plant vacuoles: where did they come from and where are they heading?

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

www.sciencedirect.com

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].


mailto:[email protected]

http://dx.doi.org/10.1016/j.pbi.2009.08.004

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


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].


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


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


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


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



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


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.


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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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.

13.�

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.



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.

30.�

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.

33.�

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-


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.

35.�

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.

40.�

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.


684 Cell Biology

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.


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.

54.�

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.


plant vacuoles: where did they come from and where are they heading?

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