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Journal of Cell Science 108, 299-310 (1995)
Printed in Great Britain © The Company of Biologists Limited 1995
Protein storage vacuoles form de novo during pea cotyledon development
Birgit Hoh, Giselbert Hinz*, Byung-Kap Jeong and David G. Robinson
Abteilung Cytologie des Pflanzenphysiologischen Instituts, Universität Göttingen, Untere Karspüle 2, D-37073 Göttingen, FRG
*Author for correspondence
SUMMARY
We have investigated the formation of protein storage
vacuoles in peas (Pisum sativum L.) in order to determine
whether this organelle arises de novo during cotyledon
development. A comparison of different stages in cotyledon
development indicates that soluble protease activities
decline and the amounts of storage proteins and the
integral membrane protein of the protein body, α-TIP,
increase during seed maturation. On linear sucrose density
gradients we have been able to distinguish between two
separate vesicle populations: one enriched in α-TIP, and
one in TIP-Ma 27, a membrane protein characteristic of
vegetative vacuoles. Both vesicle populations possess,
however, PPase and V-ATPase activities. Conventionally
fixed cotyledonary tissue at an intermediate stage in
cotyledon development reveals the presence of a complex
tubular-cisternal membrane system that seems to surround
the pre-existing vacuoles. The latter gradually become
compressed as a result of dilation of the former membrane
system. This was confirmed immunocytochemically with
the TIP-Ma 27 antiserum. Deposits of the storage proteins
vicilin and legumin in the lumen, and the presence of αTIP in the membranes of the expanding membrane system
provide evidence of its identity as a precursor to the protein
storage vacuole.
INTRODUCTION
rounded by a cisternal, tubular membrane system that already
contains deposits of storage proteins. Expansion of this
membrane network leads to the formation of the PSV. These
results are supported by subcellular fractionation results, which
have enabled us to recognize two distinct vacuole populations.
Whereas the final respository for storage proteins in cereals,
the protein body (PB), develops directly from the endoplasmic
reticulum (ER) by dilation and vesiculation (Larkins and
Hurkman, 1978; Oparka and Harris, 1982; Lending and
Larkins, 1992; Galili et al., 1993), the origin of the protein
storage vacuole (PSV) in maturing legume seeds is a matter of
controversy. Some workers have suggested that the large
vacuoles present in the young, parenchymatous, cotyledonary
cells disappear during development and become replaced by a
second set, which are ultimately transformed into PB (e.g. see
Neumann and Weber, 1978). Others (see Craig et al., 1979;
and references therein) have maintained that only one population of vacuoles exists: these gradually fill up with storage
proteins and then subdivide to form tens of thousands of PB
(diameter around 1 µm). A further possibility, expressed by
Harris and Boulter (1976) and Adler and Müntz (1983), represents a combination of both points of view, i.e. simultaneously with the subdivision of the pre-existing vegetative
vacuole (VV), additional, new protein storage vacuoles are
formed either from the ER (Adler and Müntz, 1983) or by
enlargement of putative Golgi vesicles (Harris and Boulter,
1976).
In this paper we provide evidence from subcellular fractionation and conventional electron microscopy of two sequential populations of vacuoles during cotyledonary development
in maturing pea seeds. Ultrastructural-immunocytochemical
data indicate that the original VV appears to become sur-
Key words: immunocytochemistry, pea cotyledon, protein storage
vacuole, vacuolar replacement, vegetative vacuole
MATERIALS AND METHODS
Plant material
Pea plants (Pisum sativum L. var. Haubners Exzellenz, and var.
Kleine Rheinländerin) were grown hydroponically in a greenhouse.
The former variety, because it had reduced amounts of starch, was
used exclusively for the immunocytochemistry and ultrastructural
investigations. Flowers were marked at anthesis and pods were
harvested at various intervals. Pea seeds do not develop synchronously in a pod. Moreover, the cells in individual legume cotyledons
do not develop synchronously, but rather respond at different times
to factors emanating from the embryonic axis in a wave-like manner
(Perez-Grau and Goldberg, 1989). Environmental factors, e.g. temperature, also influence the duration of developmental events leading
to seed maturation (compare for example data presented by Craig et
al., 1979, 1980; Craig and Millerd, 1981; with data from Craig and
Goodchild, 1984; Craig, 1986). Clearly the parameters ‘days after
flowering’ or ‘days post anthesis’ are not reliable guides to cotyledon
development. For this reason we have used in addition, seed fresh
weight and diameter.
