Journal of Experimental Botany, Vol. 58, No. 5, pp. 1059–1070, 2007
doi:10.1093/jxb/erl267 Advance Access publication 17 January, 2007
RESEARCH PAPER
Protein storage vacuole acidification as a control of
storage protein mobilization in soybeans
Fanglian He*,†, Feilei Huang*,‡, Karl A. Wilson and Anna Tan-Wilson§
Department of Biological Sciences, State University of New York at Binghamton, Binghamton,
NY 13902-6000, USA
Received 28 July 2006; Revised 24 October 2006; Accepted 13 November 2006
Abstract
Soybean protease C1 (EC 3.4.21.25), the subtilisin-like
serine protease that initiates the proteolysis of seed
storage proteins in germinating soybean [Glycine max
(L.) Merrill], was localized to the protein storage
vacuoles of parenchyma cells in the cotyledons by
immunoelectron microscopy. This was demonstrated
not only in germination and early seedling growth as
expected, but also in two stages of protein storage
vacuole development during seed maturation. Thus,
the plant places the proteolytic enzyme in the same
compartment as the storage proteins, but is still able
to accumulate those protein reserves. Since soybean
protease C1 activity requires acidic conditions for
activity, the hypothesis that the pH condition in the
protein storage vacuole would support protease C1
activity in germination, but not in seed maturation, was
tested. As hypothesized, acridine orange accumulation
in the protein storage vacuole of storage parenchyma
cells was detected by fluorescence confocal microscopy in seedlings before the onset of mobilization of
reserve proteins as noted by SDS-PAGE. Accumulation of the dye was reversed by inclusion of the weak
base methylamine to dissipate the pH gradient across
the vacuolar membrane. Also as hypothesized, acridine orange did not accumulate in the protein storage
vacuole of those parenchyma cells during seed maturation. These results were obtained using cells separated by pectolyase treatment and also using
cotyledon slices.
Key words: Acidity, cotyledon, enzyme regulation, protease
C1, protein storage vacuole, proteolytic enzyme, seed
maturation, soybean [Glycine max (L.) Merrill], storage protein
mobilization, subcellular localization.
Introduction
Seeds of diverse plant species employ a variety of regulatory mechanisms to ensure that proteins accumulate
during seed development; and that the reserves are mobilized during germination and early growth. Reserve
proteins are accumulated in membrane-bound protein storage vacuoles. In soybeans, the two main types of storage
proteins are the 11S glycinins and the 7S b-conglycinins,
both types being simultaneously present in all protein
storage vacuoles (Horisberger et al., 1986). The glycinins
are hexameric structures; each subunit being composed of
disulphide-bonded acidic and basic polypeptide chains
that arise from proteolytic processing of single-chain
precursor proteins (Nielsen et al., 1989).
The b-conglycinins are glycoprotein trimers composed
of homologous a#, a, and b subunits in various combinations (Thanh and Shibasaki, 1977). The N-terminal 141
and 124 amino acid residues of the a# and a subunits,
respectively, are lacking in the b subunit and are often
referred to as the N-terminal extensions (Sebastiani et al.,
1991). Wilson et al. (1986) showed that the glycinin
acidic chains and the b-conglycinin a# and a subunits are
mobilized first in seedling growth. Glycinin acidic chains
lose 1–2 kDa in mass while a# and a subunits of bconglycinin are degraded in their N-terminal extensions
through successive steps losing about 2–9 kDa in mass
each time, suggesting the result of endoprotease action.
Moreover, the appearance and subsequent disappearance
* These authors contributed equally to this work.
y
Present address: Department of Biology, University of Pennsylvania, Philadelphia, PA 19104, USA.
z
Present address: Proteomics Core, COBRE Center for Cancer Research Development, Rhode Island Hospital, Providence, RI 02903, USA.
§
To whom correspondence should be addressed. E-mail: [email protected]
ª The Author [2007]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.
For Permissions, please e-mail: [email protected]
1060 He et al.
of discrete polypeptide chains indicate that cleavage is at
specific sites. Those early proteolytic events within the Nterminal extensions of the a# and a subunits of soybean bconglycinin were shown to be catalysed by protease C1
(Qi et al., 1992, 1994; Tan-Wilson et al., 1996), a serine
protease belonging to the subtilisin family (Liu et al.,
2001). This was demonstrated by the enzyme’s in vitro
specificity for the a# and a subunits, but not the b subunit
of soybean b-conglycinin; as well as the temporal pattern
of appearance, the sizes and the N-terminal sequences of
the proteolytic intermediates showing cleavage only at
sequences rich in contiguous Glu residues (Qi et al., 1992,
1994; Tan-Wilson et al., 1996). These matched observations of the intermediates in vivo, down to the 50 kDa and
48 kDa core regions of the a# and a subunits, respectively
(Qi et al., 1992; Kawai et al., 1997). Moreover, the
specificity is consistent with the cleavage specificity of
protease C1 on synthetic peptides (Boyd et al., 2002).
