plant vacuoles: where did they come from and where are they heading?
TRANSCRIPT
Available online at www.sciencedirect.com
Plant vacuoles: where did they come from and where are theyheading?Jan Zouhar and Enrique Rojo
Genetic and technical advances of the past few years have
allowed us to test some of the vacuolar trafficking and vacuole
biogenesis models that had been previously proposed mainly
on the basis of morphological and immunolocalization studies.
We have now tools to start answering some fundamental
questions such as: How are vacuoles formed? Are all vacuoles
formed similarly? Do different types of vacuoles coexist in a
cell? How are proteins sorted to the vacuole? How many
trafficking pathways to vacuoles exist? Can there be trafficking
to two types of vacuoles simultaneously? Last but not least,
how do vacuoles balance the continuous flow of new materials,
cargo and membrane, and maintain their volume? We will
review recent data trying to answer these questions and
propose some models that accommodate the results obtained.
Address
Departamento de Genetica Molecular de Plantas, Centro Nacional de
Biotecnologıa, Consejo Superior de Investigaciones Cientıficas, E-28049
Madrid, Spain
Corresponding author: Rojo, Enrique ([email protected])
Current Opinion in Plant Biology 2009, 12:677–684
This review comes from a themed issue on
Cell Biology
Edited by Jirı Friml and Karin Schumacher
Available online 23rd September 2009
1369-5266/$ – see front matter
# 2009 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.pbi.2009.08.004
IntroductionUpon seed dispersal, siblings with essentially the same
genetic instructions must be able to grow and reproduce
in the diverse microenvironments where they land. More-
over, once germinated, they will have to explore the
surrounding media, while autonomously producing all
the materials and energy required for growth. To main-
tain certain cellular homeostasis compatible with their
genetic constitution and to achieve cost-effective growth,
plants have evolved a different cellular architecture from
metazoans. A main feature of this is the presence of large
vacuoles that make up most of the cell volume. These
large vacuoles act as buffering compartments and they
allow cellular growth at lower costs, given their low
density of organic compounds. In addition, vacuoles serve
other plant specific functions such as storing proteins to
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be used by the next generation during germination.
These properties set the plant vacuoles apart from the
metazoan vacuoles/lysosomes, which are small and dense
compartments that mainly serve a degradative role.
These peculiarities mean that to understand plant
growth, development and adaptation to the environment,
we need to learn how vacuoles are formed and dynami-
cally maintained. In this review we summarize recent
results addressing the biogenesis, trafficking and
dynamics of plant vacuoles.
Biogenesis of vacuolesTwo main types of vacuoles can be found in plant cells
(Box 1), the lytic vacuole (LV) and the protein storage
vacuole (PSV), which appear sequentially during embry-
ogenesis. After fertilization a large LV forms in the zygote
and localizes basally. The zygote then divides asymme-
trically giving rise to a vacuolated basal cell that will
develop into the suspensor and a non-vacuolated apical
cell that will generate the embryo proper. Large LVs
develop then in suspensor cells and afterwards in the
embryo proper, in cells outside the meristems. It is not
known whether these LVs are formed de novo or through
enlargement of small vacuoles pre-existing in the egg cell
or in the embryonic cells. However, LVs can rapidly
regenerate in evacuolated protoplasts [1�], suggesting that
they could also form de novo during normal plant de-
velopment.
The VCL1 gene is required for LV biogenesis in embryos.
VCL1 has homology to yeast VPS16p and forms part of a C-
VPS complex that may regulate the SYP21 and SYP22
SNAREs to execute membrane fusion at the prevacuolar
compartment (PVC) and the tonoplast [2]. In the vcl1mutant the biogenesis of LVs in the embryo is blocked
and, interestingly, large numbers of autophagosomes
accumulate instead [3]. A possible explanation for this
phenotype is that the C-VPS complex in Arabidopsis is
required for de novo formation of LVs from autophago-
somes. This is consistent with the role of the C-VPS
complex in autophagosome to vacuole fusion in yeasts
and mammals. Early observations of the biogenesis of
vacuoles in meristems also suggested an autophagic origin
of the plant LV [4]. On the basis of morphological evidence
it was proposed that vacuole formation in the meristems
occurs through dilation and fusion of autophagosomes,
forming a cage-like structure that engulfed portions of
the cytosol and finally coalesced to form the LV. However,
meristematic cells contain small vacuoles, which are parti-
tioned during mitosis between the daughter cells [5].
Current Opinion in Plant Biology 2009, 12:677–684
678 Cell Biology
Box 1 Vacuole diversity: the case of Arabidopsis
Plants contain different types of vacuoles that may co-exist in certain
cell types [10]. A paradigmatic case found in many plants including
Arabidopsis is the presence of lytic vacuoles (LVs) and protein
storage vacuoles (PSVs) in cells of the developing embryo. This
separation of lytic and storage functions constitutes a plant
adaptation for stable accumulation of large amounts of proteins to be
used by the germinating seedling. The existence of these two
vacuoles with distinct contents implies that separate trafficking
pathways for their respective cargo must exist. It is not yet settled
whether separate LVs and PSVs are generally present in plant cells
[37], or only in specialized tissues such as those of the embryo
[10,38,44]. That said, the prevailing view now is that in most
vegetative cells a single class of vacuole is present, the central
vacuole that has LV characteristics. Nonetheless, this single LV from
vegetative cells appears to be served by different trafficking
pathways, which may be also used for separate transport to PSVs or
LVs in seed cells.
