Plant Physiol. (1999) 119: 1361-1370
Antifreeze Proteins in Winter Rye Leaves Form Oligomeric
Complexes1
Xiao-Ming Yu and
Marilyn Griffith*
Department of Biology, University of Waterloo, Waterloo, Ontario,
Canada N2L 3G1
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ABSTRACT |
Antifreeze proteins (AFPs) similar to
three pathogenesis-related proteins, a glucanase-like protein (GLP), a
chitinase-like protein (CLP), and a thaumatin-like protein (TLP),
accumulate during cold acclimation in winter rye (Secale
cereale) leaves, where they are thought to modify the growth of
intercellular ice during freezing. The objective of this study was to
characterize the rye AFPs in their native forms, and our results show
that these proteins form oligomeric complexes in vivo. Nine proteins were separated by native-polyacrylamide gel electrophoresis from apoplastic extracts of cold-acclimated winter rye leaves. Seven of
these proteins exhibited multiple polypeptides when denatured and
separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After isolation of the individual proteins, six were shown by immunoblotting to contain various combinations of GLP, CLP, and TLP in
addition to other unidentified proteins. Antisera produced against
individual cold-induced winter rye GLP, CLP, and TLP all dramatically
inhibited glucanase activity in apoplastic extracts from
cold-acclimated winter rye leaves, and each antiserum precipitated all
three proteins. These results indicate that each of the
polypeptides may be exposed on the surface of the protein complexes. By
forming oligomeric complexes, AFPs may form larger surfaces to interact with ice, or they may simply increase the mass of the protein bound to
ice. In either case, the complexes of AFPs may inhibit ice growth and
recrystallization more effectively than the individual polypeptides.
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INTRODUCTION |
Winter rye (Secale cereale) is an overwintering plant
that survives freezing to temperatures less than 
20°C by forming
ice in intercellular spaces (Pearce, 1988
; Brush et al., 1994).
As winter rye plants acclimate to low temperatures, they secrete proteins into the leaf apoplast, where ice forms. These apoplastic proteins accumulate to levels of about 0.3 mg protein
g
1 leaf fresh weight after 7 weeks of cold
acclimation, and they decrease dramatically in concentration within a
few days if the plants are returned to 20°C to deacclimate (Marentes
et al., 1993). Many of these are AFPs, which modify the growth of ice
crystals and inhibit the recrystallization of ice when assayed in vitro (Griffith et al., 1992; Hon et al., 1994; Griffith and Antikainen, 1996). Although the AFPs are an important component of winter survival
in winter cereals (Chun et al., 1997), we know little about how these
proteins function in vivo. The objective of this study was to
characterize rye AFPs in their native forms.
When the apoplastic proteins from cold-acclimated winter rye leaves are
denatured and separated by SDS-PAGE, six major polypeptides with
molecular masses ranging from 16 to 35 kD are present. All six
polypeptides exhibit antifreeze activity when assayed individually (Hon
et al., 1994). However, these six polypeptides are not unique proteins,
as shown by amino-terminal amino acid sequencing, immunoblotting, and
assays of enzymatic activity (Hon et al., 1995). Two of the apoplastic
polypeptides were identified as GLPs, two were CLPs, and two were TLPs.
GLPs, CLPs, and TLPs are also known as pathogenesis-related proteins
because they can be induced to accumulate in plants by many plant
pathogens, and the presence of pathogenesis-related proteins is
positively correlated with disease resistance (Carr and Klessig,
1989; Stintzi et al., 1993). Although 
-1,3-endoglucanase and
endochitinase activities are present in crude apoplastic extracts of
both nonacclimated and cold-acclimated leaves, only apoplastic extracts
obtained from cold-acclimated leaves have antifreeze activity (Hon et
al., 1995). In fact, a native chitinase purified from cold-acclimated
leaves exhibits both antifreeze activity and endochitinase activity
(Hon et al., 1995). Because the apoplastic proteins that accumulate at
cold temperature in winter rye have both antifreeze and enzymatic
activities, these proteins may play dual roles in freezing tolerance
and resistance to low-temperature diseases.
