Download The Metamorphosis of the Aleurone Protein Storage Vacuole

Annals of Botany 82 : 399–412, 1998
Article No. bo980702
REVIEW ARTICLE
From Storage Compartment to Lytic Organelle : The Metamorphosis of the
Aleurone Protein Storage Vacuole
P A U L C. B E T H KE*, S A R A H J. S W A N S O N*, S T E F A N H I L L M E R† and R U S S E L L L. J O N E S*‡
* Department of Plant and Microbial Biology, UniŠersity of California, Berkeley, CA 94720
and † Plant Physiology Institute, UniŠersity of GoX ttingen, D-37037 GoX ttingen, Germany
Received : 4 May 1998
Accepted : 25 May 1998
Protein storage vacuoles are found in a variety of tissues but are especially abundant in the storage organs of fruits
and seeds. In this review, we focus on the protein storage vacuoles of cereal aleurone. In the mature grain, these
organelles are repositories for reserve nitrogen, carbon and minerals. Following imbibition, protein storage vacuoles
of cereal aleurone change from storage compartments to lytic organelles. Changes in protein storage vacuole structure
and enzymatic activity during this transition are discussed. It is emphasized that protein storage vacuoles are poised
for reserve mobilization, and that gibberellin perception by the aleurone cell initiates a signalling cascade that
promotes acidification of the vacuole lumen and activation of enzymes and transporters.
# 1998 Annals of Botany Company
Key words : Protein storage vacuole, cereal aleurone, gibberellin, abscisic acid, protein body, endosperm reserves.
INTRODUCTION
Vacuoles are ubiquitous organelles in plant cells. They
perform myriad functions and take on a variety of forms
(Matile, 1975 ; Boller and Wiemken, 1986). In this review we
focus our attention on a specialized class of vacuole, the
protein storage vacuole. Protein storage vacuoles are found
in a wide variety of tissues, but they are especially abundant
in the storage organs of fruits and seeds (Chrispeels, 1985 ;
Muntz, 1989 ; Shewry, 1995). This organelle stores proteins,
carbohydrates, neutral lipids and minerals that are used to
support early growth of the seedling (Bewley and Black,
1994). Protein storage vacuoles are typically 1–5 µm in
diameter in mature seeds and grain, and a single plant cell
may accumulate hundreds of protein storage vacuoles.
Protein storage vacuoles have been intensively investigated in the cotyledons of legumes and in the endosperm of
cereals. Despite their functional similarities, differences in
protein storage vacuole structure and biogenesis exist
between species and within the same tissue of a given species
(Chrispeels, 1985 ; Muntz, 1989 ; Okita and Rogers, 1996 ;
Robinson and Hinz, 1997). In general, protein storage
vacuoles are formed either by budding from the endoplasmic
reticulum (ER), or by fragmentation of the central vacuole.
The former mechanism is common in the starchy endosperm
of cereals and the latter in the cotyledons of dicots, but
many exceptions exist. In the starchy endosperm of rice, for
example, two types of protein storage vacuole are formed,
one containing prolamines, the other glutelins. Rice prolamines are stored in vacuoles formed directly from the ER,
‡ For correspondence.
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but glutelins are stored in Golgi-derived vacuoles (Okita
and Rogers, 1996). In the starchy endosperm of wheat yet
another mechanism appears to exist for the formation of
protein storage vacuoles. Galili et al. (1996) have shown
that protein storage vacuoles can arise directly from ER or
by a process in which ER-derived vesicles containing storage
protein are incorporated into the protein storage vacuole by
autophagy.
A feature of all protein storage vacuoles is that stored
materials accumulate either as large polymers, which is the
case for stored carbon and nitrogen, or as complex salts,
which is the case for sequestered minerals (Bewley and
Black, 1994). Because of the economic and nutritional
importance of endosperm and cotyledon storage proteins,
much attention has been focused on their synthesis and
intracellular deposition within protein storage vacuoles
(Shewry, Napier and Tatham, 1995 ; Galili et al., 1996 ;
Richard et al., 1996). Much less attention has been focused
on the mobilization of these reserves. In cereals, mobilization
of protein storage vacuole reserves from dead starchy
endosperm cells is accomplished by enzymes secreted into
the starchy endosperm by the aleurone or scutellum, and
by enzymes released from starchy endosperm cells prior
to grain maturation (Fincher, 1989 ; Jones and Jacobsen,
1991), and this topic is not covered here. Mobilization of
stored reserves from living cells is unlike the mobilization of
starchy endosperm reserves and is preceded by a change in
the function of the protein storage vacuole. The protein
storage vacuole abandons its role as a storage compartment
and becomes an acidic, lytic organelle wherein stored
proteins, lipids, carbohydrates and phytin are degraded. In
aleurone cells, this change from storage compartment to
lytic organelle is hormonally regulated. The dramatic
# 1998 Annals of Botany Company
400
Bethke et al.—Protein Storage Vacuoles in Cereal Aleurone
changes in structure and enzymatic activity that occur in
protein storage vacuoles of barley aleurone during the phase
of reserve mobilization are the topics of this review.
MATURE ALEURONE PROTEIN STORAGE
VACUOLES ARE POISED FOR RESERVE
MOBILIZATION
The cereal aleurone cell is an excellent model for studying
the action of the plant growth regulators gibberellic acid
(GA) and abscisic acid (ABA), but more recent investigations have focused on how these hormones regulate gene
expression (Fincher, 1989 ; Jacobsen, Gubler and Chandler,
1995 ; Bethke, Schuurink and Jones, 1997). This approach
has been eminently successful, and a great deal is known
about how GA and ABA affect the transcription of genes
for abundantly transcribed enzymes such as α-amylase. GAand ABA-responsive elements have been identified in the
promoters of several cereal aleurone genes, and functional
trans-acting factors have been found (Jacobsen et al., 1995).
Much less is known about other aspects of hormonal
regulation in the aleurone cell. GA and ABA receptors have
not been identified, and signal transduction pathways that
engage cellular targets are only beginning to be described
(Bethke et al., 1997).
Many key enzymes and transporters in the aleurone cell,
however, are not regulated by transcription or translation.
These polypeptides are synthesized in the developing grain,
but remain inactive. Following imbibition or GA perception,
these proteins become active, and the aleurone cell quickly
begins the process of synthesizing and secreting hydrolytic
enzymes. These enzymes degrade starchy endosperm reserves, making them available for uptake by the scutellum.
Since reserves within the barley aleurone cell must be
mobilized before the more abundant reserves in the starchy
A
endosperm can be utilized, protein storage vacuoles must be
poised for rapid reserve mobilization. Several lines of
experimental evidence support this hypothesis. As described
in detail below, all of the transporters that have been
identified in the protein storage vacuole tonoplast appear to
be present in the dry grain and their amounts are relatively
unaffected by GA or ABA. Likewise, some of the enzymes
that are needed to mobilize protein storage vacuole reserves
are also present in the dry grain. When new protein
synthesis is required, it may begin with translation from preexisting mRNAs. Activation of protein storage vacuoles,
therefore, may require little more than water and cytosolic
signals. A diagram illustrating known components of cereal
aleurone protein storage vacuoles is presented in Fig. 1.