In addition to PSVs and LVs, other case of co-existing vacuoles has
been reported in Arabidopsis. During leave senescence the
senescence-associated vacuoles (SAVs) are formed de novo. SAVs
are characterized by a higher cysteine-protease activity and a lower
pH than the LVs [45]. They are smaller in size and lack a g-TIP
tonoplast aquaporin, which is highly abundant in the membrane of
the LVs from the same cells. How SAVs are formed is unknown. They
contain aggregates that resemble partially degraded cytosolic
material and their tonoplast lack the g-TIP aquaporin, similarly to root
autolysosomes [46]. However, their size is smaller that regular
autophagosomes suggesting that they are not originated directly by
autophagy.
Therefore, at least in meristematic cells, it is more likely
that LVs are not formed de novo but are instead inherited
and then enlarge in differentiating cells of the meristem
(Figure 1) through a process that may involve autophagy.
Indeed, evidence has been presented for constitutive
autophagy contributing to vacuole expansion in the root
meristem [6]. Moreover, using a GFP-AtAtg8 marker, it
has been shown that appearance of autophagosome-like
structures correlates with the start of vacuolation and cell
elongation [7]. In the regeneration assay in evacuolated
protoplasts, biogenesis of the LV was also parallelled by
autophagic uptake of cytosolic contents [1�]. Interest-
ingly both vacuole regeneration and uptake of cytosolic
materials were insensitive to inhibitors that block
starvation-induced autophagy, suggesting that it involves
a special type of autophagy. This could explain why
mutants impaired in regular autophagy are not impaired
in vacuole formation. Another mutant with a very specific
defect in LV generation during embryogenesis has been
reported. In the grv2/kam2 mutant, the first division of
the zygote is normal, but then a large LV forms in the
apical cell. This large LV alters the plane of the next
division of the apical cell and is inherited by one of the
resulting cells [8]. The resulting large vacuolated cell
persists until the heart stage, but does not affect the
development of the rest of the cells that give rise to an
apparently normal embryo. GRV2/KAM2 is localized
into an uncharacterized compartment, and is required
Current Opinion in Plant Biology 2009, 12:677–684
for proper endosome organization and trafficking to LVs
and PSVs, also in the adult plant [9].
The prevailing evidence suggests that PSVs form de novoat late stages of embryogenesis. The PSV first develops as
a tubular structure that encircles the pre-existing LV, and
then expands and may even incorporate the LV into their
lumen [10]. Interestingly, it has been shown that the PSV
from tobacco and tomato seeds is actually a compound
organelle: within the limiting tonoplast there is a matrix
that contains the storage proteins and also membrane
bound compartments with lytic characteristics, the glo-
boids [11]. The origin of the globoids is not known, but a
possibility is that they correspond to the LVs that are
engulfed during PSV formation. Arabidopsis seeds also
contain globoid-like compartments that store phytate
crystals, but it is unclear whether they are also membrane
bound [12]. Arabidopsis mutants devoid of PSVs have not
been isolated, but several mutants with altered PSV
morphology have been characterized. The grv2/kam2mutant displays distorted PSV morphology and secretes
storage proteins [9]. The vamp727syp22 double mutant is
partially affected in transport of storage proteins in seeds
and displays fragmented vacuoles with internal mem-
branes [13�]. VAMP727, SYP22, VTI11 and SYP51 form
a complete SNARE complex that may execute hetero-
typic fusion between the PVC and the PSV, which is
consistent with defective delivery of vacuolar cargo and
with the smaller size of PSVs in vamp727syp22 seeds. In
addition, the fragmented PSV phenotype indicates that
homotypic vacuole fusion is also altered in the mutant.
While VAMP727 is localized in the PVC, SYP22, VTI11
and SYP51 have a dual localization in the PVC and the
vacuole where they may mediate homotypic fusion as part
of a SNARE complex with a different R-SNAREs, prob-
ably from the vacuolar VAMP71 group [14]. VCL1 inter-
acts with SYP22 and is probably also required to mediate
membrane fusion at the PSV tonoplast. Unfortunately,
vcl1 embryos arrest before the formation of PSVs, so the
role of the C-VPS complex in PSV de novo biogenesis has
not yet been tested. Interestingly mutants in two com-
ponents of a putative plant retromer complex, VPS29 and
VPS35 also display fragmented PSVs [15,16]. This is a
surprising result, as the retromer complex is thought to
function in recycling from the PVC to the TGN [17], not
in vacuole fusion. In yeast, for instance, vps29 and vps35mutants have wild type vacuolar morphology. The effects
on PSV fusion may be an indirect effect of perturbing the
function of sorting nexins, which are part of the retromer
complex but may have other roles in trafficking. Indeed,
yeast mutants in vps5 (the snx1 orthologue) display frag-
mented vacuoles. Moreover, altering sorting nexin func-
tion by over expression has drastic effects on the
endomembrane system in animals and plants [18]. This
indirect effect on SNX functionality may also explain the
defective polar transport of PIN proteins in vps29 mutant
plants [19].