Subcellular fractionation
All procedures were performed at 0-4°C. Isolation of endomembrane
fractions was usually carried out with 0.5-2 g testa-free cotyledons.
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B. Hoh and others
Cotyledon tissue was hand-chopped with a razor blade for 15 minutes
in a Petri dish with 5 ml of a medium containing 40 mM HEPES, pH
7.4, 250 mM sucrose, 3 mM MgCl2, 0.1 mM EDTA, 1 µM DTT, 2
µM leupeptin, 0.7 µM pepstatin, 1 mM o-phenanthroline. After
filtering through 1 layer of Miracloth, the homogenate was then precentrifuged at 1,000 g for 10 minutes. The supernatant was centrifuged at 100,000 g for 60 minutes to give a total membrane fraction
(tmf). This pellet was then resuspended in 1 ml of homogenizing
medium (without sucrose) and layered onto a linear (15% to 50%
(w/w), 16 or 38 ml) sucrose gradient, which was then centrifuged in
a swing-out rotor (Sorvall AH-627) at 80,000 g for 16 hours. Fractions
(1-1.5 ml) were collected and analyzed for marker enzyme activities
or for the presence of certain antigens by western blotting.
Marker enzyme assays
Triton-stimulated inosine diphosphatase (IDPase, EC 3.6.1.6) and
antimycin A-insensitive NADH-cytochrome c reductase (CCR, EC
1.6.99.3) were measured as described by Sauer and Robinson (1985).
Anion-stimulated, nitrate-inhibitable H+-ATPase (V-ATPase, EC
3.5.1.3) and cation-stimulated inorganic pyrophosphatase (PPase; EC
3.6.1.1) were assayed exactly according to the protocols given by
Robinson et al. (1994). NADH-malate dehydrogenase (MDH, EC
1.1.1.37) was determined on soluble extracts of cotyledons (see
below) by adding 100 µl samples to a reaction mixture containing 520
µl 100 mM MOPS, pH 7.5, 10 µl 20 mM NADH, 20 mM oxaloacetate. NADH oxidation was measured as ∆ ε366 nm (molar absorption
coefficient of NADH at 366 nm was 3.34 mM−1 cm−1).
Measurement of protease activities
Soluble extracts of pea cotyledons at different developmental stages
were prepared by homogenizing cotyledons in a medium containing
100 mM Tris-HCl, pH 7.5, 1 mM EDTA, 10% (w/w) sucrose. After
Miracloth filtration and precentrifugation at 1,000 g for 2 minutes the
cell extract was centrifuged at 80,000 g for 2 hours. The supernatant
of this centrifugation contained soluble proteins such as storage
proteins, vacuolar proteases and cytosolic enzymes, e.g. malate dehydrogenase (MDH). Endopeptidase activity in soluble extracts (50 µg
protein per assay) was determined at pH 4.5 (potassium acetate buffer)
using azocasein as substrate, as previously described (Demmer et al.,
1993). Exopeptidase activity (also determined on 50 µg soluble
protein extracts) was measured exactly as described by Nishimura and
Beevers (1978) using carboxy-benzoxyphenylalanine as substrate.
lead citrate when necessary, and observed with a Philips CM 10
electron microscope.