STRUCTURE OF PROTEIN STORAGE
VACUOLES
Barley aleurone cells from mature grain contain hundreds
of spherical protein storage vacuoles of about 1–5 µm
diameter (Fig. 2 A). These vacuoles have also been referred
to as protein bodies or aleurone grains. We use the term
protein storage vacuole here to emphasize the many features
these organelles share with other plant vacuoles. This
organelle is easily recognized by light and electron microscopy because of the numerous inclusions in the vacuole
lumen and the oleosomes embedded between the inner and
outer leaflets of the tonoplast (Figs 2 and 3). Freezefractured (Fernandez and Staehelin, 1985 ; Cornejo et al.,
1988) and high-pressure-frozen, freeze-substituted (J. Lonsdale and R. Jones, unpubl. res.) tissue observed by electron
microscopy show that protein storage vacuoles are filled
with electron-dense material. Histochemical staining of
high-pressure-frozen, freeze-substituted tissue shows that
the electron-dense material that fills the protein storage
B
Protein storage vacuole
Globoid
crystal
+
2+
(K , Ca
2+
Mg
and P)
Protein
crystalloid
Ca2+
SV
channel
OA–
Organic anion
transporter
Nuclease
Phytase
GS-X
Protease
H2O
7S globulins and
other storage proteins
(amino acids)
+
H
Lipase
α-TIP
V-ATPase
pH 7.2
PM
GSH conjugate
transporter
H+
pH 5.5
Oleosomes
(triglycerides)
2+
+
Ca -H
antiport
H+
Protein storage vacuole
V-PPase
PM
F. 1. Diagram of some of the constituents within cereal aleurone protein storage vacuoles. A, Storage reserves and the enzymes that mobilize
these reserves ; B, transporters present in the protein storage vacuole tonoplast.
Bethke et al.—Protein Storage Vacuoles in Cereal Aleurone
F. 2. Differential interference contrast and fluorescence micrographs
of barley aleurone protoplasts. A, Freshly isolated protoplast filled
with protein storage vacuoles. Note that each vacuole contains
numerous inclusions. B, Barley aleurone protoplast 5 d after treatment
with GA. In this cell, the smaller vacuoles have coalesced to form one
large protein storage vacuole (PSV). C, Pseudocoloured confocal image
of three protoplasts whose vacuoles have been loaded with the pHsensitive, fluorescent probe BCECF. Pseudocolour indicates intensity
of fluorescence, where blue is minimum and red is maximum intensity
(Reprinted from Swanson and Jones, 1996). D, Fluorescent image of a
protoplast whose protein storage vacuoles contain the proteolysis
product of the fluorogenic protease substrate ZFR-CMAC. For details
see Swanson, Bethke and Jones, 1998. Bars 10 µm.
vacuole lumen is likely to be protein. Electron micrographs
of storage protein vacuoles in aleurone tissue prepared by
chemical fixation which show an organelle only partially
filled with protein (e.g. Jones, 1969 a ; Swift and O’Brien,
1972 ; Lott, 1980) are misleading because chemical fixation
and solvent dehydration of aleurone tissue extract storage
proteins.
The lumen of the barley aleurone protein storage vacuole
contains 7S globulins, which are the major storage proteins.
Yupsanis et al. (1990) characterized the salt-soluble proteins
of aleurone protein storage vacuoles from the Himalaya
cultivar of barley and found that the major storage proteins
included a group of immunochemically related globulins of
50-, 40- and 25-kDa. These peptides had a common Nterminal amino acid sequence which was similar to that in
the N-terminus of pea and bean vicilins, as well as cottonseed
7S globulins. Additional peptides of 70 000 kDa also
cross-reacted with the anti-oat 7S globulin antibody, but
these did not co-purify with the other peptides. This led the
authors to suggest that the 70 000 kDa peptides might be
part of a separate holoprotein similar to the convicilins in
pea and bean. In marked contrast, the predominant storage
proteins in the protein storage vacuoles of barley starchy
endosperm cells are the alcohol-soluble hordeins, members
of the prolamine class of storage proteins (Shewry, 1995).
Hordeins have not been found in the aleurone layer.
At least two types of inclusions are found in the protein
401
storage vacuoles of barley aleurone cells (Jacobsen, Knox
and Pyliotis, 1971 ; Lott, 1980). Histochemistry (Jacobsen et
al., 1971) and electron microscopy (Pomeranz, 1973 ; Liu
and Pomeranz, 1975 ; Stewart, Nield and Lott, 1988 ; Batten
and Lott, 1996) have identified globoid crystals and protein
crystalloids in these vacuoles. X-ray microanalysis suggests
that the globoid crystal consists of phytin (K, Mg and Ca
salt of myo-inositol hexakisphosphate) (Stewart et al.,
1988), and electron microscopy shows that the globoid is
surrounded by an enveloping membrane of unknown
composition (Jones, 1973). The globoid crystal of the
aleurone cell is the principal site of K, Mg and P in the
mature grain. X-ray microanalysis has shown that 75 % of
grain P and K, and 60 % of Mg are in the endosperm. Over
80 % of endosperm P, K and Mg is found in the aleurone
layer, largely in the globoid crystals (Liu and Pomeranz,
1975 ; Stewart et al., 1988). The distribution of Ca in
Himalaya barley grain differs from that of K, Mg and P
in that this element is distributed almost equally between
endosperm and embryo (Stewart et al., 1988). Over 80 % of
endosperm Ca is found in the protein storage vacuole of the
aleurone layer (Stewart et al., 1988).
The other type of inclusion in the protein storage vacuole
of barley aleurone, the protein crystalloid (Lott, 1980), also
referred to as the protein}carbohydrate body (Jacobsen et
al., 1971), can be distinguished based on its histochemical
staining properties and electron density. Little is known
about the composition of this inclusion. In wheat, oat and
barley, an inclusion within aleurone protein storage vacuoles
is the major site of niacin deposition (Fulcher, O’Brien and
Wong, 1981). This inclusion is likely to be the protein
crystalloid.
Protein storage vacuoles in barley aleurone cells are also
a site of neutral lipid storage. Triglycerides are stored in
oleosomes that are embedded within the tonoplast (Fig. 3).
The presence of oleosomes in the tonoplast provides valuable
clues to the origin of the barley aleurone protein storage
vacuole. There is widespread agreement that triglycerides
are synthesized on ER membranes and deposited in
oleosomes as they form between the inner and outer leaflets
of smooth ER (Fig. 3 ; Huang, 1992). Oleosomes are
therefore unique organelles in that they are surrounded
by a half-unit membrane (Huang, 1992). There is disagreement, however, about how oleosomes become associated with protein storage vacuoles. Whereas one model
suggests that newly formed oleosomes become detached
from the surface of the ER and fuse with the vacuolar
membrane (Fernandez and Staehelin, 1985 ; Huang, 1992 ;
Napier, Stobart and Shewry, 1996), we propose an
alternative model. In this model oleosomes do not become
detached from the ER. Rather, the ER membrane that
synthesizes the neutral lipid also serves as the site of storage
protein accumulation (Fig. 3). This latter model is supported
by observations made using electron microscopy which
show that oleosomes in mature barley aleurone cells remain
attached to the ER, often by long extensions of the outer
leaflet of the ER membrane (Fig. 3 D). This model is also
consistent with observations regarding lipid mobilization.