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Plant vacuoles: where did they come from and where are they heading? Zouhar and Rojo 679
Figure 1
The meristem, a model for studying vacuole biogenesis. The image illustrates the progressive formation of large vacuoles as cells enter the transition
zone of the meristem (arrowhead) where cell differentiation and elongation starts. The tonoplast is labelled with a dTIP-GFP construct (green signal)
while cell walls are labelled with propidium iodide (red signal). Lateral root cap cells (asterisks). The synchronization of vacuole enlargement in
differentiating cells of the meristem constitutes a very powerful system to study how vacuole biogenesis occurs and how it is regulated during
development. For example, by analyzing the high-resolution map of the transcriptome of Arabidopsis roots [47], we found that the gene ontology terms
endomembrane system, intracellular protein transport and vacuolar membrane are significantly enriched in longitudinal pattern gene clusters with
maximums of expression in the elongating cells of the transition zone (patterns 14, 15, 18, 22, 28, 29 and 34 in [47]). Functional analysis of the
trafficking genes found in these clusters may reveal their function in LV formation.
Targeting to vacuoles and the receptorsinvolvedTo serve their multiple functions, vacuoles must contain a
precise complement of proteins and lipids. Proteins,
lipids and even organelles are transported to the vacuole
through biosynthetic, endocytic and autophagic path-
ways. Plant autophagy has been the focus of a recent
excellent review [20], while endocytic trafficking will be
covered by other reviews in this issue. Thus, we have
focused here on summarizing recent developments in the
mechanisms of biosynthetic trafficking to the vacuole.
Although certain vacuolar cargo is sorted in the endoplas-
mic reticulum and transported directly to the vacuole
[21], most proteins in the biosynthetic pathway are sorted
in the Golgi apparatus [22]. In plants, positive sorting is
required for separating soluble vacuolar cargo from the
rest of the proteins that will be secreted by the default
bulk flow pathway. Initial work in vacuolar trafficking was
focused in identifying the sorting signals that marked
cargo for their sorting into the vacuolar route. Two main
types of vacuolar sorting determinants (VSDs) have been
found, the sequence-specific VSD (ssVSD) and the C-
terminal VSD (ctVSD). The ssVSDs show sequence
conservation, in particular they contain an Ile/Leu residue
that is essential for vacuolar targeting, while the ctVSDs
lack any obvious sequence conservation but have in
common their strict localization to the C-terminus of
the protein and the over representation of hydrophobic
amino acids. This classification may have some integra-
tive value, as VSDs from the same class have some similar
biological properties. For example, ctVSDs have been
found only in vacuolar storage proteins, and may explain
how this type of cargo can be differentiated in the Golgi
and targeted to the PSV. Some proteins even combine
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ssVSDs and ctVSDs that may serve for dual targeting to
the PSV matrix and the globoids [23]. It is important to
note that relatively few VSDs have been characterized
and new types may remain to be discovered. Moreover,
although Arabidopsis is now established as the primary
model for intracellular trafficking studies, only a few
endogenous vacuolar cargo have been studied and even
fewer VSDs have been characterized.
Similarly to yeast and animal models, the plant VSDs may
be recognized by sorting receptors that direct the cargo to
specific vesicles. Alternatively, ‘sorting by retention’ has
also been postulated as a possible mechanism for vacuolar
targeting of certain storage proteins, which form aggre-
gates that may preclude their exit in secretory vesicles
[24,25], although no proof for this hypothesis is yet
available. Importantly, VSDs are not only necessary for
vacuolar sorting but also sufficient to target chimeric
proteins to the vacuole. Thus, rather than particular
physical properties of the cargo, it is the specific recog-
nition of those VSDs that targets proteins to vacuoles.
Indeed, two families of putative vacuolar sorting recep-
tors have been identified in plants. The Vacuolar Sorting
Receptor (VSR) family contains seven members in Ara-
bidopsis, and the Receptor Homology-transmembrane-
RING H2 domain (RMR) family contains six members
(Figure 2). Initially it was thought that VSRs were recep-
tors for cargo with ssVSDs and destined for lytic vacuoles,
while RMRs were the long-sought receptors for storage
vacuole cargo with ctVSDs. However, this simple dicho-
tomy is probably not correct and the nature of the recep-
tors for different cargo is a matter of strong debate [26].