Immunocytochemistry
Slices of cotyledon tissue (1 mm thick) were immersed in primary
fixative (2% (w/v) paraformaldehyde, 1% (v/v) glutaraldehyde in 50
mM sodium cacodylate, pH 7.4) for 2 hours at room temperature, and
then, after renewing the fixative, for a further 16 hours at 4°C. The
tissue slices were then washed in 50 mM cacodylate buffer (3× 10
minutes) and subsequently placed in freshly prepared secondary
fixative (1% OsO4, 0.8% (w/v) K4Fe[CN]6.3H2O in 50 mM sodium
cacodylate, pH 7.4) for 2 hours at room temperature. After washing
with cacodylate buffer (2× 10 minutes) and distilled water (2× 10
minutes) the tissue slices were treated with an aqueous borohydride
solution (0.5% (w/v) KBH4; 30 minutes) and then washed again in
distilled water (3× 10 minutes) before dehydrating (10 minutes in
30%, 50%, 70%, 90%, 3× 10 minutes in 100% ethanol) and infiltrating with LR White hard grade. Polymerisation (in the absence of O2)
took place at 60°C for 20-24 hours. Thin sections were picked up on
formvar-coated nickel grids, held on Tris-buffered saline (TBS) for
up to 30 minutes before blocking (30 minutes) with either: (a) fresh
skimmed (1.5% fat) milk (for α-TIP); or (b) a solution containing 3%
(w/v) BSA + 0.2% (v/v) BSA-C (Aurion, Biotrend, Köln, FRG) in
TBS (for the other antisera). Then the sections were incubated for 1
hour at room temperature on primary antibody solutions, washed in a
solution containing 1% (w/v) BSA + 0.07% (v/v) BSA-C in TBS (4×
5 minutes) and exposed to 10 nm gold-conjugated secondary antibody
(Biocell, Cardiff, UK; diluted 1:30 in washing solution). The grids
were then washed again (2× 5 minutes in washing solution, 2× 5
minutes in double-distilled water) and poststained in uranyl acetate
and lead citrate.
Antibodies and their dilutions
The antibodies employed in this investigation were as follows
(dilutions for western blotting are given in parentheses), [dilutions for
immunogold labelling are given in square brackets]: (1) affinitypurified polyclonal antibodies raised against legumin from Pisum
sativum seeds (Casey, 1979); diluted (1:3000), [1:200]. (2) affinitypurified polyclonal antibodies raised against vicilin from Pisum
sativum seeds (Croy et al., 1980); diluted (1:1000), [1:2 vol. pea leaf
extract : 1 vol. washing solution]. (3) polyclonal antibodies raised
Protein determinations
Protein was measured according to Peterson (1977).
SDS-PAGE and western blotting
SDS-PAGE (on 10 or 12% minigels), silver staining, electrophoretic
transfer of polypeptides and incubation with primary antibodies was
as previously described (Demmer et al., 1993). Visualisation of bound
antibodies was with an ECL kit (Amersham-Buchler, Braunschweig,
FRG).
Conventional electron microscopy
Segments (1 mm3) of cotyledon tissue were prefixed for 2 hours at
room temperature in 2% (v/v) glutaraldehyde in 50 mM sodium
phosphate, pH 7.0, washed 4× 15 minutes in the same buffer and then
either postfixed for 3 hours at room temperature in 2% (w/v) OsO4 or
postfixed for 2 hours at room temperature in 2% (w/v) OsO4 containing 0.8% (w/v) K4Fe(CN)6.3H2O. The tissue was subsequently
washed in phosphate buffer, then in water and stained en bloc in
aqueous 2% (w/v) uranyl acetate for 16 hours at 4°C. Dehydration
was either in an acetone series, followed by embeddment in Spurr’s
resin, or in an ethanol series, followed by embeddment in London
Resin White (Hard Grade). Thin sections were picked up on Formvarcoated, 100- or 200-mesh grids, poststained with uranyl acetate and
Fig. 1. Activity of vacuolar proteases as a function of cotyledon age.
(d) Endoproteases; 100% corresponds to 2.1 units min−1 mg
protein−1. (s) Exopeptidases; 100% corresponds to 0.74 µmol min−1
mg protein−1. (m) NADH-dependent malate dehydrogenase; 100%
corresponds to 7.2 mmol min−1 mg protein−1. DAF, days after
flowering.
Protein storage vacuoles in peas
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Fig. 2. Thin sections through developing pea cotyledon cells: (a) 4/5 mm diameter cotyledon. Storage protein synthesis is not yet underway.
Numerous vegetative vacuoles (VV) are visible; (b) 7 mm diameter cotyledon (200 mg fwt). Low magnification micrograph showing storage
protein deposits (arrowheads) surrounding groups of VV, which are pressed together. (c, d) High magnification micrographs demonstrating that
the storage protein deposits (arrowheads) lie outside the VV and within a tube-like membrane system (arrows). a, ×3,240; b, ×2,700 (bars, 5
µm); c, ×24,840; d, ×34,470; bars, 0.5 µm.
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Fig. 3. Later stages in the development of the protein storage vacuole (PSV) seen in cells from 8 mm pea cotyledons. (a) Micrograph depicting
the dilation of the tube-like PSV precursor. (b) Penultimate stage in PSV development. Internal VV are very small. The PSV has enlarged
considerably and contains storage protein aggregates in its lumen as well as at its periphery (arrowheads). (a) ×23,100; bar, 0.5 µm; (b)
×41,800; bar, 0.25 µm.