Empty oleosome ‘ ghosts ’ are not observed when lipids are
mobilized from oleosomes, indicating that the oleosome
402
Bethke et al.—Protein Storage Vacuoles in Cereal Aleurone
A
B
C
Globoid crystal
Oleosome
Storage protein
Oleosome
Oleosome
Protein crystalloid
Outer ER
membrane leaflet
ER lumen
Storage
protein
ER lumen
Oleosome formation
Storage protein deposition
Mature protein storage vacuole
D
F. 3. A model for protein storage vacuole formation in barley aleurone and electron micrographs of a barley aleurone layer. A, Protein storage
vacuole formation begins with the production of oleosomes in the endoplasmic reticulum (ER) membrane. B, Storage proteins are deposited
within the lumen of the ER and oleosomes are attached to the ER by a stalk whose membrane is contiguous with the outer leaflet of the ER
membrane. C, A mature protein storage vacuole containing storage protein, a globoid crystal, and a protein crystalloid, and surrounded by
oleosomes. D, Stereo pair of electron micrographs from a barley aleurone layer showing the attachment of oleosomes to the ER by long extensions
of the outer leaflet of the ER membrane. Thick sections (gold) were viewed in an intermediate voltage electron microscope (JEOL 4000).
membrane reverts to the ER or tonoplast when lipid is
removed from the oleosome.
PROTEIN STORAGE VACUOLE
STRUCTURE DURING RESERVE
MOBILIZATION
When growth of the seedling is initiated or GA is supplied
to de-embryonated grains, isolated aleurone layers, or
aleurone protoplasts of barley, a cascade of events is
initiated that includes the release of minerals from the
aleurone cell and the synthesis and secretion of acid
hydrolases (Fincher, 1989 ; Bethke et al., 1997). The protein
storage vacuoles, which for months or years have functioned
as nutrient repositories, become lytic organelles, rapidly
hydrolyzing the stored polymers in their lumen, often with
the use of pre-existing enzymes.
Mobilization of protein storage vacuole polymers is
accompanied by dramatic changes in the structure of this
organelle (Jones, 1969 b ; Jones and Price, 1970 ; Ory and
Henningsen, 1975). As illustrated in Fig. 2 A and B, protein
storage vacuoles increase in size but decrease in number
following incubation in GA. When secretory enzyme
synthesis has ceased, one large vacuole occupies almost the
entire volume of the cell (Fig. 2 B ; Swanson, Bethke and
Bethke et al.—Protein Storage Vacuoles in Cereal Aleurone
Jones, 1998). The large (approx. 40 µm diameter) vacuole in
GA-treated cells is likely to form from the coalescence of
smaller protein storage vacuoles (Jones and Price, 1970).
Several pieces of evidence from electron microscopy studies
support this idea. First, protein storage vacuoles are not
separate organelles, but are linked by tonoplast connections
(Jones and Price, 1970). Second, the large vacuole formed
after GA treatment contains remnants of the phytin globoids
found in the smaller protein storage vacuoles. Since there is
no evidence that remnants of the phytin globoid leave the
protein storage vacuole and enter enlarging vacuoles, we
conclude that the latter form from the former. Third, the
tonoplast of large vacuoles, like that of smaller protein
storage vacuoles, has oleosomes embedded between the
inner and outer leaflets of the membrane (Jones and Price,
1970) and contains proteins recognized by anti-αTIP
antibodies (Schuurink, Chan and Jones, 1996).
The formation of a large vacuole from smaller protein
storage vacuoles brings about a substantial reduction in
tonoplast surface area and a concomitant reduction in the
number of oleosomes. Gluconeogenesis and the synthesis of
sucrose occurs in barley aleurone cells in response to GA
(Chrispeels, Tenner and Johnson, 1973). GA also brings
about an increase in glyoxylate cycle enzymes in barley
(Jones, 1972) and germinating wheat (Doig et al., 1975)
aleurone cells. These observations suggest that neutral lipids
stored in oleosomes are metabolized via the glyoxylate cycle
to produce sucrose and other sugars. The ability of the
aleurone layer to convert fat to sugar may appear to be an
anomaly considering that this tissue lies adjacent to a large
store of carbohydrate. However, for starch in the endosperm
to be converted to sugars, the aleurone cell must synthesize
mRNAs for secretory proteins such as α-amylase. Production of α-amylase and other mRNAs requires a large
pool of available ribose, which is likely to come from the
pentose phosphate pathway via gluconeogenesis. The ability
of the aleurone cell to make sugars at the expense of stored
fat would make this tissue independent of other tissues in
the grain.
As protein storage vacuoles coalesce in response to GA,
there is a change in the structure of the phytin globoid and
protein crystalloid inclusions. Globoids and crystalloids in
aleurone cells from mature grain are electron-dense structures (Jones, 1969 a), but following incubation in GA these
inclusions lose their electron opacity, and in the case of the
globoid, only the limiting envelope remains in the large
protein storage vacuole (Jones and Price, 1970). This loss of
electron density coincides with the hydrolysis of phytin.
Other materials accumulate in the central vacuole of GAtreated cells, including membrane fragments and lipid
droplets of unknown composition (Jones and Price, 1970 ;
Jones, 1987). We speculate that these membrane fragments
represent the remnants of organelles that are broken down
by autophagy in the protein storage vacuole (see below).
Aubert et al. (1996) observed the accumulation of phosphoryl choline in the vacuole of sucrose-starved, cultured
sycamore cells. They speculated that phosphoryl choline
accumulates in these cells as a result of membrane
degradation in the vacuole of starved cells undergoing
autophagy (Aubert et al., 1996).
403
ENZYMES THAT MOBILIZE PROTEIN
STORAGE VACUOLE RESERVES
Many hydrolytic enzymes are required for the mobilization
of protein storage vacuole reserves (Fig. 1). Storage proteins
are degraded by proteases (Bethke, Hillmer and Jones,
1996), and density labelling of newly synthesized proteins
with H O") or D O suggests that storage proteins are almost
#
#
completely hydrolyzed to amino acids (Filner and Varner,
1967 ; Chrispeels and Varner, 1973). Amino acids resulting
from storage protein breakdown become incorporated into
newly synthesized secretory proteins at the ER membrane.
Phytate is hydrolyzed by phytase (Gabard and Jones, 1986),
and the solubilized minerals are released from the aleurone
cell as cations and anions (Jones, 1973). Triglycerides are
degraded by lipases, and the resulting free fatty acids are
used to produce sugars and ATP. Active nucleases are also
present in protein storage vacuoles, but a precise role in
DNA or RNA cleavage has not been established.
Proteases
The mobilization of storage proteins is brought about by
proteases, and multiple protease activities have been found
in aleurone protein storage vacuoles. In barley, both aspartic
and cysteine protease activities have been identified in these
organelles (To$ rma$ kangas et al., 1994 ; Bethke et al., 1996).
As shown in Fig. 4, these enzymes have pH optima around
pH 4 and have no activity at pH 6±5 or greater. Since the pH
of protein storage vacuoles in freshly prepared aleurone
protoplasts is approx. pH 7 (Swanson and Jones, 1996),
these enzymes are unlikely to be active in the dry grain. Ingel assays suggest that as the protein storage vacuole lumen
is acidified to pH 5±5 or below (Swanson and Jones, 1996),
a sequential activation of proteases may occur (Bethke et
al., 1996). In the proteolytic activity gels shown in Fig. 4, for
example, no protease activities are detectable at pH 6±5 in
extracts of purified protein storage vacuoles from protoplasts treated with GA for 17±5 h (see Bethke et al., 1996 for
details of the assay). At pH 5±5, however, two aspartic
protease activities (AP1 and AP2) are seen in the lower half
of the gel. Further acidification to pH 4±5 results in activation
of two slowly migrating cysteine proteases (CP1 and CP2).
A third cysteine protease activity (CP3), which is seen
between the two aspartic protease activities, is observed at
pH 4±5, and this activity is greater at pH 3±5. The consequences of staggered protease activation in barley aleurone
are unknown, but in maize starchy endosperm, sequential
protease activation promotes efficient hydrolysis of zeins
(Bewley and Black, 1994).