Functional genetic analysis has provided several pieces of
evidence supporting a role for VSRs as sorting receptors
Current Opinion in Plant Biology 2009, 12:677–684
680 Cell Biology
Figure 2
Domain structure of VSRs and RMRs. VSRs (A) and RMRs (B) are type I
membrane proteins with a conserved lumenal protease-associated
domain (PA, IPR003137), which in VSRs is involved in binding to VSDs
[48]. In addition, VSRs contain a lumenal growth factor receptor domain
(EGF, IPR009030). By contrast, the cytosolic domains of VSRs and
RMRs are not conserved. VSRs have a short cytosolic tail (T) that
interacts with the mA-adaptin subunit of an AP-1 adaptor complex [49]
and may be involved in coat recruitment at the TGN. RMRs have a large
cytosolic region that contains a RING-H2 zinc finger (RING, IPR001841)
and a serine rich domain (S) of unknown functions. RING finger domains
are generally found in E3-ligases. Moreover, RNF13, a mouse
endosomal protein with a similar domain structure as RMRs (PA-TMD-
RING-Serine Rich) has E3-ligase activity and probably ubiquitinates itself
[50]. Importantly, RMRs are found in the inner vesicles of the
multivesicular PVC, and internalization of membrane proteins at the PVC
is thought to be triggered by ubiquitination. Therefore, we propose that
the RMRs may autoubiquitinate themselves to get internalized in PVC for
degradation in the vacuole. Interestingly, RMR1 targeting to a putative
vacuolar compartment was enhanced in the presence of phaseolin [31],
suggesting that cargo can regulate RMR turnover.
for storage proteins. An Arabidopsis vsr1 mutant was
shown to missort endogenous seed storage proteins to
the apoplast [27]. Components of a putative retromer
complex (VPS29 and VPS35) and of a TGN localized
SNARE complex (VTI12, VPS45), which may be involved
in recycling of VSRs [28,29], are also required for proper
targeting of storage proteins [15,16,29,30�]. By contrast,
the function of RMRs still remains obscure, as trafficking
defects have only been observed by over expression of
dominant negative and chimeric RMR proteins [31,32].
Storage proteins are segregated to the rims of Golgi cis-
ternae already at the cis side of the Golgi both in pea and
Arabidopsis [33�]. They are then transported in dense
vesicles (DVs), which form at the trans side of the Golgi
stacks and are morphologically distinct form typical cla-
thrin coated vesicles that may transport lytic cargo to the
vacuole. The major argument against VSR being storage
protein sorting receptors was that in pea cotyledons they
are spatially segregated (albeit partially) from storage cargo,
as assayed by immunoelectron microcopy and subcellular
fractionation studies [34]. However, a recent paper addres-
sing the localization of VSRs during storage protein depo-
sition in Arabidopsis seeds has challenged the spatial
separation in this species [35�]. In Arabidopsis seeds,
storage PSV cargo (cruciferins and napins) are spatially
segregated from their processing proteases (aspartic pro-
tease A1 and b-VPE) at the trans side of the Golgi,
consistent with their independent trafficking to the PSV.
Importantly, VSRs colocalize with the storage cargo in the
Current Opinion in Plant Biology 2009, 12:677–684
rims of the cisternae and in DV, consistent with the genetic
data implying a role of VSRs as storage cargo receptors. The
relative distribution of VSRs, RMRs and cruciferins in
Arabidopsis seeds was also recently studied [33�]. In their
study, VSRs were also found in DVs and in the PVC
together with storage proteins. However, storage proteins
segregated to the cisternal rims already at the cis side of the
Golgi stacks together with RMRs, but not with VSRs that
preferentially label the trans side of the Golgi. These
results by Hinz and co-workers, and the previous ones
they obtained in pea cotyledons, indicate that VSRs do not
participate in the initial segregation to the periphery of the
cisternae, but they are compatible with VSRs mediating
sorting into DV at the trans side. An appealing possibility is
that VSRs and RMRs may act as co-receptors for storage
proteins. RMRs could act first by nucleating storage
protein at the cis side of the Golgi, while in the trans side
VSRs may participate in sorting the cargo into DVs. Inter-
estingly, the targeting of VSRs is affected by co-expression
of RMR [32], suggesting a close functional link between
these proteins in vivo.
Is there simultaneous trafficking to differenttypes of vacuoles?Although different vacuoles clearly coexist in certain
cells, it does not automatically follow that active traffick-
ing to two different vacuoles can occur simultaneously in
the cell. In Arabidopsis seeds, where LVs and PSVs are
found in the same cell, storage cargo and their processing
proteases are segregated in the Golgi but then converge
into the same PVC [35�]. This confluence in the PVC is
consistent with results in tobacco BY-2 cells showing that
storage and lytic markers co-localize in the same popu-
lation of PVCs [36]. A question begs as to why cargo is first
separated and then brought together en route to the
vacuole. The segregation in the Golgi may be necessary
to prevent processing, which may interfere with packa-
ging of storage aggregates into DVs for vacuolar targeting.