Protein storage vacuoles in peas
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Fig. 4. (a) Immunocytochemistry of pea cotyledon cells: control section prepared for immunogold labelling without an incubation in primary
antiserum. (b, c) Imunogold localisation of vicilin in the tube-like PSV precursor (b) and in the peripheral storage protein deposits of the
dilating young PSV (c). CW, cell wall; PB protein body. (a) ×20,700; (b) ×25,875; (c) ×28,800; bar, 0.5 µm.
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Fig. 5. Immunogold localisation of the protein body integral membrane protein α-TIP. This antigen is not present in VV (a) but is recognisable in
the dilating young PSV (b) and, of course, in the membrane of mature protein bodies (a, c). (a) ×53,000; (b) ×60,000; (c) ×57,000; bars, 0.25 µm.
against the 25 kDa integral membrane protein (α-TIP) of Phaseolus
vulgaris protein bodies (Johnson et al., 1989); diluted (1:2000), [1:2
vol. pea leaf extract : 1 vol. fresh skimmed milk; Melroy and Herman
(1991)]. (3) Polyclonal antibodies raised against the 27 kDa integral
membrane protein (TIP-Ma 27) of Beta vulgaris storage root
parenchyma vacuoles (Marty-Mazars et al., 1995); diluted (1:2000),
[1:200]. (4) Polyclonal antibodies raised against the 73 kDa pyrophosphatase subunit (PPase) of Phaseolus mungo hypocotyl tonoplast
(Maeshima and Yoshida, 1989); diluted (1:250), [1:20]. (5) Polyclonal antibodies raised against the holoenzyme of the tonoplastic H+ATPase from Kalanchoe daigremontiana (Bremberger et al., 1988;
Haschke et al., 1989); diluted [1:100].
RESULTS
The deposition of storage proteins during the maturation of pea
cotyledons is accompanied by a dramatic reduction in proteolytic activities (Fig. 1). This is a specific vacuolar event, since
cytosolic enzyme activities, e.g. malate dehydrogenase, do not
behave in the same manner. The question therefore arises as to
whether this feature of cotyledon development is a result of the
turnover of the proteolytic enzymes or of the vacuoles. The
results that we present below suggest that the latter is the more
likely possibility.
Conventional electron microscopy
Thin sections prepared from pea cotyledons (var. Haubners
Exzellenz) fixed before storage protein synthesis had begun (45 mm cotyledons) revealed profiles of numerous vegetative
vacuoles per cell (Fig. 2a). At intermediate stages of development, i.e. during active synthesis of storage proteins (7/8 mm
Protein storage vacuoles in peas
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Fig. 6. Immunolocalisation of TIP-Ma 27, a VV membrane protein. (a) Low magnification overview necessary for orientation of a VV lying
within an expanding PSV. (b, c) High magnification details of positively labelled VV and unlabelled PSV membranes. (a) ×13,000; bar, 1 µm;
(b) ×50,000; bar, 0.25 µm; (c) ×37,000; bar, 0.25 µm.
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Fig. 7. Immunogold localisation of tonoplastic PPase and V-ATPase, proteins normally present in VV. (a, b) Detection of PPase in the
membranes of developing PSV (a) and mature protein bodies (b). (c, d) As for a and b, but with V-ATPase antiserum. (a, b) ×44,100;
(c) ×37,000; (d) ×34,000; bars, 0.25 µm.
diameter cotyledons) we have observed structures hitherto not
reported in cells of this tissue. Storage protein deposits are
highly osmiophilic, as documented in all previous ultrastructural investigations on legume cotyledons. The first recognizable deposits of this type are visible in 8 mm, 200 mg cotyledons. At low maginification (Fig. 2b) storage protein clumps
are often seen at the periphery of groups of VV which seem to
be pressed together. Higher magnifications (Fig. 2c,d) show
that the storage protein deposits are actually contained in a
tenuous, tube-like, membrane system. In conventional, glutaraldehyde/osmium-fixed tissue these membranes are thinner
and have less contrast than the tonoplast of the VV.