Our first attempts at isolating the native AFPs from apoplastic
extracts of cold-acclimated winter rye leaves showed that the
antifreeze polypeptides coeluted during gel-filtration chromatography (Griffith et al., 1992), which suggested that they may be associated in
complexes. To elucidate the possible synergistic role of AFPs in the
mechanism of freezing tolerance of the winter rye plant, we
characterized winter rye AFPs accumulated during cold acclimation in
their native forms using three different approaches: (a) separation of
cold-induced apoplastic proteins by native-PAGE and chitin-affinity chromatography; (b) examination of each NP by SDS-PAGE and
immunoblotting; and (c) immunoinhibition of glucanase activity and
immunoprecipitation of AFPs in cold-acclimated rye apoplastic extracts.
Our results indicate that the winter rye AFPs form oligomeric complexes
in vivo.
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MATERIALS AND METHODS |
Plant Materials and Growth Conditions
Winter rye (Secale cereale L. cv Musketeer) seeds were
surface-sterilized in a 0.3% sodium hypochlorite solution for 5 min, rinsed with distilled water several times, planted in 15-cm pots of
coarse vermiculite, and germinated at 20°C/16°C (day/night) with a
16-h daylength and a PPFD of 300 µmol m2
s1 for 1 week. Nonacclimated plants were grown
under the same conditions for an additional 2 weeks. Cold-acclimated
plants were transferred to 5°C/2°C (day/night) with an 8-h
daylength and a PPFD of 300 µmol m2
s1 for an additional 7 weeks. Nonacclimated
plants grown at 20°C/16°C for 3 weeks are similar in physiological
age to cold-acclimated plants grown at 5°C/2°C for 7 weeks (Krol et
al., 1984; Griffith and McIntyre, 1993). Plants were watered as needed
with modified Hoagland solution (Huner and Macdowall, 1976).
Apoplastic Protein Extraction
Apoplastic proteins were extracted by vacuum infiltrating the
leaves with extraction buffer containing 20 mM ascorbic
acid and 20 mM CaCl2, followed by
centrifugation at 900g to recover the proteins (Hon et al.,
1994). Total protein was measured using the Bradford (1976) method, as
modified by Bio-Rad, with BSA as the standard protein. Diluted crude
apoplastic extracts were concentrated about 2-fold for cold-acclimated
samples and 10-fold for nonacclimated samples, as needed, by
ultrafiltration (Centriprep-10, Amicon, Beverly, MA).
Protein Electrophoresis and Purification
Apoplastic proteins extracted from rye leaves were separated with
an 8% (w/v) continuous native-PAGE gel using the Mini-Protein II cell
and a single-well preparative comb according to the manufacturer's instructions (Bio-Rad). The gel buffer was 30 mM -Ala
and 20 mM lactic acid, pH 3.8. To locate the position of
each protein band, a 0.5-cm gel strip was cut from each of the two
longitudinal edges of the gel immediately after electrophoresis,
stained with 0.1% (w/v) Coomassie brilliant blue R-250 in 40% (v/v)
methanol and 10% (v/v) acetic acid (for 10 min), destained with 40%
(v/v) methanol and 10% (v/v) acetic acid (for 20 min), and then
carefully matched to the remaining gel. Gel pieces corresponding to the individual proteins shown on the two Coomassie blue-stained gel strips
were cut from the remaining gel. The gel pieces of each NP were placed
in 5-fold-diluted gel buffer and homogenized using a gel nebulizer
(Amicon). The homogenized gel slurries were sonicated overnight to
allow the proteins to diffuse out of the gel. After centrifugation at
14,500g for 10 min, the NPs were recovered from the
supernatant and concentrated in one step with Micropure separators and
Microcon microconcentrators (Amicon). These procedures were carried out
at 4°C. Each of the NPs from the apoplastic extracts was denatured,
and the component polypeptides were separated by SDS-PAGE (15% [w/v]
acrylamide) and stained with Coomassie brilliant blue R-250 according
to the method of Laemmli (1970).
Immunoblotting
Isolated NPs and polypeptides were transferred onto 0.45-µm
nitrocellulose membranes (Bio-Rad) using the Mini Trans-Blot cell (Bio-Rad) according to the manufacturer's instructions. A solution of
0.7% (v/v) acetic acid, pH 3.8, was used to transfer NPs, and a buffer
composed of 25 mM Tris, 192 mM Gly, and 20%
(v/v) methanol, pH 8.3, was used to transfer polypeptides. The blots
were probed with the anti-GLP antiserum (dilution, 1:2,000), the
anti-CLP antiserum (dilution, 1:2,000), or the anti-TLP antiserum
(dilution, 1:10,000) produced against isolated winter rye AFPs similar
to GLPs, CLPs, and TLPs, respectively (Antikainen et al., 1996). The
immunoreactions were detected by alkaline phosphatase conjugated to
goat anti-rabbit IgG (Sigma) with 5-bromo-4-chloro-3-indolyl phosphate-toluidine salt (BioShop, Burlington, Ontario, Canada) and
nitroblue tetrazolium (Sigma) as substrates.