However, upon reaching the PVC, targeting to the
vacuole may be irreversible so processing of the cargo
may begin, as has been reported [35�]. If the trafficking
pathways for lytic and storage cargo converges in a unique
PVC in seeds, it is then likely that the cargo is transferred
to a single class of vacuole. If one assumes that trafficking
is redirected from LVs to PSVs after these latter orga-
nelles first appear in the cell, this could explain how
during seed maturation PSVs are gradually enlarged while
LVs shrink. But how would the vacuolar trafficking
machinery favour transport to PSVs? The reasons may
be purely physical. In the initial stages, PSV develops as a
tubular structure that encircles LVs and may limit access
to the inner LV, thus favouring fusion with the outer PSV.
Alternatively, fusion with the PSV may be favoured owing
to the presence of specific proteins in the PSV tonoplast.
Some reports have suggested that different types of PVCs
and independent trafficking to two types of vacuoles may
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Plant vacuoles: where did they come from and where are they heading? Zouhar and Rojo 681
Figure 3
Chemical genomics can be used to study endomembrane system in a regulated manner that might not achievable by classical genetics. (A) Screens
can be set up to search for chemicals inducing scorable phenotypes in seedlings [51], yeast [52] or pollen [53]. (B) Follow-up screens using
mutagenized Arabidopsis seeds to identify resistant (2) and hypersensitive (3) mutants may identify the drug targets [54�] or unravel connections
between several cellular pathways [55].
be present simultaneously in cells. In Arabidopsis proto-
plasts, RMR1 localizes to a compartment proposed to act as
a PVC for storage cargo such as phaseolin that is separate
from the SYP21-labelled PVC [31]. Moreover, phaseolin is
then targeted to a small disc shaped compartment labelled
by the aquaporins DIP and a-TIP, which may represent
the PSV of vegetative tissues [37] and is distinct from the
large LV targeted by other vacuolar proteins [29]. How-
ever, these studies were based on transient expression in
protoplasts, and may not reflect the in vivo situation.
Indeed, the presence of the a-TIP-labelled disc shaped
compartment was not observed when stable transgenic
plants were analyzed [38]. Moreover, in seeds, endogenous
RMRs and VSRs colocalize with storage proteins in what
appears to be the same PVC [33�]. Thus, we currently
favour a model where in most cells, if not in all, vacuolar
proteins arrive at unique PVCs and are then transferred to a
single vacuole type. However as shown in seeds, different
pathways may lead to this common destination, whether it
is the LV during early embryogenesis or the PSV at later
stages. Moreover, targeting to the single vacuole of vege-
tative tissues may also occur through independent path-
ways, similarly to the situation in yeast [39]. Indeed, the
vti12 and vps45 mutants are defective in trafficking of
storage proteins in vegetative tissues, while the trafficking
of other vacuolar cargo such as CPY, TTG or VPEg is not
altered [29,30�]. By contrast, the vti11 mutant and
AtSNX2b over expression plants show perturbed vacuolar
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targeting of an Aleurain-GFP lytic marker while storage
cargo appears not to be affected [18,30�]. It should be noted
that the blockage of protein trafficking in those mutants is
incomplete so it cannot be excluded that other vacuolar
cargo are less sensitive to weak alterations of transport (i.e.
they have higher affinity for sorting receptors and may
tolerate partial depletion at the TGN). To unequivocally
prove the presence of distinct pathways complete blockage
should be attained. Newly developed genetic screens for
storage vacuole trafficking genes [30�,40�] may allow us to
obtain such mutants, although complete inhibition may be
lethal to the plant. To circumvent this caveat, chemical
genomics (Figure 3) can be used to reversibly knockout
essential genes and test the effect in trafficking of the
different cargo.
Vacuole dynamicsCells must somehow dispose of the excess of vacuole
membranes that are delivered along with cargo. More-
over, under certain conditions or developmental stages,
vacuoles undergo major rearrangements in shape and size
that will require biogenesis or removing of tonoplast
membranes. In dividing shoot meristem cells the
vacuoles fragment and their total volume is reduced by
80% (the surface by 50%) from prometaphase to early
telophase [5]. The fragmentation may be necessary for
cell plate positioning/formation or for an even vacuole
distribution into the daughter cells, as occurs in yeast.
Current Opinion in Plant Biology 2009, 12:677–684
682 Cell Biology
Dramatic changes in vacuole size and morphology are also
observed during opening and closure of stomata [41] or
during elicitor-induced cell death [42]. In closing stomata,
the plasma membrane surface decreases concomitantly to
an increase in tonoplast surface, which becomes highly
convoluted with intravacuolar structures [43]. The new
tonoplast membrane could originate from the endocy-
tosed plasma membrane that would travel through endo-
somes to be transferred to the vacuole. But how is the
reciprocal loss of tonoplast membrane during stomata
opening achieved? The SNARE protein SYP22/VAM3
has been implicated in the disappearance of intravacuolar
structures during stomatal opening [41]. Gao et al. inter-
preted that the intravacuolar structures were fragmented
vacuoles and they proposed that SYP22 was involved in
their homotypic fusion. However, this would not explain
how tonoplast membrane is lost during stomata opening.
