Plant Cell Vacuoles
Secondary article
Article Contents
Jean-Marc Neuhaus, University of Neuchâtel, Neuchâtel, Switzerland
Enrico Martinoia, University of Neuchâtel, Neuchâtel, Switzerland
. Introduction
. Vacuolar Constituents
Most plant cells contain one or several vacuoles, which may occupy up to 95% of the
cellular space. The vacuoles often appear empty under a light microscope (hence their
name), except when they contain pigments or precipitated substances. The vacuole is
delimited from the cytosol by the vacuolar membrane, which is also called tonoplast.
Introduction
The vacuole is the largest compartment of a mature plant
cell and may occupy up to 95% of the total cell volume. In
such mature cells, the cytosol is visible only as a thin layer,
which is separated from the cell wall by the plasma
membrane, and from the vacuolar sap (cell sap) by the
vacuolar membrane (tonoplast). The constituents of the
cell sap are mainly inorganic salts and water. The vacuole,
therefore, enables the plant to reach a large size and a large
surface area by accumulating salts from the environment,
which drive further water uptake osmotically. Thus only a
minimum of energy is diverted from the energy-consuming
synthesis of metabolites. The requirement for cytosolic
ions to be in homeostasis is also met by the vacuole as it
serves as an internal reservoir of metabolites and nutrients
(Matile, 1978; Boller and Wiemken, 1986; Martinoia,
1992). It can also be used for storage of other compounds
that would be problematic in the cytosol, such as ions,
pigments, storage compounds or toxic substances.
Vacuolar Constituents
Vacuolar constituents may vary between and within plant
species, depending on the environmental conditions.
Primary metabolites such as carbohydrates, amino acids
and organic acids, as well as inorganic ions such as
chloride, nitrate, potassium and sodium, are among the
solutes usually present in the cell sap. Most of these solutes
are stored only temporarily in the vacuole. In contrast,
many compounds of secondary plant metabolism are
thought to be sequestered definitively within the vacuole.
Since many of these compounds are toxic and often play a
role in plant defence, detoxification of the cytosol and
storage of weapons against predators can be regarded as
further functions of the vacuole. However, recent results
indicate that even these compounds may be at least
partially recycled. A third class of substances found within
the vacuole are man-made chemicals (xenobiotics) that
have been modified by the plant to cope with these
potentially toxic compounds.
. Plant Cells Can Possess More Than One Type of Vacuole
. Vacuole Biogenesis
. Transport Processes across the Vacuolar Membrane
In most cases, the enzymatic activities found in the
vacuole are restricted to hydrolases, which are mainly
localized in this compartment (Matile, 1978; Boller and
Wiemken, 1986). It has therefore been suggested that the
vacuole corresponds to the lysosomes of animal cells.
Additionally, several transferases are present in the
vacuole. The best investigated so far are involved in
fructan synthesis. Specialized vacuoles (protein bodies)
from grains accumulate storage proteins as nitrogen source
for the seedlings as well as phytate as phosphate store.
Plant Cells Can Possess More Than One
Type of Vacuole
Plant biochemists have been puzzled by the large number
of compounds found in vacuoles, some of which seemed
incompatible. This can be explained by the existence of
different vacuoles with different functions, such as lytic or
storage compartments. In certain specialized cells different
types of vacuoles can be distinguished optically because
only one of the vacuoles contains a pigment or a
precipitated substance, as in the cells of Mimosa leaves.
Sometimes the difference is only visible in the electron
microscope (Figure 1). It has recently become possible to
label two different vacuolar compartments of single cells
with antibodies recognizing two different tonoplast intrinsic proteins (TIP) or two different soluble vacuolar
proteins (Paris et al., 1996). In many cells however,
proteins of different types of vacuoles can also be found
in the same large vacuole. This could be due to fusion of
different types of vacuoles.