Although adjacent cells often show these structures at presumably different developmental stages the sequence of events
that we now present seems, in our opinion, to be the most likely
one. The next stage in our reconstruction of storage protein vacuologenesis appears to entail a dilation of the tube-like
membrane system (Fig. 3a). Since the storage protein deposits
are always found appressed to the outer surface of the tubes,
the enlargement takes place through an inwardly directed
expansion. As a result the VV appear to get smaller and can be
recognized in section as a ‘two-membrane compartment’ (Fig.
3a). The impression was often gained that the VV is completely
engulfed by the expanding tubes, but we have also seen images
that suggest that the VV is expelled into the cytoplasm. Dilation
of the tube system continues until it reaches vacuole-like proportions (Fig. 3b). At this stage only small profiles of internal
membranes are visible and the lumen is seen to contain small
clumps of storage protein in addition to the larger deposits at
the periphery. These are clearly the protein storage vacuoles
(PSV), previously described by Craig and coworkers (e.g. see
Craig et al., 1979, 1980) in developing pea cotyledons.
Immunogold localisation of vacuolar markers
In order to be able to identify, and therefore differentiate
between, the VV and the PSV we have performed postembedding immunogold labelling with antisera directed against
several characteristic VV and PSV proteins. Control sections
treated with gold-coupled secondary antibodies without a prior
incubation in primary antibody solution revealed virtually no
gold labelling (Fig. 4a). Using vicilin antibodies we were able
to confirm that the osmiophilic deposits in the tube-like
Protein storage vacuoles in peas
Fig. 8. Total cell membranes of young pea cotyledons (3-4 mm
diameter; 8-15 mg fr. wt) separated by isopycnic centrifugation on
linear sucrose density gradients. The distribution of marker enzyme
activities for ER (CCR), GApp (IDPase) and the vacuolar marker VATPase across the gradient are presented together with profiles of
western blots for TIP-Ma 27 and PPase. At this stage α-TIP and
legumin are not expressed in cotyledon tissue.
membrane system do indeed contain storage proteins, both at
the early (‘chain of beads’) and late (dilation) stages (Fig.
4b,c). Similar labelling was obtained with legumin antibodies
(data not shown).
Antibodies prepared against the integral membrane protein
of bean seed PBs (α-TIP; Johnson et al., 1989) label the
membrane of the tube-like PSV precursor (Fig. 5b) as well as
the membrane of fully developed PBs in pea cotyledons (Fig.
5c) but not VV (Fig. 5a). Despite the use of a mixture of pea
leaf extract and low fat milk as a blocking reagent (recommended by Melroy and Herman, 1991) we have found the
degree of non-specific labelling with α-TIP antiserum to be
higher than with any of the other antisera used in this study.
Antibodies raised against an integral tonoplast polypeptide of
the γ-TIP family, TIP-Ma 27 (Marty-Mazars et al., 1995),
labelled the internal remnants of presumptive VV membrane
within the dilating PSV (Fig. 6b). By contrast gold label is
completely absent from the membrane of the PSV (Fig. 6a,c).
In addition to labelling the membranes of VV (data not shown),
antibodies directed against PPase and V-ATPase also labelled
the membranes of developing PSV (Fig. 7a,c) as well as mature
PBs (Fig. 7b,d). Positive labelling of the PB membrane was
also observed with an antiserum prepared against the 70 kDa
subunit of the V-ATPase (Mandala and Taiz, 1986; data not
shown). Thus immunocytochemical discrimination between
PSV and VV membranes was not possible with these two types
of antisera.
Subcellular fractionation
In order to confirm our ultrastructural and immunocytochemical data we have performed subcellular fractionation with pea
307
Fig. 9. As for Fig. 8 but for pea cotyledons at an intermediate
developmental stage (7 mm; 150 mg). In addition to TIP-Ma 27 and
PPase, western blot profiles for the PSV membrane protein α-TIP
and luminal protein legumin (prolegumin polypeptides at 70/60 kDa;
mature legumin at 40 kDa; gel run under reducing conditions) are
presented.
cotyledon (var. Kleine Rheinländerin) tissue at three different
developmental stages. ER and GApp (Golgi apparatus)
membranes were identified in isopycnic sucrose density
gradients by measuring the marker enzymes CCR and IDPase,
respectively. The activities of the two tonoplast enzymes PPase
and V-ATPase were also measured in gradient fractions, but,
of the two, only profiles for V-ATPase are presented, since
PPase produced a broad smear across the gradient. This is
reflected in western blots using PPase antiserum.