Moreover, using a tonoplast marker, Tanaka et al. showed
that the intravacuolar structures remained connected to
the limiting tonoplast. It is possible that these structures
are pinched off during stomatal opening and degraded in
the vacuole lumen in the same way inner vesicles from
the multivesicular PVC are transported into the vacuole
for degradation. Alternatively, there may be a mechanism
to link directly removal of tonoplast and gain of plasma
membrane, such as direct fusion of vacuoles to the plasma
membrane.
ConclusionsResearch in intracellular trafficking in plants is gaining
momentum. Markers for different pathways and compart-
ments of the endomembrane system are now available,
and genetic dissection of biosynthetic, endocytic and
autophagic pathways is underway. We can now study
these pathways in much greater detail and start to define
a mechanistic map of vesicle trafficking in plant cells. As
we complete this chart, we can start asking new questions
such as how these different pathways are organized and
connected in a given cell, and how they are regulated in
response to developmental or environmental cues. This
will help us to understand the contribution that vacuoles
make to plant growth and adaptation, which considering
the architecture of plant cells is bound to be large.
AcknowledgementsThis work was supported by a grant from the Spanish Ministerio deEducacion y Ciencia (BIO2006-11150 to ER), and by a postdoctoral I3Pfellowship to JZ from the Consejo Superior de Investigaciones Cientificas-CSIC.
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10. Frigerio L, Hinz G, Robinson DG: Multiple vacuoles in plant cells:rule or exception? Traffic 2008, 9:1564-1570.
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12. Otegui MS, Capp R, Staehelin LA: Developing seeds ofArabidopsis store different minerals in two types of vacuolesand in the endoplasmic reticulum. Plant Cell 2002, 14:1311-1327.
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Ebine K, Okatani Y, Uemura T, Goh T, Shoda K, Niihama M,Morita MT, Spitzer C, Otegui MS, Nakano A et al.: A SNAREcomplex unique to seed plants is required for protein storagevacuole biogenesis and seed development of Arabidopsisthaliana. Plant Cell 2008, 20:3006-3021.
The authors define a complete SNARE complex that may be required formembrane fusion between the PVC and the tonoplast. They demonstratephysical and genetic interaction between VAMP727 and SYP22 in med-iating trafficking of storage cargo to the vacuole and in plant develop-ment.
14. Uemura T, Ueda T, Ohniwa RL, Nakano A, Takeyasu K, Sato MH:Systematic analysis of SNARE molecules in Arabidopsis:dissection of the post-Golgi network in plant cells. Cell StructFunct 2004, 29:49-65.
15. Shimada T, Koumoto Y, Li L, Yamazaki M, Kondo M, Nishimura M,Hara-Nishimura I: AtVPS29, a putative component of a retromercomplex, is required for the efficient sorting of seed storageproteins. Plant Cell Physiol 2006, 47:1187-1194.
16. Yamazaki M, Shimada T, Takahashi H, Tamura K, Kondo M,Nishimura M, Hara-Nishimura I: Arabidopsis VPS35, a retromercomponent, is required for vacuolar protein sorting andinvolved in plant growth and leaf senescence. Plant Cell Physiol2008, 49:142-156.
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Plant vacuoles: where did they come from and where are they heading? Zouhar and Rojo 683
17. Kleine-Vehn J, Leitner J, Zwiewka M, Sauer M, Abas L, Luschnig C,Friml J: Differential degradation of PIN2 auxin efflux carrier byretromer-dependent vacuolar targeting. Proc Natl Acad Sci U SA 2008, 105:17812-17817.
18. Phan NQ, Kim S-J, Bassham DC: Overexpression ofArabidopsis sorting nexin AtSNX2b inhibits endocytictrafficking to the vacuole. Mol Plant 2008, 1:961-976.
19. Jaillais Y, Santambrogio M, Rozier F, Fobis-Loisy I, Miege C,Gaude T: The retromer protein VPS29 links cell polarity andorgan initiation in plants. Cell 2007, 130:1057-1070.
20. Bassham DC: Plant autophagy—more than a starvationresponse. Curr Opin Plant Biol 2007, 10:587-593.
21. Tamura K, Yamada K, Shimada T, Hara-Nishimura I: Endoplasmicreticulum-resident proteins are constitutively transported tovacuoles for degradation. Plant J 2004, 39:393-402.
22. Robinson DG, Oliviusson P, Hinz G: Protein sorting to thestorage vacuoles of plants: a critical appraisal. Traffic 2005,6:615-625.
23. Nishizawa K, Maruyama N, Utsumi S: The C-terminal region ofalpha’ subunit of soybean beta-conglycinin contains twotypes of vacuolar sorting determinants. Plant Mol Biol 2006,62:111-125.
24. Castelli S, Vitale A: The phaseolin vacuolar sorting signalpromotes transient, strong membrane association andaggregation of the bean storage protein in transgenictobacco. J Exp Bot 2005, 56:1379-1387.
25. von Lupke A, Schauermann G, Feussner I, Hinz G: Peripheralmembrane proteins mediate binding of vacuolar storageproteins to membranes of the secretory pathway ofdeveloping pea cotyledons. J Exp Bot 2008, 59:1327-1340.