Vacuole Biogenesis
Origin of vacuoles
Vacuoles are part of the secretory system of plants, and are
ultimately derived from the endoplasmic reticulum (ER),
as are the nuclear envelope, the Golgi apparatus and the
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1
Plant Cell Vacuoles
Transport of soluble proteins to the vacuoles
Soluble proteins are transported to preexisting vacuoles
via the Golgi apparatus, as indicated by the typical
modifications of glycans. Vesicles containing vacuolar
proteins have also been visualized in the process of budding
from the Golgi apparatus. Vacuolar proteins harbour
positive sorting information that diverts them from the
default pathway for soluble secretory proteins, which
would otherwise result in secretion into the extracellular
space.
Vacuolar sorting signals
Figure 1 Electron microscopic view of apple leaf cells possessing two
different types of vacuoles (V1 containing a network of thick strands and a
bright fringe, and V2). The other labelled structures are cell wall (CW),
plasma membrane (PM), nucleus (N) with nucleolus (Nu), nuclear
envelope (NE) with pores (Po), multivesicular body (MB) and dictyosomes
(D). This figure was reprinted from Michel M, Gnägi H and Müller M (1992)
Diamonds are a cryosectioner’s best friend. Journal of Microscopy 166: 43–
56, with permission of the Royal Microscopical Society.
plasma membrane. Protein storage bodies of various
cereals can be seen by electron microscopy to bud directly
from the ER as storage proteins aggregate (Galili et al.,
1993). In legumes, storage vacuoles are formed near
preexisting lytic vacuoles, deriving apparently from a
tubular-cisternal membrane system. In some meristematic
cells, lytic vacuoles also seem to form by fusion and
enlargement of ER or ER-derived tubules. Vacuoles can
fuse to a single huge central vacuole during tissue
differentiation, but they are also able to form smaller
vacuoles by fission or blebbing. Fusion of different types of
vacuoles can also occur, mixing storage and digestive
proteins.
2
Analysis of several vacuolar proteins indicates that they are
often synthesized as larger precursors with N- or Cterminal propeptides. Truncation of these propeptides
results in secretion. Addition of the propeptides to different
secreted proteins results in their vacuolar localization.
These propeptides thus contain vacuolar sorting signals
(VSS). Other vacuolar proteins appear to contain internal
VSS.
Comparison of sequences revealed that N-terminal
propeptides of aleurain and sporamin contained a
conserved motif NPIR (one-letter code for amino acids),
and mutation analysis indicated that the I was the most
important determinant, but the neighbouring positions
were also involved, as not every substitution was tolerated.
This sequence-specific VSS (ssVSS) is not necessarily
located in an N-terminal propeptide but also functions
when added to the C terminus of a reporter protein. This
means that an ssVSS might also be located within a surface
loop of a vacuolar protein, where it would be much more
difficult to identify. Proteins with ssVSS appear to be
targeted to lytic vacuoles.
A putative vacuolar sorting receptor (VSR) for this first
type of VSS was purified and cloned. Binding of the VSR
was pH-dependent and showed a similar sequence
specificity as found by the mutation analysis. Using
VSR-binding as a test, a further ssVSS was tentatively
identified in the C-terminal region of the 2S albumin from
Brazil nuts. Analysis of peptides derived from the pumpkin
2S albumin identified two motifs, one of which contained a
motif NLPS that is conserved in this protein family and
also found in the N-terminal propeptide of several proteins
related to sporamin.
A second type of VSS was found in C-terminal
propeptides of cereal lectins and of chitinases. Mutation
analysis indicated little sequence specificity for these Cterminal VSS (ctVSS), except for the accessibility from the
C-terminus. The terminus could be blocked by the addition
of an N-glycosylation site or by glycines. Comparison of
many C-terminal propeptides indicates the preference for a
long side-chain at the terminal position and for a negative
charge in one of the preceding positions. No sorting
receptor has yet been identified for these ctVSS. Wortmannin, an inhibitor of phosphatidylinositol kinases,
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Plant Cell Vacuoles
γ
α
γ
PVC
α
α
SDV
2
γ
γ
PV
γ
δ
δ
δ
δ
δ
Golgi
Delta
vacuole
δ
Default
ER
3
δ
γ
?