In gradients of total cell membranes from young cotyledons
(3/4 mm, 8-15 mg) neither storage nor membrane intrinsic
proteins of PSV were detectable (Fig. 8). By contrast, the VV
membrane marker, TIP-Ma 27, was found in fractions (3032%, w/w, sucrose) that were slightly denser than those containing ER and GApp membranes. As cotyledons mature the
equilibrium density of both ER and GApp membranes shows
a progressive increase to a value of around 30%, w/w, sucrose
(compare Fig. 8 with Figs 9 and 10). This feature is not shown
by TIP-Ma-27-carrying membranes. At the crucial intermediate stage of development (7 mm, 150 mg cotyledons) the TIPMa 27 peak falls in a trough between fractions of lower (2026%) and higher (36-45%) sucrose concentration which
contain α-TIP-bearing membranes (Fig. 9). In mature cotyledons (10 mm, 250 mg) there is still a major peak of α-TIPbearing membrane at an equilibrium density of around 3644%, w/w, sucrose, but the lighter α-TIP fractions seem to
have disappeared (Fig. 10). A closer look at the marker enzyme
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B. Hoh and others
are detected in the first two or three fractions (see especially
Fig. 9). In mature cotyledons the cells are full of PBs that,
because of their extreme density, are pelleted out either in the
preliminary centrifugation step or in the gradients, thereby
reducing this effect. As an explanation for the predominance
of pro- rather than mature legumin in PSV-containing fractions
we would like to point out that these two forms of legumin
have different solubility properties. Whereas legumin can be
effectively removed from PBs with 1 M NaCl (Mäder and
Chrispeels, 1984), this treatment is only capable of solubilizing about one half of the endomembrane-bound prolegumin (R.
Jung, personal communication; and our own unpublished
data).
DISCUSSION
Fig. 10. As for Fig. 8 but for mature pea cotyledons (10 mm; 250
mg).
profiles in Figs 9 and 10 suggest, however, that, whilst the
amount of α-TIP in the lighter fraction decreases, this fraction
also shifts to a higher density in parallel with the behaviour of
the ER and GApp membranes.
Profiles for V-ATPase activity show two peaks of activity
in isopycnic gradients prepared from all three stages of
cytoledon development. The most prominent of the two lies at
a density of 36-43% sucrose and therefore coequilibrates with
α-TIP-bearing membranes. The other coincides with those
fractions containing TIP-Ma 27. In mature cotyledons this
latter peak is considerably reduced. Our results therefore point
strongly to the existence of two populations of vacuoles, since
the different isopycnic densities of TIP-Ma 27- and α-TIP-containing membranes preclude the possibility of these two
antigens belonging to the same membrane. Further evidence
against the idea that storage proteins accumulate in VV is the
observation that the major prolegumin (70 and 60 kDa
polypeptides)-containing fractions in density gradients
prepared from both intermediate and mature pea cotyledons
have densities different from that of the TIP-Ma 27-containing
fractions.
In gradients prepared from mature cotyledons there is poor
agreement on the positions of prolegumin- and α-TIP-containing fractions (Fig. 10). One would expect PSV to contain
more legumin than prolegumin and this should be reflected in
the presence of 40 and 20 kDa polypeptides in western blots
of reducing gels. Their absence in PSV-membrane-containing
fractions suggests that these polypeptides have been released
during suspension of the total membrane fractions prior to
layering onto the gradients. They then enter the gradient and
The vacuole of plant cells is usually regarded as a multifunctional organelle, although it would be incorrect to assume that
all vacuoles are identical in either a single cell or tissue (see
Wink, 1993, for a recent review). In this respect there is an
increasing body of evidence that demonstrates the distinctive
character of vacuoles in mature seeds. Not only are copious
amounts of nitrogen-rich proteins deposited in them, hence the
name protein storage vacuole (PSV), but their tonoplast is
characterized by the presence of a particular integral membrane
protein of molecular mass 25 kDa, named α-TIP (Johnson et
al., 1989, 1990). By contrast the tonoplast of vegetative tissues
in higher plants is characterized by the presence of an isoform
of TIP, termed γ-TIP (Höfte et al., 1992), as well as the
presence of two H+-pumps, a V+-ATPase (Ward and Sze,
1992) and a PPase (Rea and Poole, 1993). In this study we have
been able to confirm the presence of α-TIP in the PSV of
maturing pea cotyledons, but have shown, for the first time,
that PSV also possess PPase and V-ATPase polypeptides. It
has therefore been possible, through immunocytochemistry
and subcellular fractionation, to distinguish between two populations of vacuoles in the cotyledons of maturing pea seeds.