Glucanase Activity Assay and Immunoinhibition
Total -1,3-glucanase (EC 3.2.16) activity was assayed
colorimetrically using laminarin (Sigma) as a substrate and
dinitrosalicylic reagent to detect the reducing sugars produced,
according to the method of Abeles and Forrence (1970), with some
modifications. Crude apoplastic extract (50 µL) was added to 50 µL
of 1% (w/v) laminarin in the extraction buffer and then incubated at
37°C for 10 min. The reaction was stopped by adding 300 µL of
dinitrosalicylic reagent and heating at 95°C for 5 min. The resulting
colored solution was cooled to room temperature and diluted 1:10 with
distilled, deionized water, and the A500
was read using a microplate reader (model EL308, Bio-Tek, Burlington,
VT). The blank was a mixture of 50 µL of crude extract, 50 µL of
1% laminarin, and 300 µL of dinitrosalicylic reagent. The specific
enzyme activity was defined as the amount of enzyme that produced
reducing sugar at a rate of 1 nmol Glc equivalents
s1 mg1 protein.
For the experiment involving immunoinhibition of -1,3-glucanase
activity, crude apoplastic extracts from cold-acclimated plants were
incubated (apoplastic extract:antiserum, 2:1 [v/v]) with the
preimmune serum, anti-GLP antiserum, anti-CLP antiserum, or anti-TLP
antiserum at 25°C for 20 min, and then total -1,3-glucanase activity was assayed as described above.
Immunoprecipitation of AFPs
Crude apoplastic extract from cold-acclimated rye leaves (500 µL) was mixed with 200 µL of protein A-Sepharose CL-4B (Sigma) preswollen in extraction buffer containing 20 mM ascorbic
acid and 20 mM CaCl2 to remove
apoplastic proteins that bind nonspecifically to the beads. After
gentle shaking at room temperature for 4 h and centrifugation at
17,300g for 10 min, the beads were discarded. The
supernatant was mixed with preimmune serum, anti-GLP antiserum, anti-CLP antiserum, or anti-TLP antiserum (100 µL) and shaken gently
at room temperature for 2 h. Fifty microliters of protein A-Sepharose CL-4B (Sigma) preswollen in extraction buffer containing 20 mM ascorbic acid and 20 mM
CaCl2 was added to the extract-antiserum mixture.
After the sample was shaken for 2 h at room temperature and
centrifuged at 17,300g for 5 min, the supernatant was
discarded and the pellet was washed five times with extraction
buffer to remove unbound proteins. The washed pellet was denatured with SDS reducing buffer (Laemmli, 1970) at 90°C for 5 min and centrifuged at 17,300g for 10 min. The supernatant was analyzed by
SDS-PAGE and immunoblotting.
Purification of Chitinase
The native rye chitinase was purified from apoplastic extracts of
cold-acclimated winter rye leaves by affinity chromatography using
colloidal chitin as the column substrate (Huynh et al., 1992; Hon et
al., 1995). The purity of chitinase was examined on 8% native-PAGE and
15% SDS-PAGE gels stained with Coomassie brilliant blue R-250,
followed by immunoblotting in which antisera produced against
cold-induced rye CLP, GLP, and TLP were used as probes.
Antifreeze Activity Assay
Antifreeze activity was assayed qualitatively by examining the
morphological characteristics of ice crystals (DeVries, 1986) grown in
apoplastic extracts of cold-acclimated rye leaves, in solutions of
individual proteins eluted from native gels, and in chitinase fractions
purified by chitin-affinity chromatography. The growth of ice crystals
in each sample was controlled by a nanoliter osmometer (Clifton
Technical Physics, Hartford, NY), and the morphological characteristics
of ice crystals were examined using a phase-contrast photomicroscope
(model BHT, Olympus). A rating system was developed to quantify and
compare the effects of different apoplastic extracts on ice-crystal
growth (Chun et al., 1997). The disc-like crystals grown in water were
rated 0 because they have no antifreeze activity. Hexagonal discs grown in very low (nanomolar) concentrations of AFPs or in solutions of AFPs
with low specific activity were rated 1. Hexagonal columns grown in
dilute (micromolar) solutions of AFPs or in solutions of AFPs with
moderate specific activity were rated 3. Crystals forming complex
hexagonal bipyramids in high concentrations (
100 µM) of
AFPs or in solutions of AFP with high specific activity were rated 5.