γ
LV
CCV
Transport of tonoplast proteins to the
vacuoles
Much less is known about the pathway of tonoplast
proteins from the ER to the vacuole. The most abundant
tonoplast proteins are the tonoplast intrinsic proteins
(TIPs or aquaporins). Since different TIPs are localized in
different vacuolar compartments, they have to be sorted by
different mechanisms. The C-terminal transmembrane
segment of a-TIP was sufficient to target a reporter protein
to a tonoplast, with or without its cytosolic tail. Thus either
this segment contains a tonoplast sorting signal or a
tonoplast (which type of vacuole?) is the default destination for membrane proteins, as suggested for yeast. The Cterminal cytosolic tail of a-TIP actually prevented movement of a reporter protein from the ER to the Golgi, but
α
α
α
α
1
γ
2
γ
α
PSV
δ
Electron microscopic analysis has detected the formation
of two different types of vesicles containing vacuolar
proteins from the Golgi: clathrin-coated vesicles (CCV)
and smooth dense vesicles (SDV). Clathrin-coated vesicles
are well known from animal and yeast cells to participate in
receptor-mediated transport, either from the trans-Golgi
or from the cell surface to the endosomal compartment, an
intermediate on the way to the lysosomes. The putative
VSR for ssVSS was indeed isolated from pea CCVs.
Smooth dense vesicles of pea cotyledons, however, were
shown by immunocytochemistry to contain storage
proteins in an aggregated form. The transport vehicle for
vacuolar proteins with a ctVSS is unknown.
Both transport pathways appear to involve prevacuolar
compartments. Smooth dense vesicles carry storage
proteins to multivesicular bodies, a kind of vacuole with
numerous internal vesicles similar to endosomes of animal
cells. For the lytic pathway, antibodies against the VSR
detected both the Golgi and prevacuoles (approximately
250 nm) clustered near large vacuoles. These prevacuoles
are likely to be the acidified compartment where the VSR
releases its ligand and from which it recycles back to the
Golgi.
α
Vesicles transporting proteins to vacuoles
not to a vacuole. Finally, vacuolar sorting of soluble and
membrane proteins was found to be differentially sensitive
to inhibitors (monensin, brefeldin A), a further indication
of different sorting pathways.
The abundance of TIPs in tonoplasts beyond the
presumable need for aquaporins (30–50% of total
tonoplast proteins) even suggests a structural role. TIPs
could be involved in the biogenesis of new vacuoles from
the ER and could later function as identity markers for
each type of vacuole. The plant cell could then prevent or
allow the fusion of vacuoles of a given type with each other
or with vacuoles of a different type and also with Golgiderived protein-containing vesicles. This model reconciles
the vacuole biogenesis directly from the ER with the traffic
of soluble proteins via the Golgi (Figure 2).
α
caused secretion of this type of proteins, at a concentration
with no effect on the ssVSS-harbouring proteins, at least in
tobacco BY-2 cells. Proteins with ctVSS appear to be
targeted to nonlytic or storage vacuoles.
The third type of VSS is protein-structure-dependent
(psVSS) and has been found in several seed proteins. These
tend to aggregate, once modifications in the Golgi
apparatus have made them more hydrophobic. Aggregation is a possible nonreceptor-mediated sorting mechanism
known to occur in specialized animal cells (secretory
granules). Aggregation occurring in the ER seems to cause
the formation of protein bodies in cereals. The effect of
wortmannin on this transport system is unknown.