In barley aleurone protein storage vacuoles isolated
17±5 h after aleurone protoplasts had been treated with GA,
two aspartic and three cysteine protease activities were
prominent on activity gels (Fig. 4 ; Bethke et al., 1996). One
of these activities, the aspartic protease HvAP (Hordeum
Šulgare aspartic protease) has been well characterized
(Sarkkinen et al., 1992 ; Runeberg-Roos et al., 1994 ;
To$ rma$ kangas et al., 1994). The identities of the other
activities are unknown. HvAP is an aspartic protease
similar to mammalian cathepsin D. Purified HvAP exists as
404
P
Bethke et al.—Protein Storage Vacuoles in Cereal Aleurone
L
PSV
P
pH 6.5
P
L
L
PSV
pH 5.5
PSV
P
L
PSV
CP1
CP2
AP1
CP3
AP2
pH 4.5
pH 3.5
F. 4. Proteolytic activity gels reveal multiple protease activities in
protein storage vacuoles isolated from GA-treated protoplasts. Samples
in each of the four gels were : purified protoplasts (P), a lysate depleted
of protein storage vacuoles (L) and isolated protein storage vacuoles
(PSV). In-gel assays were done essentially as described in Bethke et al.,
1996. Denatured haemoglobin was incorporated into each gel to serve
as a substrate for proteolysis. Following electrophoresis, gels were
incubated for 3 h at 37 °C in solutions buffered to pH 6±5, 5±5, 4±5 or 3±5
and then stained with Coomassie Brilliant Blue. Dark regions are
stained haemoglobin, light bands are regions of proteolytic activity.
Three cysteine proteases (CP1, CP2 and CP3) and two aspartic
proteases (AP1 and AP2) are indicated by arrowheads. Note that
proteolytic activity increases with decreasing pH. Bethke and Jones,
unpubl. res.
two heterodimeric isoforms and it was shown that both
isoforms are derived from a single gene by differential
processing of the protein precursor (Sarkkinen et al., 1992).
The mRNA for HvAP was localized in developing and
germinating grain using in situ hybridization (To$ rma$ kangas
et al., 1994). HvAP mRNA was not detectable in the starchy
endosperm later than 13 d after anthesis, but was detectable
in the cells of the mature aleurone layer. In germinating
grains, HvAp mRNA was found in the seedling, scutellum
and aleurone.
Active HvAP has been found in isolated vacuoles from
barley leaf protoplasts (Runeberg-Roos et al., 1994), and
anti-HvAP antibodies recognize proteins in barley aleurone
protein storage vacuoles (Bethke et al., 1996). The location
of HvAP protein has been determined for developing and
germinating barley grain (To$ rma$ kangas et al., 1994). HvAP
protein appeared in the aleurone layer concomitantly with
the development of aleurone cells (8–15 d after anthesis).
Between 20 and 28 d after anthesis, the amount of
immunologically detectable HvAP in the aleurone layer
decreased, such that at 28 d after anthesis no signal was
present. Before the third day after imbibition, however,
HvAP was again seen in aleurone cells. To$ rma$ kangas et al.
(1994) suggested that HvAP mRNAs might be stored in the
mature grain and are available for translation immediately
upon hydration of the grain. Protein blots probed with the
anti-HvAP antibody confirmed that HvAP was present in
the aleurone layer (Bethke et al., 1996). Aleurone layers
imbibed in the absence of hormone and protoplasts
incubated for 17±5 h in either ABA or GA contained much
greater amounts of HvAP than dry aleurone layers. Since
there was little difference in the amount of HvAP protein in
protoplasts incubated in ABA or GA, it is likely that the
synthesis of HvAP in aleurone layers is stimulated by
imbibition not by GA.
A role for HvAP in protein processing has been proposed.
In Šitro, HvAP removes 13 of the 15 amino acids in the Cterminal pro sequence of barley lectin (Runeberg-Roos et
al., 1994). Since immunoelectron microscopy showed that
HvAP and barley lectin were colocalized in barley vacuoles,
it was suggested that HvAP participates in the processing of
barley lectin (Runeberg-Roos et al., 1994). Additional in
ŠiŠo substrates for HvAP are likely to exist but have not
been characterized.
Aleurone cells synthesize numerous cysteine proteases
which are secreted into the starchy endosperm, and some of
these effectively proteolyze hordeins (Koehler and Ho,
1988, 1990 a, b). Other cysteine proteases are not secreted,
but are found within the protein storage vacuoles of the
aleurone (Fig. 4 ; Bethke et al., 1996 ; Swanson et al., 1998).
Cysteine protease activities were first identified in barley
aleurone protein storage vacuoles using an in-gel activity
assay (Bethke et al., 1996). More recently, this localization
of cysteine proteases to protein storage vacuoles has been
confirmed in ŠiŠo using a fluorogenic protease substrate
that is transported into the vacuole and then proteolyzed
(Fig. 2 D ; Swanson et al., 1998). When this substrate was
conjugated to glutathione, isolated, intact vacuoles were
able to take it up in an ATP-dependent manner and the
fluorescent proteolysis product accumulated in the vacuole
lumen. Accumulation of the proteolyzed product, however,
was inhibited by E-64 and leupeptin, two cysteine protease
inhibitors. These last two pieces of data suggest that there
are active cysteine proteases in the protein storage vacuoles
of barley aleurone protoplasts.
Unlike the aspartic protease activities in barley aleurone
protein storage vacuoles, at least some of the cysteine
protease activities are strongly upregulated by GA treatment
of barley aleurone protoplasts. Protein storage vacuoles
isolated from freshly prepared protoplasts show only one
band of cysteine protease activity in the activity gel assay
(Bethke et al., 1996). Vacuoles from cells incubated in GA
for 17±5 h, however, showed greatly increased activity in two
slowly migrating bands of cysteine protease activity.
Whether this increase in activity is the result of new protein
synthesis, as it is for HvAP, is not known. An equally likely
hypothesis is that cysteine protease zymogens are processed
to their active form in a GA-dependent manner. Processing
and activation of zymogens could be coupled to the
acidification of the protein storage vacuole lumen.
The amount of the cysteine protease aleurain is also
upregulated by GA. Aleurain, however, is not localized to
Bethke et al.—Protein Storage Vacuoles in Cereal Aleurone
the protein storage vacuoles, but is found in a smaller
organelle termed the aleurain-containing vacuole (Holwerda
et al., 1990). The function of the aleurain-containing vacuole
is unknown, as is its relationship to the protein storage
vacuole. Aleurain has been cloned and the sequence encodes
a cysteine protease of 361 amino acids. Aleurain is
synthesized as a 42 kDa propeptide which is subsequently
cleaved in a post-Golgi compartment to the mature 32 kDa
form (Holwerda et al., 1990 ; Holwerda and Rogers, 1992).
The increase in aleurain activity that occurs following GAstimulation results from increased transcription of the
aleurain mRNA and subsequent translation and posttranslational processing (Holwerda et al., 1990 ; Holwerda
and Rogers, 1992). Because purified aleurain functions as an
aminopeptidase, it has been suggested that aleurain functions as a processing enzyme.