Purified protease C1 also catalyses hydrolysis of the acidic
subunits of the glycinins, reducing each by 1–2 kDa (Qi
et al., 1994). The presence of contiguous acidic amino
acid residues near their C-termini suggests the general
location of these cleavage reactions. The cleavage specificity of protease C1, combined with the limited presence
in the storage proteins of the type of sequence at which it
can act, makes this protease suitable for early, but limited,
degradation of protein stores, leaving the unprocessed
portions of the storage proteins for utilization at a later
stage of seedling development.
As the first proteolytic enzyme to initiate protein reserve
mobilization, study of its regulation would contribute to
understanding how plants switch from synthesis and
accumulation of storage proteins during development to
hydrolysis of those proteins upon germination. The
regulation of protease C1 activity is not accomplished by
delaying transcription, translation, or processing to active
enzyme until germination. Transcription of the gene coding
for protease C1 occurs even during seed development, as
shown by RNA gel blot analysis, and developing seeds
have protease activity with the cleavage specificity characteristic of protease C1 (Liu et al., 2001). The possibility of
inhibition by the abundant Kunitz and Bowman–Birk
trypsin inhibitors that also accumulate in the protein
storage vacuoles (Diaz et al., 1993) is ruled out, as
protease C1 activity is not affected by these protease
inhibitors in vitro (Qi et al., 1992). Two other hypotheses
remain to be investigated. The localization of protease C1
in lytic vacuoles (Jiang and Rogers, 1998; reviewed by
Robinson et al., 2005), separated from the seed storage
proteins during seed development, could prevent protease
C1 from acting on the storage proteins. Alternatively, the
enzyme may be in the protein storage vacuoles along with
the storage proteins, but conditions in the vacuoles of
maturing seed are not conducive to protease C1 activity. A
likely regulating condition would be pH, as the in vitro
activity of the enzyme is known to decrease precipitously
at pH values above the optimum pH range of 3.5–4.5. At
pH 6, depending on whether the substrate is the a or the
a# subunit of soybean b-conglycinin, protease C1 activity
is only 10–20% of the pH 4 optimum activity. At pH 7, it
is 0–10% (Qi et al., 1992).
To explore the two hypotheses for regulation of protease
C1 activity (separation from the substrate storage proteins
during seed development, or localization in the same
compartment as its substrate proteins, but at a pH condition
not conducive to activity) the subcellular localization
of protease C1 and the pH conditions in protein storage
vacuoles in the parenchyma cells of seedling cotyledons
and maturing seeds have been examined.
Materials and methods
Plant materials
Soybean seeds [Glycine max (L.) Merrill] were imbibed in distilled
water and grown on moist filter paper for 2 d at 21 C in the dark.
Time was reckoned from the beginning of imbibition. For longer
periods of seedling growth, plants were grown in the growth
chamber with a cycle of 16 h at 25 C and 8 h at 20 C in
continuous darkness. For production of developing seeds, soybeans
were planted in potting soil and grown under greenhouse conditions
with a 16 h photoperiod, with the temperature regulated at 23–
27 C in the light and 18–22 C in the dark.
SDS-PAGE analysis and western blot
Extracts were prepared by homogenizing cotyledons in 50 mM
TRIS-Cl, 100 mM NaCl, pH 8.0, using 1.5 ml of buffer per
cotyledon. Dry seeds were first ground to a fine powder using a seed
mill before homogenization with buffer. To prevent proteolysis
during extraction, a protease inhibitor cocktail consisting of 2 mM
4-(2-aminoethyl) benzenesulphonyl fluoride, 1 mM EDTA, 133 lM
bestatin, 14 lM E-64, 1 lM leupeptin, and 0.3 lM aprotinin was
added to the extraction buffer. The homogenates were centrifuged at
40 000 g for 30 min and the clear supernatant collected. SDS-PAGE
was performed by the method of Laemmli (1970) using 12%
acrylamide. The separated polypeptides were detected by staining
with Coomassie Brilliant Blue G250. For quantitation of the stained
polypeptide band intensities, the gel was scanned and the image
analysed using 1D Image Analysis software from Kodak Digital
Science (Rochester, NY).
For western blot analysis polypeptides separated by SDS-PAGE
were electrophoretically transferred to a nitrocellulose membrane
(BA83, Schleicher and Schuell, Keene, NH) by the method of
Towbin et al. (1979). Protease C1 was immunostained with affinitypurified protease C1-specific antibody (1.12 mg ml 1) diluted
1:250, followed by detection with goat anti-rabbit IgG–alkaline
phosphatase conjugate as described by Barnaby et al. (2004).
Immunoelectron microscopy
Small pieces (0.530.532 mm) cut from the cotyledons were fixed
for 24 h at 4 C in a mixture of freshly prepared 4% (w/v)
paraformaldehyde, 1% (v/v) glutaraldehyde in 0.1 M potassium
phosphate buffer (pH 7.2). The tissue blocks were then washed with
the same buffer twice for 1 h each at 4 C. The blocks were then
dehydrated in a graded ethanol series (30, 50, 70, and 80% (v/v)
Soybean protease C1 1061
ethanol for 20 min each; then 90% and 100% ethanol for 1 h each).