26. Craddock CP, Hunter PR, Szakacs E, Hinz G, Robinson DG,Frigerio L: Lack of a vacuolar sorting receptor leads to non-specific missorting of soluble vacuolar proteins in Arabidopsisseeds. Traffic 2008, 9:408-416.
27. Shimada T, Fuji K, Tamura K, Kondo M, Nishimura M, Hara-Nishimura I: Vacuolar sorting receptor for seed storageproteins in Arabidopsis thaliana. Proc Natl Acad Sci U S A 2003,100:16095-16100.
28. Oliviusson P, Heinzerling O, Hillmer S, Hinz G, Tse YC, Jiang L,Robinson DG: Plant retromer, localized to the prevacuolarcompartment and microvesicles in Arabidopsis, may interactwith vacuolar sorting receptors. Plant Cell 2006, 18:1239-1252.
29. Zouhar J, Rojo E, Bassham DC: AtVPS45 is a positive regulatorof the SYP41/SYP61/VTI12 SNARE complex involved intrafficking of vacuolar cargo. Plant Physiol 2009, 149:1668-1678.
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Sanmartin M, Ordonez A, Sohn EJ, Robert S, Sanchez-Serrano JJ,Surpin MA, Raikhel NV, Rojo E: Divergent functions of VTI12 andVTI11 in trafficking to storage and lytic vacuoles inArabidopsis. Proc Natl Acad Sci U S A 2007, 104:3645-3650.
The authors report a novel screen for mutants that secrete the vacuolarstorage cargo VAC2. VAC2 is a fusion of CLV3 to the vacuolar sortingdeterminants of barley lectin. Secretion of VAC2 in the trafficking mutantsleads to early termination of shoot and floral meristems, a phenotypeeasily detected. They then show that the SNARE VTI12 is required fortransport of VAC2 as well as of other storage cargo, while VTI11 may berequired for transport of lytic cargo.
31. Park M, Lee D, Lee GJ, Hwang I: AtRMR1 functions as a cargoreceptor for protein trafficking to the protein storage vacuole.J Cell Biol 2005, 170:757-767.
32. Park JH, Oufattole M, Rogers JC: Golgi-mediated vacuolarsorting in plant cells: RMR proteins are sorting receptors forthe protein aggregation/membrane internalization pathway.Plant Sci 2007, 172:728.
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Hinz G, Colanesi S, Hillmer S, Rogers JC, Robinson DG:Localization of vacuolar transport receptors and cargoproteins in the Golgi apparatus of developing Arabidopsisembryos. Traffic 2007, 8:1452-1464.
This immunoelectron microscopy study of the localization of putativereceptors and cargo in Arabidopsis seeds and the study of Otegui and co-
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workers [35�] complement the genetic data obtained in this model plant.The authors show that storage cargo first segregates to the rims of thecisternae at the cis side of the Golgi, similarly to what they had reported inpea seeds. Importantly, RMRs but not with VSRs are found at the rims ofthe cisternae at the cis side, indicating that this initial segregation of cargomay be dependent on RMRs but not on VSRs.
34. Hinz G, Hillmer S, Baumer M, Hohl II: Vacuolar storage proteinsand the putative vacuolar sorting receptor BP-80 exit the golgiapparatus of developing pea cotyledons in different transportvesicles. Plant Cell 1999, 11:1509-1524.
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Otegui MS, Herder R, Schulze J, Jung R, Staehelin LA: Theproteolytic processing of seed storage proteins inArabidopsis embryo cells starts in the multivesicular bodies.Plant Cell 2006, 18:2567-2581.
In this study, double immunolabelling was used to analyze the localizationof different types of cargo and of VSRs in Arabidopsis seeds. The authorsdemonstrate that VSRs are present in dense vesicles together withstorage cargo, which supports a role of VSRs as receptors for storageproteins. Moreover, they show that storage cargo and processing pro-teases are first separated in the Golgi but then converge in the PVC,where processing starts.
36. Miao Y, Li KY, Li HY, Yao X, Jiang L: The vacuolar transport ofaleurain-GFP and 2S albumin-GFP fusions is mediated by thesame pre-vacuolar compartments in tobacco BY-2 andArabidopsis suspension cultured cells. Plant J 2008, 56:824-839.
37. Park M, Kim SJ, Vitale A, Hwang I: Identification of the proteinstorage vacuole and protein targeting to the vacuole in leafcells of three plant species. Plant Physiol 2004, 134:625-639.
38. Hunter PR, Craddock CP, Di Benedetto S, Roberts LM, Frigerio L:Fluorescent reporter proteins for the tonoplast and thevacuolar lumen identify a single vacuolar compartment inArabidopsis cells. Plant Physiol 2007, 145:1371-1382.
39. Bowers K, Stevens TH: Protein transport from the late Golgi tothe vacuole in the yeast Saccharomyces cerevisiae. BiochimBiophys Acta 2005, 1744:438-454.
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Fuji K, Shimada T, Takahashi H, Tamura K, Koumoto Y, Utsumi S,Nishizawa K, Maruyama N, Hara-Nishimura I: Arabidopsisvacuolar sorting mutants (green fluorescent seed) can beidentified efficiently by secretion of vacuole-targeted greenfluorescent protein in their seeds. Plant Cell 2007, 19:597-609.