Phytases
The mobilization of phytin is brought about by phytase
(Cosgrove, 1980). Two phytase activities have been recognized by the IUPAC-UB : 3-phytase (EC 3.1.3.8) from
microbes and 6-phytase (EC 3.1.3.26) from plants. 3-Phytase
dephosphorylates phytic acid (myo-inositol-1,2,3,4,5,6hexakisphosphate ; IP ) beginning at the D-3 position, and
'
6-phytase dephosphorylates phytic acid beginning at the L6 position (Cosgrove, 1980). 6-Phytase has been isolated
from a variety of plant sources, but it is especially abundant
in seeds and pollen. Phytase has been carefully studied in
wheat bran (mostly the aleurone and testa pericarp of the
wheat endosperm), and two classes of phytase, F1 and F2,
have been identified (Cosgrove, 1980). F1 phytase is a 6phytase producing myo-inositol 1,2,3,4,5 pentaphosphate
followed by removal of phosphate from carbons 5, 4, 3}1
and 1}3 yielding myo-inositol 2-phosphate. F2 phytase is a
2-phytase that dephosphorylates IP to inositol 1-phosphate.
'
Inositol monophosphate is hydrolyzed to free inositol and
phosphate by a separate enzyme, inositol monophosphatase
(Cosgrove, 1980).
Phytase has recently been purified to homogeneity from
several higher plant tissues, most notably from the roots
and shoots of Zea mays (Laboure, Gagnon and Lescure,
1993 ; Hubel and Beck, 1996). Corn 6-phytase consists of a
dimer of two identical 38-kDa subunits (Laboure et al.,
1993 ; Hubel and Beck, 1996). The pH optimum of the corn
enzyme is around 5, confirming earlier reports that 6phytase from a variety of plant sources, including seeds, has
an acidic pH optimum (Cosgrove, 1980 ; Hubel and Beck,
1996). Northern blot analysis of 6-phytase RNA in
germinating corn kernels showed low transcript abundance
in embryos from dry kernels and in embryos from kernels
soaked in aerated water for 12 h. Phytase mRNA accumulated after 24 h of imbibition and before radicle protrusion.
Transcript amount was maximal 2 d after the beginning of
imbibition, the time when the radicle protruded (Maugenset,
Martinez and Lescure, 1997). Maugenset et al. (1997)
concluded that 6-phytase from corn was likely to be
involved in the mobilization of phosphate from phytic acid.
Although 6-phytase activity has been studied intensively
in wheat grain (Cosgrove, 1980), this gene has not been
405
cloned from the small grain cereals. We showed that phytin
globoids isolated from barley aleurone layers have associated phosphatases with high phytase activity (Gabard and
Jones, 1986). As determined by activity gel assays of density
gradient fractions, these candidate phytases were present in
the aleurone of dry grain. Their activity did not change with
imbibition in water but declined upon incubation in GA
(Gabard and Jones, 1986). Since these data are not consistent
with the observations of Maugenset et al. (1997), further
experimentation is required.
Because of the role that inositol 1,4,5-triphosphate (InsP )
$
plays in cellular signalling in eukaryotes, there is interest in
knowing the nature of the products that result from phytin
hydrolysis. There is substantial evidence that plant cells
contain InsP (Drobak, 1992 ; Crain, 1993), and recent
$
reports confirm that InsP can arise from phosphatidyl$
inositol 4,5-bisphosphate (PiP ) by the action of phospho#
lipase C (Brearley, Parmar and Hanke, 1997). There is no
convincing evidence, however, that InsP arises from the
$
hydrolysis of phytic acid in seeds or grains (Cosgrove,
1980). A detailed study of inositol phosphates in barley
aleurone layers failed to detect the presence of InsP , but
$
both inositol 1,2,3- and inositol 1,2,6-trisphosphate were
identified (Brearley and Hanke, 1996). The spectrum of
inositol phosphates in barley aleurone cells found by
Brearley and Hanke (1996) were very similar to the in Šitro
products of phytic acid hydrolysis by wheat bran phytase(s),
indicating that the inositol phosphates in aleurone extracts
were the products of phytic acid degradation. Although
attention has been focused on InsP as a signalling molecule
$
in eukaryotic cells, evidence is accumulating that other
inositol phosphates including IP can serve as signalling
'
molecules in microorganisms and mammals (Huisamen and
Lochner, 1996 ; Van Haastert and Van Dijken, 1997). As yet
a role in signal transduction for inositol phosphates other
than IP has not been identified in plant cells.
$
Lipases
Triglycerides in oleosomes are hydrolyzed to free fatty
acids by lipases. The localization of lipase in barley aleurone
was examined using sucrose density gradient centrifugation
of homogenates from aleurone layers imbibed in buffer or
treated with ABA and GA (Fernandez and Staehelin, 1987).
In freshly prepared aleurone layers, no lipase activity was
associated with purified oleosomes. Instead, lipase activity
was found in a 10 000 g pellet. GA treatment of aleurone
layers caused a shift in lipase activity from the 10 000 g pellet
to the oleosome-containing fraction. This shift in enzymatic
activity began 1 h after incubation in GA and was complete
1 h later, when approx. 75 % of all lipase activity was
associated with the oleosome-containing fraction. Total
lipase activity increased only slightly following GA treatment. Aleurone layers treated with ABA did not show this
shift in lipase activity, and the shift in activity was reduced
when layers were treated with both GA and ABA. Because
protein storage vacuole membranes could be identified in
the 10 000 g pellet, and because membrane connections
between oleosomes and protein storage vacuoles were
observed by freeze-fracture electron microscopy (Fernan-
406
Bethke et al.—Protein Storage Vacuoles in Cereal Aleurone
dez and Staehelin, 1987), the authors suggested that lipase
was transferred from protein storage vacuoles to oleosomes
following perception of a GA signal. This transfer might
occur along the continuous phospholipid layer that links
oleosomes and protein storage vacuoles. A similar situation
was found in wheat aleurone, where acid lipase activity in
freshly isolated aleurone layers was found in density gradient
fractions containing protein storage vacuoles but not in
oleosome containing fractions (Jelsema et al., 1977).
Nucleases
Nucleases are also contained within aleurone protein
storage vacuoles (Holstein et al., 1991 ; A. Fath and
R. Jones, unpubl. res.). A type I nuclease was purified from
barley aleurone (Brown and Ho, 1987 ; Brown, Mecham
and Ho, 1988), and immunoelectron microscopy localized
this enzyme to the protein storage vacuole (Holstein et al.,
1991). Protein blotting of fractions enriched in intact protein
storage vacuoles has confirmed this observation (J-Z. Huang
and R. Jones, unpubl. res.). Nuclease I is a 32 kDa
endonuclease that hydrolyzes both DNA and RNA. Based
on immunoelectron microscopy it was inferred that barley
Nuclease I was delivered to the protein storage vacuoles via
the Golgi apparatus since both ER and Golgi labelled with
anti-Nuclease I antibodies.
The existence of Nuclease I within protein storage
vacuoles raises interesting questions about its function. One
hypothesis was that it might hydrolyze the small amount of
RNA present in these organelles (Holstein et al., 1991). An
extension of this hypothesis is that following GA perception,
unwanted DNA or RNA fragments are transported into
protein storage vacuoles for destruction. Rapid hydrolysis
of nuclear DNA occurs in living aleurone cells as they
approach death (A. Fath, P. Bethke and R. Jones, unpubl.
res.), and much of the nuclease activity seen in these cells at
that time is in the protein storage vacuoles. No data exist to
suggest how nucleic acids might be transported through the
tonoplast and into the vacuole. Autophagic organelles or
vesicles, however, may fulfil the role of delivering unwanted
cytosolic macromolecules or multi-molecular structures to
the vacuole for destruction or inactivation.