This was followed by infiltration with a graded series of 100%
ethanol/LR White resin (Electron Microscopy Sciences, Philadelphia, PA) at 4 C (2:1, 1:1, and 1:2) followed by pure LR White
resin for 1 h, then with fresh resin overnight for 36 h at 56 C. The
sections (60–90 nm thick) were cut with a Reichert–Jung Ultracut
ultramicrotome and placed on Formvar-coated nickel grids.
Prior to immunostaining, sections were digested with 0.5% (w/v)
bovine trypsin (Sigma–Aldrich, St Louis, MO) in 10 mM TRISHCl, 0.1% (w/v) CaCl2, pH 7.4 at room temperature for 5 min, then
washed 435 min with TBS buffer (25 mM TRIS-Cl, 150 mM
NaCl, pH 7.4). Blocking was done with 5% (w/v) non-fat milk
powder in TBST [TBS with 0.1% (v/v) Tween-20] for 2 h. The
grids were incubated at 4 C overnight with affinity-purified
primary antibody (1.12 mg ml 1, diluted 1:75 in TBST containing
5% (w/v) non-fat milk powder). The sections were successively
washed 435 min each with TBST. This was followed by incubation
with goat anti-rabbit IgG conjugated to 10 nm gold particles
(Electron Microscopy Sciences) (1:30 dilution in TBST containing
5% non-fat milk powder) for 1 h at room temperature. After
washing, the sections were counterstained with a 2% aqueous uranyl
acetate solution stain (Watson, 1958) followed by a lead citrate stain
(Venable and Coggeshall, 1965). Controls were developed in the
same way, but using non-immune rabbit IgG (Sigma–Aldrich) in
place of the primary antibody. The specimens were viewed using
a Hitachi 7000 STEM scanning transmission electron microscope
(Ibaraki, Japan) and photographed using Kodak 4489 EM film
(Eastman Kodak Company, Rochester, NY). For structural analysis
on the electron microscope, similar tissue blocks were prepared and
post-fixed in 0.5% osmium tetroxide for 2 h at room temperature.
The blocks were dehydrated using a graded series of acetone and
infiltrated with Spurr’s resin overnight at 70 C. Thin sections (60–
90 nm) were cut, stained with uranyl acetate and lead citrate, and
viewed in the electron microscope as described above.
Maceration of cotyledon tissue
Cotyledons were harvested and cut into thin transverse slices less
than 1 mm thick with a razor blade, and suspended in sterile 10 mM
potassium 2-(N-morpholino)ethanesulphonate, 600 mM mannitol,
pH 5.5 at room temperature for 30 min with gentle shaking, then
vacuum infiltrated for 2 min with the same sterile buffer containing
10 mg ml 1 pectolyase Y-23 (Karlan Research Products, Cottonwood, AZ), 1 mg ml 1 ampicillin, and 5 mg ml 1 protease-free
bovine serum albumin (Research Organics, Cleveland, OH). After
20 h incubation in the dark with shaking, the separated cells were
recovered by passage through a 280 lm stainless steel screen
followed by centrifugation at 65 g for 6 min. The pelleted cells were
washed by suspending in the same buffer containing 0.1 mM
disodium EDTA, repelleted by centrifugation for 4 min, and then
resuspended in a minimal volume of the same wash solution.
Acridine orange treatment and confocal fluorescence
microscopy
Acridine orange (Sigma–Aldrich) was added to one aliquot of cell
suspension to a final concentration of 50 lM. Another aliquot of
cell suspension was treated with 50 lM acridine orange and 30 mM
methylamine hydrochloride. After incubation at room temperature
in the dark for 100 min, the cells were washed twice and suspended
in a minimal volume for examination by confocal microscopy. To
study cotyledon slices, transverse sections approximately 0.5 mm
thick were cut free-hand with a razor blade. The sections were
incubated for 2 h at room temperature with gentle shaking either in
50 lM acridine orange dissolved in water or in 50 lM acridine
orange with 30 mM methylamine hydrochloride. The slices were
then briefly washed with water and incubated in water or 30 mM
methylamine hydrochloride for 1 h at room temperature with gentle
shaking. Confocal microscopy was performed with a Zeiss LSM
510 confocal microscope. Excitation was at 488 nm using an argon
laser. Fluorescence emission was visualized at a series of wavelengths in the 582–655 nm range with a Plan-Neofluar 403 oil
immersion objective (1.3 NA) for single cells, and either a PlanApochromat 35 or 310 objective (NA 0.16 and NA 0.13,
respectively) for cotyledon slices. To allow direct comparison of
fluorescence intensity, care was taken to use the same confocal
microscope settings when studying all separated cells. Similar care
was taken when studying all cotyledon slices, with the additional
care to set the plane of focus at approximately one half of the
thickness of the slice.