The authors report an efficient screen for mutants that secrete in seeds astorage vacuole marker, SP-GFP-CT24, which consists on GFP with asignal peptide fused to the vacuolar sorting determinant from b-conglyci-nin. Secretion of SP-GFP-CT24 stabilizes the protein and the mutant seedsare highly fluorescent. Through this screen the authors have found novelalleles of VSR1 and a membrane-associated protein of unknown function.
41. Gao XQ, Li CG, Wei PC, Zhang XY, Chen J, Wang XC: Thedynamic changes of tonoplasts in guard cells are importantfor stomatal movement in Vicia faba. Plant Physiol 2005,139:1207-1216.
42. Higaki T, Goh T, Hayashi T, Kutsuna N, Kadota Y, Hasezawa S,Sano T, Kuchitsu K: Elicitor-induced cytoskeletalrearrangement relates to vacuolar dynamics and execution ofcell death: in vivo imaging of hypersensitive cell death intobacco BY-2 cells. Plant Cell Physiol 2007, 48:1414-1425.
43. Tanaka Y, Kutsuna N, Kanazawa Y, Kondo N, Hasezawa S, Sano T:Intra-vacuolar reserves of membranes during stomatalclosure: the possible role of guard cell vacuoles estimated by3-D reconstruction. Plant Cell Physiol 2007, 48:1159-1169.
44. Olbrich A, Hillmer S, Hinz G, Oliviusson P, Robinson DG: Newlyformed vacuoles in root meristems of barley and peaseedlings have characteristics of both protein storage andlytic vacuoles. Plant Physiol 2007, 145:1383-1394.
45. Otegui MS, Noh YS, Martinez DE, Vila Petroff MG, Staehelin LA,Amasino RM, Guiamet JJ: Senescence-associated vacuoleswith intense proteolytic activity develop in leaves ofArabidopsis and soybean. Plant J 2005, 41:831-844.
46. Moriyasu Y, Hattori M, Jauh GY, Rogers JC: Alpha tonoplastintrinsic protein is specifically associated with vacuolemembrane involved in an autophagic process. Plant CellPhysiol 2003, 44:795-802.
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47. Brady SM, Orlando DA, Lee JY, Wang JY, Koch J, Dinneny JR,Mace D, Ohler U, Benfey PN: A high-resolution rootspatiotemporal map reveals dominant expression patterns.Science 2007, 318:801-806.
48. Cao X, Rogers SW, Butler J, Beevers L, Rogers JC: Structuralrequirements for ligand binding by a probable plant vacuolarsorting receptor. Plant Cell 2000, 12:493-506.
49. Happel N, Honing S, Neuhaus JM, Paris N, Robinson DG,Holstein SE: Arabidopsis mu A-adaptin interacts with thetyrosine motif of the vacuolar sorting receptor VSR-PS1. PlantJ 2004, 37:678-693.
50. Bocock JP, Carmicle S, Chhotani S, Ruffolo MR, Chu H,Erickson AH: The PA-TM-RING protein RING finger protein 13 isan endosomal integral membrane E3 ubiquitin ligase whoseRING finger domain is released to the cytoplasm byproteolysis. FEBS J 2009, 276:1860-1877.
51. Surpin M, Rojas-Pierce M, Carter C, Hicks GR, Vasquez J,Raikhel NV: The power of chemical genomics to study the linkbetween endomembrane system components and thegravitropic response. Proc Natl Acad Sci U S A 2005,102:4902-4907.
Current Opinion in Plant Biology 2009, 12:677–684
52. Zouhar J, Hicks GR, Raikhel NV: Sorting inhibitors (Sortins):chemical compounds to study vacuolar sorting inArabidopsis. Proc Natl Acad Sci U S A 2004,101:9497-9501.
53. Robert S, Chary SN, Drakakaki G, Li S, Yang Z, Raikhel NV,Hicks GR: Endosidin1 defines a compartment involved inendocytosis of the brassinosteroid receptor BRI1 and theauxin transporters PIN2 and AUX1. Proc Natl Acad Sci U S A2008, 105:8464-8469.
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Rojas-Pierce M, Titapiwatanakun B, Sohn EJ, Fang F, Larive CK,Blakeslee J, Cheng Y, Cutler SR, Peer WA, Murphy AS et al.:Arabidopsis P-glycoprotein19 participates in the inhibitionof gravitropism by gravacin. Chem Biol 2007,14:1366-1376.
This work illustrates an importance of Arabidopsis as a model organism inboth classical and chemical genetics. Using a mutant collection, an auxintransporter was identified as a target of a potent gravitropism inhibitor.
55. Norambuena L, Zouhar J, Hicks GR, Raikhel NV: Identification ofcellular pathways affected by Sortin2, a synthetic compoundthat affects protein targeting to the vacuole in Saccharomycescerevisiae. BMC Chem Biol 2008, 8:1.
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