TRANSPORT THROUGH THE TONOPLAST
Transport through the tonoplast is an essential feature of
aleurone protein storage vacuoles. Most of the reserves
stored there must pass through the vacuolar membrane
before they benefit either the aleurone cell or the embryo. At
the same time , harmful or surplus compounds may be
transported into protein storage vacuoles for storage,
detoxification or destruction. Although much remains to be
learned about the transporters in the protein storage vacuole
tonoplast, it is clear that an extensive, carefully regulated
system exists. By using biochemical, immunological and
biophysical approaches, researchers have identified several
transporters. These include the aquaporin αTIP, the slow
vacuolar (SV) channel, a Ca#+}nH+ antiporter, the H+ATPase and H+-PPase, as well as at least two ATP-binding
cassette (ABC) transport activities (Fig. 1 B). Significantly,
the transporters that are used for the export of nutrient
reserves from the vacuole to the cytosol have not been
characterized.
Tonoplast intrinsic protein
αTIP (α-tonoplast intrinsic protein) is an integral tonoplast protein having six transmembrane helices that was first
purified from bean cotyledons, where it constitutes approx.
2 % of total extractable protein (Johnson, Herman and
Chrispeels, 1989). αTIP is highly expressed in the tonoplast
of seed storage tissues but is absent or present in very low
amounts in other membranes and tissues (Maurel, 1997).
αTIP is a member of the MIP family of transporters, and
can function as a water channel when expressed in Xenopus
oocytes. The water channel activity of αTIP in oocytes was
dependent on phosphorylation (Maurel et al., 1995). Oocytes
injected with αTIP cRNA had a water permeability that was
four to eight-fold greater than that of control oocytes.
Treatment of injected oocytes with compounds that increased the activity of endogenous protein kinase A
increased water permeability by an additional 80–100 %.
When putative phosphorylation sites in the αTIP cRNA
were disrupted by site-directed mutagenesis, injection of
mutant cRNA caused less of an increase in permeability of
the oocyte membrane than injection of wild type cRNA.
Since αTIP is phosphorylated in plant vacuoles, it was
suggested that phosphorylation of αTIP might be a means
for regulating tonoplast osmotic permeability (Maurel et
al., 1995).
αTIP is present in the dry barley aleurone (Bethke et al.,
1996), as well as in ABA- or GA-treated aleurone layers or
protoplasts (Schuurink et al., 1996). On protein blots, αTIP
appears as a doublet of approx. 25 kDa, and the relative
amount of the upper band with respect to the lower band
varies with hormone treatment and duration. Although
αTIP is an abundant protein, its function is unclear. A role
in water transport is presumed (Schaffner, 1998), but it is
not known if this is to facilitate grain desiccation or
rehydration. What is clear is that protein storage vacuoles
that are rapidly hydrolyzing stored proteins and solubilizing
stored minerals may have a relatively large requirement for
water if they are to remain iso-osmotic with the cytosol.
αTIP may also function to buffer the osmolality of the
cytosol from changes in water potential in the free space of
the wall (Maurel et al., 1993). In this way, TIPs in the
tonoplast would ensure that water entry into the protein
storage vacuole was at least as rapid as water entry into the
cytosol. This might minimize the osmotic shock experienced
by other organelles following imbibition.
The slow Šacuolar channel
Another abundant tonoplast transporter found in aleurone cells is the slow vacuolar (SV) channel. This class of
channel is ubiquitous in the tonoplast of plants and was
found in the tonoplast of both ABA- and GA-treated barley
aleurone protoplasts (Bethke and Jones, 1994). The SV
channel is a voltage-regulated, cation channel that is selective
for Ca#+ but also readily transports K+. Like αTIP, the role
Bethke et al.—Protein Storage Vacuoles in Cereal Aleurone
of the SV channel in the aleurone is unknown. A role in
facilitating calcium-induced calcium release has been proposed for the SV channel in guard cells (Ward and
Schroeder, 1994), but this remains controversial. Under one
set of experimental conditions, calcium release through the
SV channel was found to be thermodynamically unfavourable (Pottosin et al., 1997).
Although the function of the aleurone SV channel remains
unknown, its regulation has been studied in detail. The SV
channel in aleurone is voltage regulated, requiring a positive
(cytosol-vacuole) membrane potential of approx. 20–40 mV
for opening (Bethke and Jones, 1994). The conditions under
which this occurs are not known. Channel opening is also
promoted by calcium and calmodulin (CaM) (Bethke and
Jones, 1994). Cytosolic calcium concentrations greater than
about 1 µ are required for opening, and in the absence of
added CaM the probability of the channel being in the open
state increases with cytosolic free calcium up to at least
100 µ. Plant CaM, e.g. Chenopodium rubrum (Weiser,
Blum and Bentrup, 1991) or spinach (Bethke and Jones,
1994), but not bovine brain CaM, also stimulated channel
opening and increased the sensitivity of the channel toward
calcium. It seems likely, therefore, that the effects of calcium
on channel opening are at least partly the effect of
Ca#+ interacting with membrane-bound CaM. Immunolocalization of CaM in barley aleurone protoplasts has
shown strong binding of an anti-CaM antibody to an
antigen in the barley tonoplast.
Activation of the SV channel by increases in Ca and CaM
suggests that the transport activity of this channel may be
upregulated by GA. Protein storage vacuoles isolated from
barley aleurone protoplasts treated with GA had whole
vacuolar SV currents that were 240 % of currents in protein
storage vacuoles isolated from ABA-treated protoplasts
(Bethke and Jones, 1994). One explanation for this difference
is that protein storage vacuoles isolated from GA-treated
cells have more CaM associated with them than those
isolated from ABA-treated cells. GA treatment of barley
aleurone layers increased the amount of CaM mRNA and
protein (Schuurink et al., 1996). In barley and wheat, GA
treatment resulted in increased cytosolic calcium concentrations (Bush, 1995).
SV channel opening, however, may be inhibited by GAinduced acidification of the vacuole lumen. This could
provide another link to GA signalling. It is not known,
however, if SV channel activity in aleurone is decreased by
vacuolar acidification as it was in Vicia faba and Beta
Šulgaris (Schultz-Lesdorf and Hedrich, 1995).
The barley aleurone SV channel is also regulated by
protein phosphorylation (Bethke and Jones, 1997). When
protein storage vacuoles isolated from ABA-treated barley
aleurone protoplasts were treated with the protein phosphatase inhibitor okadaic acid, the activity of the SV
channel decreased. This decrease could be overcome by
addition of an okadaic-acid-insensitive protein phosphatase.
SV channel activity could also be inhibited by the addition
of 200 µ ATP, and this inhibition was prevented by the
protein kinase inhibitor H-7. These experiments suggest
that phosphorylation can decrease the activity of the
channel, and that the protein phosphatase and protein
407
kinase that regulate this activity are localized to the protein
storage vacuole tonoplast. The effect of ATP addition,
however, was dependent on its concentration. At 2 m,
ATP increased the activity of the SV channel. Two
millimolar ATP is close to the presumed cytosolic concentration of ATP in most plant cells. Addition of
recombinant CDPK in the presence of either 200 µ or
2 m ATP strongly stimulated activity of the channel.
These data were interpreted to mean that there are at least
two phosphorylation sites that regulate the activity of the
SV channel. Increased phosphorylation of the first site
relative to the second led to an increase in channel activity
(Bethke and Jones, 1997). The regulation of the SV channel
is complex, and it is worth noting that with the exception of
Ca#+ most of the known regulatory components for this
channel are not cytosolic but rather are located on the
tonoplast. It will be interesting to learn if other tonoplast
transporters in protein storage vacuoles are accompanied by
a similar suite of regulatory elements.