For accompanying structural analysis, sections 0.5–0.75 lm thick
were cut and stained with 1% toluidine blue in a 1% sodium borate
aqueous stain solution, gently heated on a hot plate then carefully
rinsed with distilled water (Richardson et al., 1960). The stained
sections were photographed with an Olympus BX-60 microscope
with a 340 0.75 NA UPlanF1 objective and an Optronics digital
microscope camera system.
Results
SDS-PAGE analysis of storage protein proteolysis
An analysis of the time-course of storage protein
degradation was carried out to provide a reference point
for this investigation on the proteolytic enzyme that
initiates the mobilization of storage proteins. Proteins in
extracts of dry seed and of soybean cotyledons harvested
on different days after seed imbibition were separated on
an SDS-PAGE gel stained with Coomassie Blue. Protein
amounts in the lanes were equalized with respect to
cotyledon number. Figure 1A shows the resulting gel
pattern with the stained bands corresponding to the a#, a,
and b subunits of b-conglycinin, and the acidic and basic
polypeptides of glycinin, indicated at the side. Also
marked are 50 kDa and 48 kDa polypeptides, shown
previously (Wilson et al., 1986) to be in vivo proteolytic
breakdown products of the a# and a subunits of bconglycinin, respectively, as well as final products of
protease C1 action on purified b-conglycinin (Qi et al.,
1992; Tan-Wilson et al., 1996). Intensities of the stained
a#, a, and b subunits of b-conglycinin; and the acidic and
basic polypeptides of the glycinins were plotted in Fig.
1B. The stain intensities are presented as percentages of
the intensities in the day-1 sample rather than dry seed
because preparation of the protein extracts from dry seed
is different from the procedure used for the seedling
cotyledons. The data show that the proteolytic processing
of the a# and a subunits of b-conglycinin and of the
acidic polypeptides of the glycinins occur following the
second day, counting from the time the seeds are imbibed
with water. The b subunit of soybean b-conglycinin and
the basic polypeptides of the glycinins are mobilized later.
Figure 1C shows the change in intensity for the 50 kDa
and 48 kDa products of protease C1 action. It is common
1062 He et al.
to see small amounts of these proteins in dry seed; and
this is evident in this analysis. These proteins increase as
the b-conglycinin a# and a subunits lose their N-terminal
extensions until after day 6. Their decrease after day 6,
along with the decrease of the b-conglycinin b subunit,
has been attributed to proteolysis by protease C2,
a cysteine protease that is synthesized only upon germination (Seo et al., 2001).
Subcellular localization of soybean protease C1 in
seedling cotyledons
For protease C1 to be able to initiate hydrolysis of the
storage proteins, it would have to be in the protein storage
vacuoles by 2 d after imbibition, just before evidence of
its proteolytic action is noted. To test this, ultrathin
sections of soybean seedling cotyledons 2 d after imbibition were made and subjected to analysis by immunoelectron microscopy. The primary antibody used was
polyclonal antibody raised in rabbits immunized with
a synthetic peptide with the sequence GDAPNKGEGIDGSSSRY. This represents a sequence in protease C1
that was not present in any other of the sequenced
soybean, thale cress [(Arabidopsis thaliana (L.) Heynh.],
tomato (Lycopersicon esculentum Mill.), or muskmelon
(Cucumis melo L.) subtilases, or in other soybean proteins
with known sequence. The antibody, referred to as antiPC1, was previously described and characterized by
Barnaby et al. (2004) as anti-SBPC2. It binds to purified
protease C1 (70 kDa) and to a single 70 kDa band in
western blots of SDS-PAGE of soybean cotyledon extract
(Fig. 2). The secondary antibody was goat anti-rabbit IgG
conjugated to 10 nm gold particles. As shown in Fig. 3A,
with representative enlarged areas (a–d) of the image
below it, gold particle labelling was observed in the
protein storage vacuole, which appears as a membranebound vacuole uniformly filled with protein. The majority
of the gold particles appear singly as in areas c and d. No
label was seen in the protein storage vacuole when, as
a control, the same concentration of non-immune rabbit
IgG was used in place of anti-PC1 (Fig. 3B, with an
enlarged image to show the absence of gold particles).
Figure 3C shows a cotyledon cell with several protein
storage vacuoles surrounded by numerous oil bodies in
a section from a 2 d seedling cotyledon that was post-fixed
with osmium tetroxide to preserve membrane structures.
Fig. 1. (A) Coomassie Blue-stained SDS-PAGE gel loaded with protein
extracts of soybean seed and of seedling cotyledons. From left to right:
S, seed; M, molecular mass standards as listed to the right of the gel; 1,
2, 4, 6, 8 refer to days after water imbibition when cotyledons were
taken for protein extraction. Protein loads were normalized with respect
to the number of cotyledons. To the left of the gel, the stained bands
corresponding to the polypeptide chains of b-conglycinin (a’, a, b) and
the acidic and basic chains of the glycinins (A, B) are indicated; as are
the 50 kDa and 48 kDa proteolytic intermediates of b-conglycinin
digestion. (B, C) Staining intensities of the major storage protein
polypeptide chains are plotted versus days after imbibition.