δ
CW
Figure 2 Vacuole biogenesis in plants. Compartments of the secretory
pathway are indicated: endoplasmic reticulum (ER); Golgi; cell wall (CW);
and three types of vacuoles: protein storage vacuole (PSV, a-TIP, blue), lytic
vacuole (LV, g-TIP, red) and ‘d vacuole’ (d-TIP, violet). The transport
pathway from ER to Golgi involves concentration of proteins (grey
spheres). Storage proteins (blue) aggregate in the Golgi near the rim of the
cisternae and leave the Golgi in smooth dense vesicles (SDV) to a
prevacuolar compartment (PVC) that fuses with the PSV. Clathrin-coated
vesicles carry soluble proteins (red) selected by a receptor protein to a
prevacuole (PV) from which the receptor recycles to the Golgi, while the
cargo proteins continue to the LV. The pathway to ‘d vacuoles’ is unknown.
The proteins lacking specific sorting determinants (green) are transported
by default to the plasma membrane. Possible additional pathways are
indicated: (1) direct transport of a-TIP from ER to PSV; (2) fusion of PSV and
LV; and (3) exchanges or fusion between LV and ‘d vacuole’. Picture drawn
by John Rogers.
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Plant Cell Vacuoles
Transport Processes across the Vacuolar Channels and carriers
Membrane
Plant vacuoles may exercise different roles, depending on
Vacuolar proton pumps
Transport processes across membranes depend in many
cases on the proton motive force (DpH and DC) generated
by pumps residing in these membranes (Figure 3). The
tonoplast contains two different proton pumps, an ATPase
and a PPase (Rea and Sanders, 1987). V-type ATPases (VATPases) are multimeric complexes present on the
endomembrane system of eukaryotic cells (Sze et al.,
1992). They show some homologies with the F-ATPases
present on the plasma membrane of eubacteria and on the
inner membranes of mitochondria and chloroplasts and
also form a characteristic ‘head and stalk’ structure. The VATPases can be distinguished from other ATPases by their
highly specific inhibition by some antibiotics such as
bafilomycin. V-ATPases are also found on other plant
endomembranes such as the Golgi apparatus or ER, but it
is still uncertain whether they are functional. The tonoplast
proton-pumping pyrophosphatase (H+-PPase) is found
widely distributed in higher plants as well as in algae,
liverworts, mosses and ferns. Homologous proteins have
been found in some bacteria, but so far not in animals or
fungi. The tonoplast H 1 -PPase is a monomeric enzyme of
about 87 kDa which is strictly dependent on Mg2 1 and
activated by K 1 (Rea and Poole, 1993).
GS-X
ATP
Cl–/NO3–?
Mal2–
K+/Ca2+
ADP+Pi
PPi
K+
H+
Calmodulin
Pi+Pi
Ca2+
cADPR
Ca2+
IP3
∆pH, ∆Ψ
ATP
Ca2+
H+
Na+
H+
ADP+Pi
H+
H 2O
Amino acids
Glucose Sucrose
Figure 3 Vacuolar transport systems. Pumps (red), transporters (green)
and channels (blue) of the vacuolar membrane. For malate it is unclear
whether a transporter or a channel is involved. Some channels are
regulated by calmodulin, cyclic ADP-ribose (cADPR) or inositol
trisphosphate (IP3), as indicated. GS-X, glutathion-conjugated
xenobiotics. The picture is not exhaustive; for more details see text and the
literature cited.
4
the nutritional state of a plant or whether a tissue is part of
a source organ such as a leaf, or of a storage organ like a
tuber. Therefore transport mechanisms may differ for
different types of vacuoles. A case where different vacuolar
transport mechanisms are described in different plants is
sucrose, which is accumulated within sugarbeet tuber
vacuoles by an H 1 -antiport mechanism, while facilitated
diffusion is observed in barley, tomato and in stalk tissue of
sugar cane. In barley and other fructan-accumulating
species, sucrose is readily metabolized to higher polymers
within the vacuole and trapped in this compartment. In this
case, the energy for carbohydrate accumulation derives
from the glycosidic bond between glucose and fructose and
is not due to the vacuolar proton pumps. Similar
differences as for sucrose may exist for glucose. In contrast
to the members of the fructan family, which are not
transported through the tonoplast, some members of the
raffinose family are taken up by an H 1 -antiport mechanism in plants accumulating these sugars.