Ca#+-transporters
At least three calcium-transport activities have been
identified in microsomal vesicles isolated from GA-treated
wheat aleurone layers (Bush and Wang, 1995). After
separation on an isopycnic sucrose density gradient, one of
these activities, referred to as Type II, was found in a
fraction enriched in tonoplast marker enzymes. It is likely,
therefore, that the Type II activity was localized to the
protein storage vacuole membrane. Type II calcium transport activity was stimulated by calmodulin and inhibited by
the calmodulin antagonist W7. Nitrate and compounds
such as FCCP that prevent the formation of a proton
gradient inhibited Type II calcium transport by over 60 %.
Kinetic analysis revealed that this transport activity had
both a low affinity (Km ¯ 12 µ) and a high affinity (Km ¯
0±33 µ) for Ca#+. This led the authors to suggest that Type
II activity had characteristics of both a primary Ca#+
transporter and a secondary Ca#+}nH+ antiport. Bush and
Wang (1995) were not able to determine if these two Ca#+
transport mechanisms exist in the same transporter or in
two separate transporters, but suggested that Type II
calcium transporters are likely to be important in the
regulation of cytosolic calcium concentrations.
Tonoplast proton pumps
In the cells of most higher plants the vacuole is an acidic
compartment (Boller and Wiemken, 1986 ; Kurkdjian and
Guern, 1989). The protein storage vacuole of the cereal
aleurone cell is one of a few interesting exceptions (Davies
et al., 1996 ; Swanson and Jones, 1996). The pH of the
protein storage vacuoles in mature aleurone cells from
barley (Swanson and Jones, 1996) and wild oat (Davies
et al., 1996) is near neutrality. Incubation of aleurone
protoplasts in the absence of hormone results in a gradual
decline in the pH of the PSV lumen (Davies et al., 1996), and
in barley this acidification is accelerated by incubation in
GA (Swanson and Jones, 1996).
Proton pumps contribute to the regulation of vacuolar
408
Bethke et al.—Protein Storage Vacuoles in Cereal Aleurone
A
B
440 nm
0 hr
4 hr
8 hr
+ABA
6.8
6.4
pH
ABA
GA3
+GA3
6.0
5.6
0
pH
5.0
6.0
7.0
2
4
6
Time (hr)
8
10
F. 5. Pseudocolour images of barley aleurone protoplasts loaded with the pH-sensitive probe BCECF, and quantification of vacuolar pH. A,
Protein storage vacuoles in living barley aleurone protoplasts were loaded with BCECF then treated with either ABA or GA . The fluorescence
$
emission at 440 nm is presented in the left panels. The pseudocoloured fluorescent signal from protoplasts 0, 4 and 8 h after hormone addition
is presented in the three right-hand panels and corresponds to the pH scale bar below. B, Quantification of the data in A. Note that GA treatment
resulted in acidification of protein storage vacuoles but ABA treatment did not. Reprinted from Swanson and Jones, 1996.
pH (pHV) in plants (Kurkdjian and Guern, 1989), and two
types of proton pumps, the vacuolar H+ ATPase (VATPase ; EC 3.6.1.3) and the vacuolar pyrophosphatase (VPPase ; EC 3.6.1.1) (Nelson and Taiz, 1989 ; Rea and Poole,
1993), are found in the plant tonoplast. Both V-ATPase and
V-PPase have been identified in the tonoplast of aleurone
protein storage vacuoles (Swanson and Jones, 1996).
Immunofluorescence microscopy of aleurone protoplasts
and protein blotting of purified protein storage vacuoles
established that V-ATPase and V-PPase were present in the
tonoplast of barley aleurone protoplasts. These enzymes
were found in the tonoplast of freshly isolated protoplasts,
and the amount of these pumps was not affected by
incubation of protoplasts in GA or ABA (Swanson and
Jones, 1996).
Using the ratioable, pH-sensitive dye BCECF we have
shown that pHV of the aleurone protein storage vacuole is
regulated by GA (Swanson and Jones, 1996). BCECF was
used to monitor pHV in living aleurone cells since this dye
could be loaded into protoplasts non-invasively using the
membrane-permeant AM (acetoxy methyl ester) form.
BCECF specifically accumulated in the lumen of the protein
storage vacuole (Fig. 2 C) and did not affect the ability of the
aleurone cells to respond to GA or ABA (Swanson and
Jones, 1996). In freshly isolated aleurone protoplasts pHV
was around 6±7, but following exposure to GA for 8 h, pHV
dropped to around pH 5±8 (Fig. 5 ; Swanson and Jones,
1996). Acidification of the protein storage vacuole in GAtreated protoplasts began within 2 h of adding GA and
reached a maximum about 8 h after hormone addition
(Swanson and Jones, 1996). From this we can conclude
that 2 h is the maximum time required for a GA signal
transduction cascade to reach the protein storage vacuoles.
ABA slowed the acidification of protein storage vacuoles,
and protoplasts incubated in ABA for 21 h had a pHV that
was slightly higher than the pHV of protoplasts incubated in
the absence of hormones. We have shown that pHV in ABAtreated protoplasts remains higher than that of GA-treated
protoplasts after incubation for 4 or more days (Swanson et
al., 1998).
Ratio imaging of BCECF has also shown that the VATPase and V-PPase can acidify the lumen of isolated
barley aleurone protein storage vacuoles (Swanson and
Jones, 1996). When either ATP or PPi were added to intact
isolated protein storage vacuoles, pHV fell rapidly to a new
level in less than 5 min. Acidification of the vacuole lumen
occurred regardless of whether vacuoles were isolated from
ABA-, GA- or non-hormone-treated protoplasts. Acidification following ATP addition was inhibited by NO−, and
$
acidification following PPi addition was prevented by Ca#+
suggesting that both V-ATPase and V-PPase can act to
lower pHV (Swanson and Jones, 1996). Although these
experiments demonstrate that V-ATPase and V-PPase can
lower pHV in Šitro, we have not established the contribution
of these proton pumps to regulation in pHV in ŠiŠo. Indeed,
the in ŠiŠo regulation of pHV in barley aleurone cells is likely
to be complex since vacuolar acidification is much faster in
GA-treated cells than in ABA-treated cells despite the
presence of comparable amounts of V-ATPase and V-PPase
in both (Swanson and Jones, 1996). Since acidification in
Šitro occurred in minutes and acidification in ŠiŠo required
hours, acidification cannot result simply from the activation
of the proton pumps.