(B) Intensities are presented as percentages of the stain intensities in
the day 1 sample; (open circles), a# subunit of b-conglycinin; (open
squares), a subunit of b-conglycinin; (filled diamonds), b subunit of bconglycinin; (open triangles), sum of intensities of A1, A2, A3, and A4
acidic chains of glycinin; (filled inverted triangles), basic chains of
glycinin. (C) Intensities are presented as percentages of the maximum
stain intensities of the day 6 sample; open circles, 50 kDa; open
squares, 48 kDa polypeptide chains from b-conglycinin digestion.
Soybean protease C1 1063
Fig. 2. Western blot analysis with anti-PC1 antibody as primary
antibody. Purified protease C1 (0.5 lg) and soybean seedling cotyledon
proteins (180 lg total) were separated on a 12% polyacrylamide gel and
then transferred to nitrocellulose. Lanes: M, molecular mass standards
(indicated to the left, in kDa); 1, purified protease C1; 2, seedling
cotyledon protein.
Figure 3D shows another immunostained section from a
2 d seedling cotyledon, showing the sparse labelling in the
areas outside of the protein storage vacuole, in particular
the cell wall, oil bodies, and the surrounding cytosol.
Examination of immunostained sections showed an
average of 0.24660.233 (mean 6SD) gold particles
lm 2 outside the protein storage vacuole compared with
an average of 5.03362.041 gold particles lm 2 in the
protein storage vacuoles.
Subcellular localization of soybean protease C1 in
developing seeds
The presence of protease C1 in the protein storage
vacuoles of seedling cotyledons raises the question as to
how soon in seed development the enzyme is placed
in this compartment. Cotyledons from developing seeds
12 mm and 5 mm long, i.e. ;85–90% and ;35–40%,
respectively, of the mature seed size (13–14 mm), were
studied. These cotyledons would have protein storage
vacuoles in different stages of development, starting from
their genesis from a single large central vacuole. Protein
accumulates only in the periphery during the early stages
and gradually advances toward the centre until the whole
vacuole is completely filled (Mori et al., 2004). Figure 4A
shows an overview of a cotyledon cell from a 12 mm seed
showing several protein storage vacuoles in the advanced
stage characterized by fairly uniform fill. Ultrathin
sections were treated with anti-PC1 as the primary antibody, then with goat anti-rabbit IgG conjugated to 10 nm
gold particles as the secondary antibody, and examined
at higher magnification. Figure 4B shows a portion of
a protein storage vacuole from such a section with gold
particles throughout. To show the gold particles more
clearly, the image in six areas of the two most prominent
vacuoles was enlarged in panels marked a–f. Figure 4C
shows a protein storage vacuole from a section that was
treated with non-immune rabbit IgG in lieu of primary
antibody as a control. The enlarged image (g) of the
boxed section from the non-immune rabbit IgG-stained
control in Fig. 4C focuses on a location that might at first
give the appearance of having gold particles; but has
none when viewed closely. Thus, the data indicate
protease C1 to be localized in the protein storage vacuoles
close to the end of seed fill.
To determine whether protease C1 is localized in the
protein storage vacuoles even earlier, developing seeds
that were just 5 mm long were examined. Even at this
early stage of seed maturation, Liu et al. (2001) had
demonstrated the presence of active protease C1. In these
seeds, protein storage vacuoles characterized by dense
accumulation of proteins along the inside periphery and
relatively sparse fill in the centre were found. These
represent an earlier stage of protein storage vacuole
development than those studied in Fig. 4. Figure 5A
shows a section immunolabelled with anti-PC1 as primary
antibody. Specific areas (a–e) in this section were enlarged
to show that gold particles were found throughout the
protein storage vacuole, both in the dense periphery as
well as in the dense areas within the sparsely filled
centres. Figure 5B shows a control section stained using
non-immune rabbit IgG in lieu of primary antibody. The
image of the boxed section was enlarged in panel f to
show the absence of gold particles more clearly. Collectively, the results in Figs 4 and 5 indicate that protease
C1 is localized in the protein storage vacuole during seed
formation.
Comparison of pH condition within protein storage
vacuoles in germination and in seed development
This brings up the question as to how protease C1 activity
is kept in check so that a futile cycle of storage protein
accumulation and storage protein degradation does not
occur. The possibility that pH is a factor in controlling the
1064 He et al.
Fig. 3. Immunoelectron microscopy of soybean seedling cotyledon 2 d after seed imbibition. (A, B) Portion of protein storage vacuole (PSV) in
ultrathin sections treated either with anti-PC1 antibody as primary antibody (A) or with non-immune rabbit IgG in lieu of primary antibody as control
(B) followed by goat anti-rabbit IgG conjugated to 10 nm colloidal gold particles. Below (A) to the left: areas a, b, c, and d enlarged four times to
show gold particles. The majority of the gold particles appear singly as in c and d. Below (B) to the right: boxed area in (B) enlarged four times.