Amino acid concentrations are often lower in the
vacuole than in the cytosol. A transporter modulated by
free ATP catalyses the transfer of most amino acids. A
similar transport system has been described for the
exchange of peptides, polyamines, cations and inorganic
anions. Since the free ATP level is low in the cytosol and
therefore only a very low transport activity of this
transport system can be observed under physiological
conditions, it has been suggested that it may play a role
either in cytosolic homeostasis or as an H 1 -shunt
mechanism.
Inorganic and organic anion uptake is driven by the DC.
Malate and citrate are the main organic acids accumulated
in large quantities within the vacuole. Inhibition experiments suggest that one carrier or channel is responsible for
the uptake of most di- and tricarboxylates. Patch clamp
studies indicate that a separate channel is involved in the
export of organic acids. Furthermore, inorganic anions
such as chloride and nitrate most probably use a different
transport system. In the case of nitrate it must be
postulated that an additional force is driving its vacuolar
uptake, since the observed membrane potential difference
would not be sufficient to drive the concentration
difference observed between the cytosol and the vacuole.
Chloride channels are apparently activated by a protein
kinase in stomata vacuoles.
Several cation-permeable channels have been observed
in the tonoplast. The SV (slow vacuolar) channel opens
mainly at cytosolic positive membrane potentials, is
permeable for potassium and calcium, and is activated in
the presence of Ca2 1 . The SV channels are inhibited by
calmodulin antagonists, indicating the involvement of
calmodulin. In contrast, the FV (fast vacuolar) channel has
a higher open probability at cytosolic negative membrane
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Plant Cell Vacuoles
potentials and is activated by low cytosolic Ca2 1
concentrations. Additionally, in stomata cells a K 1
channel thought to have a role in facilitating vacuolar
K 1 release is described. For different plants a Na 1 /H 1
exchange mechanism, which may play a crucial role in salt
tolerance, has been demonstrated, while a K 1 /H 1
antiport also seems to exist in some cases but has not been
described in detail.
Three different calcium channels have been reported to
reside on the vacuolar membrane: an IP3-dependent
channel; a cyclic ADP-ribose (cADPR)-dependent channel; and a Ca2 1 channel (see Figure 3). The IP3-dependent
channel allows the release of calcium and the cADPRdependent channel is also responsible for the release of
Ca2 1 from the vacuole. For both the IP3- and the cADPRdependent Ca2 1 channels, however, the spectrum of
physiological stimuli to which the vacuolar receptors
respond has yet to be elucidated. The Ca 2 1 channel is
activated by membrane hyperpolarization. This channel is
potently inhibited by La3 1 and Gd3 1 . Vacuolar calcium
uptake is mediated by a Ca2 1 /H 1 exchange mechanism
and by a Ca2 1 -ATPase.
The vacuole as detoxification compartment:
the role of ABC transporters
A classical function of the central vacuole is the role as
storage compartment for potentially toxic metabolites,
such as phenolics or alkaloids, which may serve as
repellents for herbivores or which are toxic for microorganisms. Beside such plant-generated substances, potentially toxic chemicals or pesticides (xenobiotics) can be
modified by the plant and stored within the vacuole. An
efficient detoxification requires transport mechanisms that
strongly accumulate the potentially toxic compound
within the vacuole. In several cases, as for coumarylglucosides or flavonoids, it has been shown that proton antiport
mechanisms drive the vacuolar accumulation. Furthermore, trapping mechanisms such as protonation or
conformational changes have been observed to be involved
in vacuolar accumulation of secondary compounds.