There is only limited information to suggest how VATPase and V-PPase activities might be regulated in protein
storage vacuoles (Davies, 1997). Transcriptional regulation
of the V-ATPase has been reported (Low et al., 1996 ;
Tsiantis, Batholomew and Smith, 1996), but our experiments
showing that GA or ABA had no effect on the amount of
either V-ATPase or V-PPase in aleurone protoplasts suggest
that in barley aleurone protein storage vacuoles, regulation
of H+-pump activities is likely to be post-translational. Ca#+
and protein phosphorylation are attractive candidates for
signals that control pHV in the aleurone cell. Ca#+ at low
micromolar concentrations is a potent inhibitor of the VPPase (Rea et al., 1992). Because the SV channel may
Bethke et al.—Protein Storage Vacuoles in Cereal Aleurone
409
T     1. Subcellular distribution of fluorescent probes in
barley aleurone protoplasts
Probe purpose and name
ABA-treated
protoplasts
GA-treated
protoplasts
F. 6. Fluorescence from protein storage vacuoles in ABA- or GAtreated (A and B, respectively) barley aleurone protoplasts. Protoplasts
were incubated with non-fluorescent monochlorobimane which becomes fluorescent after conjugation to cytosolic glutathione. Transport
into the vacuole by a glutathione conjugate transporter results in
fluorescence from the protein storage vacuoles. Note that the amount
of vacuolar fluorescence in ABA- and GA-treated cells is approximately
the same. This suggests that rates of uptake into these organelles are
comparable. See Swanson, Bethke and Jones, 1998 for details.
transport Ca#+ from the vacuole to the cytosol (Ward and
Schroeder, 1994), the local concentration of Ca#+ at the
tonoplast of the aleurone protein storage vacuole may be
high and this would effectively inhibit the activity of this
pump. Protein phosphorylation may also play a role in the
regulation of the V-ATPase. Martiny-Baron et al. (1991)
showed that the V-ATPase of zucchini hypocotyls is likely
to be regulated by a lysophospholipid-activated protein
kinase. Phosphorylation of V-ATPase and H+ pumping in
zucchini membranes was stimulated by phospholipid,
leading Martiny-Baron et al. (1991) to conclude that proton
pumping by the V-ATPase was regulated by phosphorylation. By analogy with the role that protein phosphorylation
plays in the regulation of the SV channel of the aleurone
protein storage vacuole, we suggest that the activity of the
V-ATPase may be regulated by protein phosphorylation.
ABC transporters
Proteins that can transport a variety of potentially
harmful toxins and naturally occurring molecules into the
vacuole are located in the tonoplast (Kreuz, Tommasini and
Martinoia, 1996 ; Coleman, Blake-Kalff and Davies, 1997 ;
Rea et al., 1998). These transporters belong to the ABC
family of transport proteins. Two subclasses of these
proteins have been identified in plants, the MDRs (multidrug
resistance proteins) and MRPs (multidrug resistance associated proteins) (Rea et al., 1998). We have demonstrated the
presence of two ABC transport activities on the tonoplast of
barley aleurone protein storage vacuoles, a glutathione (GS)
conjugates transport activity and an organic anion transport
activity (Swanson et al., 1998). These transporters were
present in freshly isolated protoplasts and, unlike the H+
pumps on the tonoplast, their activities were not markedly
affected by incubation in GA or ABA (Fig. 6). Thus,
accumulation of fluorescent probes that are transported
Glutathione}sulfhydryls
Monochlorobimane
Monobromobimane
Cell tracker green
pH
BCECF-AM
Lysosensor yellow}blue
Calcium
Indo-1 ff
Indo-1
Fluo-3
Protease substrate
ZFR-CMAC
Other fluorophores
Lucifer yellow
Oregon green
Fluorescein
Location of fluorescence
Cytoplasm then vacuole
Cytoplasm then vacuole
Cytoplasm then vacuole
Cytoplasm then vacuole
Vacuole
Cytoplasm
Cytoplasm
Cytoplasm
Vacuole
No uptake
Cytoplasm then vacuole
Cytoplasm then vacuole
Reprinted from Swanson, Bethke and Jones, 1998.
into vacuoles by one of these transporters was not altered by
incubation of protoplasts in the presence or absence of
hormones. The ability of protein storage vacuoles in freshly
isolated protoplasts to accumulate fluorescent probes is a
very useful attribute. It allows non-hormone-treated cells to
be loaded with probes that can measure subsequent
responses to hormones, as was done for vacuole acidification
(Fig. 5).
Our interest in characterizing the ABC transporters on
the tonoplast of protein storage vacuoles was sparked by the
observation that these organelles accumulate a wide variety
of fluorescent probes (Table 1). With the exception of the
Ca#+-sensitive dyes, including Indo and Fura, all other cell
permeant fluorescent dyes that we have examined are taken
up and sequestered in the vacuole of aleurone cells (Table 1,
Figs 2, 5 and 6 ; Swanson et al., 1998). By monitoring the
uptake and accumulation of dyes such as monobromobimane (MBB) and monochlorobimane (MCB) that fluoresce only after binding to sulphydryls such as glutathione
(Coleman et al., 1997), we surmised that at least one of the
ABC transport activities was a transporter of GS conjugates
(Fig. 6). Using isolated protein storage vacuoles, we
confirmed that compounds such as MCB and MBB are
taken up by ATP-dependent GS conjugates transporters,
but that other dyes, including the pH-sensitive dye BCECF,
are transported into protein storage vacuoles by ATPdependent organic anion transporters (Swanson et al.,
1998). Whereas vanadate was a potent inhibitor of both
ABC transport activities in the protein storage vacuole
membrane, probenicid inhibited only the transport activity
that facilitates BCECFs accumulation. Conversely, GSconjugate transport, but not organic anion transport activity
was competitively inhibited by other GS-conjugated substrates (Swanson et al., 1998).
The experiments showing that accumulation of fluorescent
dyes into aleurone vacuoles is driven by at least two types of
ATP-dependent transport activities allow for the rational
410
Bethke et al.—Protein Storage Vacuoles in Cereal Aleurone
design of compounds that can be targeted to the plant
vacuole. For example, cell permeable electrophiles that
readily form glutathione conjugates are likely to be taken up
into the vacuole in ŠiŠo. In Šitro conjugation of these same
compounds to glutathione provides a means for their
introduction into isolated vacuoles. The ability of probenecid to prevent the uptake of dyes into vacuoles in ŠiŠo
provides a way to alter the partitioning of dyes between
vacuole and cytoplasm. Further, an understanding of the
chemistry of compounds such as indo-1ff that accumulate in
the cytosol but not in the vacuole of aleurone cells provides
a way of targeting probes to this compartment.
CONCLUSIONS
Much has been learned about the composition and function
of the aleurone protein storage vacuole. Figure 1 illustrates
constituents of the barley aleurone protein storage vacuole,
and Fig. 7 illustrates changes that occur in these organelles
following imbibition of whole grain or GA treatment of
isolated aleurone layers or protoplasts. Although these
diagrams are only cartoon depictions of this organelle, until
recently even these rudimentary aspects of protein storage
vacuoles were unknown. Yet for each new thing that has
been learned about protein storage vacuoles, multiple unknowns exist. Globulin storage proteins have been identified
in barley aleurone protein storage vacuoles, but the amount
and identity of other storage proteins remain unknown.
Proteases exist in protein storage vacuoles, but in ŠiŠo
substrates have not been determined, and a more precise
A
characterization of protein storage vacuole proteases is
needed. Nuclease I has been localized to the protein storage
vacuole, but the number of other nucleases and their role in
aleurone physiology are unclear. Lipase activities are
associated with protein storage vacuoles, but how this
activity is regulated needs further investigation. Although it
is known that stores of protein and minerals are deposited
into protein storage vacuoles, influx and efflux transporters
for mineral reserves and efflux transporters for amino acids
or small peptides have not been identified. αTIP and the SV
channel are abundantly present in the tonoplast, yet the
function of these transporters remains speculative. And
although it is clear that the activities of the vacuole are coordinated with those of the cytosol, there is only fragmentary
evidence to suggest how this might be done. As we learn
more, we are likely to unearth new layers of complexity and
discover new levels of interaction. A complete picture of the
aleurone protein storage vacuole will not be ours for many
years, yet what we can see already suggests that it will be a
fascinating picture indeed.
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Inactive
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H
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