(C) Section post-fixed with osmium tetroxide and stained with uranyl acetate and lead citrate shows cell with protein storage vacuoles (PSV) of
different sizes surrounded by numerous smaller oil bodies (OB). (D) Immunostained section focusing on cell wall (CW), oil bodies (OB) and
surrounding cytosol. The scale bar (25 nm) shown in d also applies to panels a, b, and c.
activity of protease C1 during seed development was
investigated. Slices of cotyledons of seedlings 2 d after
imbibition were macerated using pectolyase Y-23 to yield
separated cells that were then incubated with 50 lM
acridine orange. This dye has been used as an indicator of
acidic compartments, and has the advantage of detection
under conditions not complicated by autofluorescence in
the tissues examined in this study. Acridine orange passes
freely into membrane-bound spaces but cannot leave
when protonated, as would occur if the pH in those spaces
were lower relative to the surrounding cytosol. Fluorescence is detected as acridine orange accumulates. At
higher concentrations, as acridine orange oligomers are
formed, there is a shift in emission wavelength from
530 nm to 655 nm (Millot et al., 1997). Thus by examin-
ing at a range of different wavelengths, aggregation of
accumulated acridine orange is distinguishable from
acridine orange binding to nucleic acid. To verify that the
fluorescence is due to a pH gradient, the cells were also
treated with 50 lM acridine orange in the presence of
30 mM methylamine, a membrane-permeable weak base.
According to the hypothesis that pH conditions are not
conducive to protease C1 activity until after germination,
acidified protein storage vacuoles should be detected in
seedling cotyledons but not in maturing seed. Figure 6A–
C shows three representative separated cells that had
accumulated acridine orange in most or all of the protein
storage vacuoles in the viewing plane. Not all of the cells
would be expected to be in this condition since storage
protein mobilization starts at different times depending
Soybean protease C1 1065
Fig. 4. Immunoelectron microscopy of sections from a12 mm developing soybean seed cotyledon. (A) Overview showing several protein storage
vacuoles (PSV) and a starch grain (SG). (B, C) portions of PSVs from other sections labelled either with anti-PC1 as primary antibody (B) or with
non-immune rabbit IgG in lieu of primary antibody as control (C), followed by goat anti-rabbit IgG conjugated to 10 nm colloidal gold particles,
viewed in the microscope at higher magnification; a–f, areas of the image in (B) enlarged four times with arrows to indicate gold particles; g, boxed
area of the image in (C) enlarged four times.
on location in the cotyledon (Diaz et al., 1993). Those
cells that were present but did not emit fluorescence are
evident in the corresponding differential interference contrast (DIC) images of the same fields shown below the
corresponding fluorescent image. To determine if acridine
orange accumulation was indeed due to acidic conditions,
an aliquot of the cotyledon cell preparation was treated
with both acridine orange and methylamine. Almost no
fluorescence was emitted from any of the cells, some
representatives of which are shown in Fig. 6D and the
corresponding DIC image below.
According to the hypothesis being tested, the protein
storage vacuoles in cotyledons of maturing seed would
not have the acidic pH condition manifested as acridine
orange accumulation. Many cells from maturing seed
were examined. Representatives are shown in Fig. 6E and
the corresponding DIC image below. In contrast to Fig.
6A–C, these images show no fluorescence emitted from
the protein storage vacuoles from the cotyledon cells
taken during seed maturation.
To view more cells in situ, slices of cotyledons taken
from either seedlings or developing seed were treated
similarly with acridine orange and examined in the
confocal microscope. Figure 7A shows the situation 2 d
after seed imbibition, just before substantial proteolytic
digestion was evident (Fig. 1), in and around the centre of
the adaxial surface. Fluorescence due to acridine orange
accumulation was high throughout. The image in Fig. 7B,
taken at higher magnification, clearly shows fluorescence
staining of the cell walls, vacuoles in the bundle sheath
1066 He et al.
Fig. 5. Immunoelectron microscopy of a section from a 5 mm developing soybean seed cotyledon. (A, B) Sections labelled with anti-PC1 as primary
antibody (A) or with non-immune rabbit IgG in lieu of primary antibody as control (B), followed by goat anti-rabbit IgG conjugated to 10 nm
colloidal gold particles; a–e: areas of the image in (A) enlarged eight times; f, boxed area of the image in (B) enlarged eight times.
parenchymatous cells, and protein storage vacuoles in the
storage parenchyma cells. To determine whether the
fluorescence was due to a pH gradient across the vacuolar
membrane, similar cotyledon slices were treated with both
acridine orange and methylamine. The results are shown
in Fig. 7C. Methylamine did not dispel the fluorescence
emitted from the cell walls and bundle sheath cells,
indicating that the acridine orange is binding to nonpermeant compounds, thus not making it possible to
interpret the pH condition in those locations. More
important, however, is the substantial decrease in fluorescence emitted from within the majority of the cells, which
constitute the storage parenchyma.