The failure of others (e.g. see Craig et al., 1979, 1980) to detect
a developing PSV during cotyledon maturation may have been
a consequence of inadequate representation of early developmental stages. Indeed a closer scrutiny of the PSV in Fig. 7 of
Craig et al. (1979) does reveal an internal convoluted
membrane, which could represent a remnant of a VV.
Melroy and Herman (1991) have shown that the “accumulation of α-TIP in the PSV of soybean cotyledons is not correlated with the presence or concentration of stored protein in
the organelle”. Indeed α-TIP appears to be expressed quite late
in cotyledon development. Our data are in accordance with
this, since PSV-associated V-ATPase activity is detectable at
a particular density in gradients before α-TIP is detectable at
this position. The claim of Melroy and Herman (1991) that the
GApp is implicated in the transport of α-TIP to the PSV also
finds support from our gradient experiments, in that α-TIP is
seen to be transiently present in fractions enriched in ER and
GApp membranes.
Having established that there are two populations of
vacuoles in maturing pea cotyledons there arise two immediate
questions. The first addresses the fate of the VV. In thin
sections of mature pea cotyledon tissue the cytoplasm is full
of PBs, as documented on numerous occasions by several
Protein storage vacuoles in peas
authors (see references in the Introduction). Vacuoles as we
normally recognize them appear to be absent at this stage of
development and yet our gradient data indicate that VV
membrane must still be present on the basis of the continued
detection of TIP-Ma 27. Clearly, immunocytochemistry with
anti-TIP-Ma 27 should solve this problem and we will proceed
to try and provide an answer when these antibodies again
become available.
The second, perhaps more interesting question concerns the
origin of the PSV. Two possibilities for vacuolar ontogeny
have been discussed in the literature. In one, the VV is held to
be a product of what is now considered to be the trans-Golgi
network (TGN). According to this concept a reticulate, tubular
provacuolar system is believed to emanate from the TGN
(Marty et al., 1980). In meristematic cells these are assumed
to adopt a lytic function by engulfing organelles or cytoplasm.
The other hypothesis would have vacuoles developing directly
from the ER (see Amelunxen and Heinze, 1984; Robinson,
1985, and literature cited therein). The following observations
are common to a number of investigations that support this
notion: firstly, the formation of a reticulate, smooth tubular
network, which in cross-section resembles a perforated nuclear
envelope, and in three dimensions is roughly spherical. This
gradually fuses to form large flat cisternae, which then dilate
inwardly thereby displacing the residual cytoplasm. These,
purely ultrastructural, observations are supported by histochemical data for ER and vacuolar components (Hilling and
Amelunxen, 1985). The developmental changes in the vacuolar
system of maturing pea cotyledons, as described above, are
therefore quite similar, the only difference is that VV rather
than cytoplasm become encircled by the developing PSV
membrane system.
PSV membranes do not possess the ER marker enzyme
cytochrome c reductase (CCR), but may have received luminal
ER markers such as BiP (Boston et al., 1991) or calreticulin
(Sönnichsen et al., 1994). Antisera against these, as well as
against the K(H)DEL tetrapeptide (Napier et al., 1992), may
prove to be of use in determining whether the PSV is a product
of the ER or the GApp. If the former turns out to be the case
it will not be without consequence for current concepts of the
intracellular transport of storage proteins in legumes (Müntz,
1989). In particular the role of the GApp in the deposition of
the storage protein desposits in very young, non-dilated PSV
will need to be explained.
We thank Sybille Hourticolon for the photographic work, Bernd
Rauffeisen for preparing the drawings, and and Heike Freundt for help
in processing the manuscript. We are greatly indebted to a number of
colleagues who provided us with antisera, they are: R.D. Casey,
R.R.D. Croy, M.J. Chrispeels, H.-P. Haschke, M. Maeshima, F.
Marty. This study was supported by funds from the Deutsche
Forschungsgemeinschaft.
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(Received 31 March 1994 - Accepted 7 September 1994)