Metabolism of xenobiotics to glucosides or to glutathione conjugates is usually considered as a detoxification process, but these products may exert other biological
activities. Removal of such conjugates from the cytosol
could thus represent a crucial process in xenobiotic
detoxification. Vacuolar transport of glutathione conjugates is not energized by the vacuolar proton gradient but
directly by ATP. The uptake is inhibited by vanadate and
other nucleotides can partially substitute for ATP. Very
similar kinetics have been described for animal glutathione
conjugate transporters. On the molecular level, it has been
shown that several genes can mediate glutathione conjugate transport in plants. These transporters belong to the
ABC (ATP-binding cassette) family and are highly
homologous to glutathione conjugate transporters of fungi
and animals (Rea et al., 1998). It is interesting to note that
in animals glutathione conjugates are transported at the
plasma membrane and are excreted in the surrounding
medium. In contrast to the plant-generated glucosides,
pesticides conjugated to glucose may also be transported
by a directly energized transporter, probably also of the
ABC type. Direct energization by ATP allows the creation
of a much higher gradient between the cytosol and the
vacuole and it is therefore supposed that the plant uses this
type of transporter where the potential toxicity of a
product is high. The degradation products of chlorophyll,
open-chain tetrapyrroles that are still able to absorb light,
accumulate within the vacuole by the same transporters
responsible for glutathione conjugate transport. Upon
illumination, these degradation products produce very
dangerous radicals that would be lethal in a metabolically
active compartment.
Aquaporins
Owing to large size, the central vacuole of plants plays an
important role as water reserve and hence in water stress.
Water fluxes across biological membranes were long
thought to occur only through the lipid bilayer. However,
it was recently shown that in many cases special proteins,
known as aquaporins, mediate the exchange of water
between the extracellular medium and the cell as well as
within the cell. For vacuoles different types of aquaporins
have been described, the best known being the a- and the gTIPs (tonoplast intrinsic proteins). g-TIPs can be found
mainly in the large central vacuole and are strongly
expressed in young, expanding tissue as well as in vacuoles
of moving cells like Mimosa pulvini. a-TIPs are preferentially present in storage vacuoles where they are probably
involved in dehydration. Interestingly, water permeability
of a-TIPs, but not g-TIPs, is induced by phosphorylation.
References
Boller T and Wiemken A (1986) Dynamics of vacuolar compartmentation. Annual Review of Plant Physiology 37: 137–164.
Galili G, Altschuler Y and Levanony H (1993) Assembly and transport
of seed storage proteins. Trends in Cell Biology 3: 437–442.
Martinoia E (1992) Transport of solutes in vacuoles of higher plants.
Botanica Acta 105: 232–245.
Matile P (1978) Biochemistry and function of vacuoles. Annual Review of
Plant Physiology 29: 193–213.
Paris N, Stanley CM, Jones RL and Rogers JC (1996) Plant cells contain
two functionally distinct vacuolar compartments. Cell 85: 563–572.
Rea PA, Li Z-S, Lu YP, Drozdowicz YM and Martinoia E (1998) From
vacuolar GS-X pumps to multispecific ABC transporters. Annual
Review of Plant Physiology and Plant Molecular Biology 49: 727–760.
Rea PA and Poole RJ (1993) Vacuolar H 1 -translocating pyrophosphatase. Annual Review of Plant Physiology and Plant Molecular
Biology 44: 157–180.
Rea P and Sanders D (1987) Tonoplast energization: two H 1 pumps,
one membrane. Physiologia Plantarum 71: 131–141.
ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net
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Plant Cell Vacuoles
Sze H, Ward JM and Lai S (1992) Vacuolar H 1 -translocating ATPases
from plants: structure, function and isoforms. Journal of Bioenergetics
and Biomembranes 24: 371–381.
Further Reading
Leigh RA and Sanders D (1997) The plant vacuole. In: Callow JA (ed.)
Advances in Botanical Research, vol.25. San Diego, CA: Academic
Press
6
Kreuz K, Tommasini R and Martinoia E (1996) Old enzymes for a new
job – herbicide detoxification in plants. Plant Physiology 111: 349–
353.
Maurel C (1997) Aquaporins and water permeability of plant
membranes. Annual Review of Plant Physiology and Plant Molecular
Biology 48: 399–429.
Neuhaus J-M and Rogers JC (1998) Sorting of proteins to vacuoles in
plant cells. Plant Molecular Biology 38: 127–144.
Robinson DG and 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.
ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net