To test the hypothesis that the acidic condition in the
protein storage vacuoles is gained only some time after
germination, cotyledon slices taken from seedlings 1 d
after water imbibition of the seed (Fig. 7D) were analysed,
focusing on the same central adaxial region. After acridine
orange treatment, there was accumulation in the vicinity of
the cell walls and the bundle sheath parenchymatous cells
but not within the storage parenchyma, as shown at higher
magnification of a representative slice in Fig. 7E. A fixed
and embedded section of the same tissue was stained with
toluidine blue in order to visualize the protein storage
vacuoles of these cells (Fig. 7F). Thus, these results show
that the protein storage vacuoles of the storage parenchyma cells in seedling cotyledons do undergo an increase
in acidity relative to the surrounding cytosol within the
first 48 h after seed imbibition. Similar results (not shown)
were found when the time-course study was focused on
the central abaxial region, as well as on the cotyledon
edges where abaxial and adaxial surfaces meet; except
for slight acridine orange accumulation just beginning to
be evident by the first day.
Cotyledon slices from developing seeds were treated
similarly and examined. One slice taken from a region
Soybean protease C1 1067
Fig. 6. Confocal microscopy of acridine orange-stained cells from soybean cotyledons. Fluorescent and DIC (below) images of separated cells from
cotyledons of seedlings 2 d after imbibition. (A–C) Treated with 50 lM acridine orange. (D) Treated with 30 mM methylamine and 50 lM acridine
orange. (E) Fluorescent and DIC (below) images of separated cells from maturing seed treated with 50 lM acridine orange. All fluorescent images
were taken with the same settings. Scale bars are equivalent to 20 lm.
near the central vascular bundle is shown at low and high
magnification in Fig. 7G and H, respectively. Although
acridine orange accumulated in the vicinity of the cell
walls and bundle sheath parenchyma, no fluorescence was
emitted from the protein storage vacuoles of the storage
parenchyma. This was true throughout the cotyledon. To
illustrate this further, the absence of fluorescence in the
protein storage vacuoles of the storage parenchyma cells
at another location, the central adaxial region, is shown in
Fig. 7I at higher magnification.
These experiments show how protease C1 activity
might be negatively regulated during seed maturation by
the pH condition in the protein storage vacuole. Protease
C1, even though present in the protein storage vacuole,
would not have the same acidic conditions to work in
during seed maturation as it has during seedling growth.
Discussion
Protease C1 is the first plant subtilase to be localized to
a vacuole. Most of the other subtilases that have been
localized in soybean (Beilinson et al., 2002) and other
plant species were found to be extracellular (Yamagata
et al., 1994; Hamilton et al., 2003; Schwend et al., 2003;
Tian et al., 2005). The targeting of protease C1 to the
endomembrane system was predicted when application of
the TargetP prediction program (Emanuelsson et al.,
2000) to the N-terminal sequence of protease C1 (Barnaby
et al., 2004; GenBank Acc. No. AAD02075) resulted in
a score of 0.939 and a reliability class of 1 for the
presence of the signal sequence. Within the endomembrane system, the immunoelectron microscopy analysis of
the seedling cotyledon presented here shows that protease
C1 is in protein storage vacuoles of the parenchyma cells.
This location is consistent with its biochemical function as
the proteolytic enzyme that initiates degradation of stored
protein reserves. Inspection of the protease C1 sequence
for known sequence-based vacuolar sorting determinants
(reviewed in Jolliffe et al., 2005) revealed the absence of
the NPIR vacuolar sorting determinant known to mediate
binding to BP-80-like proteins in clathrin-coated vesicles
as expected for a protein bound for the lytic vacuole
(Robinson et al., 2005). The high percentage of hydrophobicity of the C-terminus of the a# subunit of soybean
b-conglycinin (.PLSSILRAFY) (GenBank accession
number BAB64303) has been proposed as an important
characteristic of the vacuolar sorting determinant that
localizes the storage protein to the protein storage vacuole
(Nishizawa et al., 2003). It is possible that the high
percentage of hydrophobic residues in the C-terminus of
protease C1 (.RSPIVVFNTA) contributes to its localization in the protein storage vacuole. However, this is likely
to be insufficient since other subtilases such as one from
tomato that has a similarly hydrophobic C-terminus
(.VRSPI-AVVSA) (GenBank Acc. No. CAA76725)
was localized in the extracellular area (Tian et al., 2005).
