick PE. 1990. Novel regulation of vegetative storage protein
genes. The Plant Cell 2, 1–6.
Staswick PE. 1994. Storage proteins of vegetative plant tissues.
Annual Reviews of Plant Physiology and Plant Molecular
Biology 45, 303–322.
Surpin M, Raikhel NV. 2004. Traffic jams affect plant development and signal transduction. Nature Reviews of Molecular Cell
Biology 5, 100–109.
Swanson SJ, Bethke PC, Jones RL. 1998. Barley aleurone cells
contain two types of vacuoles: characterization of lytic organelles
by use of fluorescent probes. The Plant Cell 10, 685–698.
Swanson SJ, Jones RL. 1996. Gibberellic acid induces vacuolar
acidification in barley aleurone. The Plant Cell 8, 2211–2221.
Szponarski W, Sommerer N, Boyer JC, Rossignol M, Gibrat R.
2004. Large-scale characterization of integral proteins from
Arabidopsis vacuolar membrane by two-dimensional liquid
chromatography. Proteomics 4, 397–406.
Takano J, Miwa K, Yuan L, Von Wiren N, Fujiwara T. 2005.
Endocytosis and degradation of BOR1, a boron transporter of
Arabidopsis thaliana, regulated by boron availability. Proceedings
of the National Academy of Sciences, USA 102, 12276–12281.
Tamura K, Shimada T, Ono E, Tanaka Y, Nagatani A,
Higashi S-L, Watanabe M, Nishimura M, Hara-Nishimura I.
2003. Why green fluorescent fusion proteins have not been observed in vacuoles of higher plants. The Plant Journal 35, 545–555.
Tamura K, Yamada K, Shimada T, Hara-Nishimura I. 2004.
Endoplasmic reticulum resident proteins are constitutively transported to vacuoles for degradation. The Plant Journal 39, 393–402.
Thomas H, Ougham HJ, Wagstaff C, Stead AD. 2003. Defining
senescence and death. Journal of Experimental Botany 54,
1127–1132.
Tiedemann J, Schlereth A, Müntz K. 2001. Differential tissuespecific expression of cysteine proteinases forms the basis for the
fine-tuned mobilization of storage globulin during and after
germination in legume seeds. Planta 212, 728–738.
Toyooka K, Okamoto T, Minamikawa T. 2001. Cotyledon cells
of Vigna mungo seedlings use at least two distinct autophagic
machineries for degradation of starch granules and cellular
components. Journal of Cell Biology 154, 973–982.
Tse YC, Lo SW, Hillmer S, Dupree P, Jiang L. 2006. Dynamic
response of prevacuolar compartments to Brefeldin A in plant
cells. Plant Physiology 142, 1442–1459.
Ueda T, Uemura T, Sato MM, Nakano A. 2004. Functional
differentiation of endosomes in Arabidopsis cells. The Plant
Journal 40, 783–789.
van der Hoorn RAL, Jones JD. 2004. The plant proteolytic
machinery and its role in defence. Current Opinion in Plant
Biology 7, 400–407.
van Doorn WG. 2005. Plant programmed cell death and the point
of no return. Trends in Plant Science 10, 478–483.
van Doorn WG, Woltering EJ. 2004. Senescence and programmed cell death: substance or semantics? Journal of Experimental Botany 55, 2147–2154.
Vitale A, Ceriotti A. 2004. Protein quality control mechanisms and
protein storage in the endoplasmic reticulum: a conflict of
interests? Plant Physiology 136, 3420–3426.
Vitale A, Hinz G. 2005. Sorting of proteins to storage vacuoles:
how many mechanisms? Trends in Plant Science 10, 316–323.
Waterworth WM, West CE, Bray CM. 2001. The physiology and
molecular biology of peptide transport in seeds. Seed Science
Research 11, 275–284.
Weihofen A, Martoglio B. 2003. Intramembrane-cleaving proteases: controlled liberation of proteins and bioactive peptides.
Trends in Cell Biology 13, 71–78.
Winter V, Hauser M-T. 2006. Exploring the ESCRTing machinery
in eukaryotes. Trends in Plant Sciences 11, 115–123.
Wisniewski J-P, Rogowsky PM. 2004. Vacuolar H+-translocating
inorganic pyrophosphorylase (Vpp1) marks partial aleurone cell
fate in cereal endosperm development. Plant Molecular Biology
56, 325–337.
Woltering EJ. 2004. Death proteases come alive. Trends in Plant
Science 9, 469–472.
Yamada K, Matsushima R, Nishimura M, Hara-Nishimura I.
2001. A slow maturation of a cysteine protease with a granulin
domain in the vacuoles of senescing Arabidopsis leaves. Plant
Physiology 127, 1626–1634.
Yamada K, Shimada T, Nishimura M, Hara-Nishimura I. 2005.
A VPE family supporting various vacuolar functions in plants.
Physiologia Plantarum 123, 369–375.
Yashimoto K, Hanaoka H, Sato S, Kato T, Tabata S, Noda T,
Oshumi Y. 2004. Processing of ATG8s, ubiquitin-like proteins,
and their deconjugation by ATG4s are essential for plant
autophagy. The Plant Cell 16, 2967–2983.
Young TE, Gallie DR. 2000. Programmed cell death during
endosperm development. Plant Molecular Biology 44, 283–301.
Yoshida K, Kawachi M, Mori M, Maeshima M, Kondo M,
Nishimura M, Kondo T. 2005. The involvement of tonoplast
proton pumps and Na+(K+)H+ exchangers in the change of petal
color during flower opening of morning glory, Ipomea tricolor
cv. Heavenly Blue. Plant Cell Physiology 46, 407–415.