Our finding that protease C1 is present in the protein
storage vacuoles, even in the early stages of cotyledon
development, explains an observation made by ShuttuckEidens and Beachy (1985). Employing 3H-leucine to
radiolabel newly synthesized storage proteins in cotyledons in culture, they observed proteolysis of the a# and a
subunits of soybean b-conglycinin simultaneously with
synthesis of the b-conglycinin precursor and processing
to the mature polypeptides. The autoradiograph of their
1068 He et al.
Fig. 7. Confocal micoscopy of acridine orange-stained transverse cotyledon slices. (A, D, G) Slices stained with 50 lM acridine orange, taken from
cotyledons 2 d after imbibition (A), 1 d after imbibition (D), and maturing seed (G). (B, E, H) Same conditions as (A), (D), and (G), respectively, but
viewed in the microscope at higher magnification. (C) Slice from cotyledon 2 d after imbibition stained with 50 lM acridine orange in the presence of
30 mM methylamine. (F) Toluidine blue-stained cotyledon slice from 1 d seedling. (I) Slice from section of developing seed cotyledon different from
that shown in (G) treated with 50 lM acridine orange. Scale bars in (A), (D), and (G) are equivalent to 500 lm; in (B), (E), (F), (H), and (I)
equivalent to 100 lm; in (C) equivalent to 200 lm. All fluorescent images were taken with the same settings.
SDS-PAGE gels loaded with proteins, obtained by immunoaffinity isolation using antibodies to the storage proteins,
showed that the major proteolytic products had masses that
correspond to the 50 kDa and 48 kDa breakdown products
of the b-conglycinin a# and a subunits. The proteolysis was
observed in seeds that were half the length of the mature
seeds. It is interesting to note the correlation with our work.
Our localization of protease C1 in the protein storage
vacuoles in seeds that were half the size of mature seed is
consistent with that earlier finding of low-level proteolysis
in early maturing seeds. To be consistent with findings in
this report, the condition in those early developing protein
storage vacuoles would be hypothesized to be acidic,
a reasonable expectation since the vacuoles are generated
from the central vacuole. Eventually, Shuttuck-Eidens and
Beachy (1985) found the proteolysis to be considerably
diminished in seeds that had reached 85% of the size of
mature seed. Similarly, the absence of accumulated acridine
orange in maturing seed demonstrated earlier was done
using seeds close to maturation.
There is precedence for pH control of mobilization of
seed reserves. Using 31P NMR analysis of intact black
Soybean protease C1 1069
gram (Vigna mungo (L.) Hepper) cotyledons, Okamoto
et al. (1999) demonstrated a change in total vacuolar pH
from 6.04 to pH 5.47 during the first 5 d after imbibition
of this legume. It is reasonable to think that there would
be proton pumps in the protein storage vacuoles of
soybean cotyledon cells since coalescence of the emptied
vacuoles gives rise to the central vacuole. Moreover, there
is evidence for accumulation of a H+-pyrophosphatase and
an H+-ATPase in protein storage vacuoles of germinating
pumpkin (Cucurbita sp.) seeds (Maeshima et al., 1994).
There is also evidence in monocots, specifically in the
aleurone cells of barley (Hordeum vulgare L.), for
regulation of proteolysis of stored protein reserves by
acidification of the protein storage vacuole (Hwang et al.,
2003).
Having controlled proteolysis of the N-terminal extensions of the b-conglycinins at a very early period in
seedling growth can be assumed to give the plant an
advantage during the early days of growth. The Nterminal extensions of the b-conglycinin a# and a
subunits are rich in Glu, Gln, Asp, and Asn. Thus, besides
being used for synthesis of proteins for the seedling, the
amino acids can be converted to TCA cycle intermediates
to provide energy or to support the mobilization of the
lipid stores by lipolysis, b-oxidation, and the glyoxylate
cycle, processes noted to occur during early seedling
growth as well (Lin et al., 1982). The presence of protease
C1 in the protein storage vacuole assures early onset. Its
cleavage specificity results in only limited proteolysis so
that the amino acid stores needed for later seedling growth
are not depleted too early. This particular enzyme is suited
to such a role on two accounts. As demonstrated in
previous studies, this protease catalyses hydrolysis of
peptide bonds that are only found in a portion of the
storage proteins. We suggest that the narrow range of pH
in which it functions, coupled to its optimum pH being
lower than the existing vacuolar pH during the most active
period of seed fill, would allow the plant to place the
enzyme in the protein storage vacuoles during storage
protein accumulation. Already in the vacuole and packaged in the midst of the storage proteins, protease C1
would have ready access to the storage proteins especially
when compared with an enzyme that needs to be delivered
to a protein-filled vacuole. It would rapidly manifest its
activity when vacuolar proton pumps increase the acidity
within the protein storage vacuoles. It would be interesting
to determine whether the same situation applies in other
legumes, especially those that have storage protein sequences similar to the N-terminal extensions of the bconglycinin polypeptide chains such as Medicago truncatula Gaetn. (Gallardo et al., 2003), pea (Pisum sativum L.)
(Bown et al., 1988), lentil (Lens culinaris Medik.) (de
Miera and Vega, 2001), peanut (Arachis hypogaea L.)
(Yan et al., 2005), and Macadamia integrifolia Maiden &
Betche (Marcus et al., 1999).
Acknowledgements
The authors acknowledge the assistance of Henry Eichelberger with
the electron microscopy work, and of Dr Dennis McGee with the
confocal microscopy work. The work was supported by NSF MCB9722984. The confocal microscope was made available through an
NSF equipment grant DBI-0321046 to DM.
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