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RESULTS |
Separation of Apoplastic Proteins
Proteins present in the apoplastic extracts from the leaves of
nonacclimated plants and plants cold acclimated for 7 weeks were
quantified and examined under nondenaturing conditions. The concentration of extractable apoplastic proteins was about 10-fold higher in cold-acclimated apoplastic extracts (0.43 ± 0.05 mg protein mL1; n = 5) than in
nonacclimated apoplastic extracts (0.04 ± 0.01 mg
mL1; n = 4). We first tried to
separate the native apoplastic proteins on a sizing column (1 × 50 cm; Bio-Gel P-100, Bio-Rad) eluted with 30 mM
-Ala and 20 mM lactic acid, pH 3.8, at a rate
of 70 µL min1. Although BSA (67 kD),
ovalbumin (43 kD), chymotrypsinogen A (25 kD), and Cyt c
(12.4 kD) were easily separated on the column, the native apoplastic
proteins from cold-acclimated rye leaves yielded only one peak with a
trailing shoulder (data not shown). According to the standard curve,
these proteins had molecular masses well below 12 kD, which indicated
that the proteins were eluted from the column at a slower rate than
predicted by their apparent sizes on SDS-PAGE. A similar result was
reported earlier when a sizing column packed with Sephacryl 200 was
used (Griffith et al., 1992).
In contrast to the results with the open columns, good resolution and
consistent separations were obtained when a continuous native-polyacrylamide (8% [w/v]) gel system was used at pH 3.8. As
shown in Figure 1, nine NPs were
identified in apoplastic extracts from cold-acclimated rye leaves,
whereas only six NPs were present in apoplastic extracts from
nonacclimated leaves when equal amounts of apoplastic proteins were
separated by native-PAGE. Although NP2, NP4, NP5, NP6, NP8, and NP9
were present in both cold-acclimated and nonacclimated leaves, NP1,
NP3, and NP7 were detected only in cold-acclimated leaves. Moreover,
NP4, NP5, NP6, NP8, and NP9 all accumulated to higher levels in
cold-acclimated leaves, as indicated by the intensity of the Coomassie
brilliant blue stain when equal volumes of apoplastic extracts from
both cold-acclimated and nonacclimated rye leaves were loaded on the
native-polyacrylamide gel (Fig. 1). Only NP2 was found to accumulate to
high levels in the apoplast of nonacclimated leaves.
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| Figure 1.
Separation of apoplastic proteins by native-PAGE.
Apoplastic proteins were extracted from cold-acclimated (CA) and
nonacclimated (NA) rye leaves by vacuum infiltration followed by
centrifugation. The apoplastic proteins were separated from equal
volumes of unconcentrated apoplastic extracts (lanes CA and NA, 1) and
from equal amounts (10 µg) of apoplastic proteins (lanes CA and NA,
2) in an 8% continuous native-polyacrylamide gel. The gel was stained
with Coomassie brilliant blue R-250. Numbers on the left refer to
individual native apoplastic proteins.
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Comparison of Apoplastic Polypeptides
When cold-acclimated and nonacclimated apoplastic proteins were
denatured and separated by SDS-PAGE, 13 polypeptides were present in
nonacclimated apoplastic extracts, with apparent molecular masses of
144, 97, 36, 35, 34, 33, 32, 28, 26, 25, 16 (doublet), and 14 kD (Fig.
2A, lane 3), whereas only 7 polypeptides
were found in cold-acclimated apoplastic extracts, with molecular
masses of 144, 35, 32, 28, 25, 16, and 14 kD (Fig. 2A, lane 1), when equal amounts of protein were loaded on the SDS-PAGE gel. Both cold-acclimated and nonacclimated apoplastic proteins contained 32- and
35-kD GLPs, a 35-kD CLP, and a 25-kD TLP, as detected positively on
immunoblots probed with anti-GLP (Fig. 2B), anti-CLP (Fig. 2C), and
anti-TLP antisera (Fig. 2D). No GLPs, CLPs, or TLPs were detected in
nonacclimated apoplastic extracts when equal volumes of unconcentrated
cold-acclimated and nonacclimated extracts were loaded onto the gels
and immunoblots (Fig. 2, B-D, lanes 1 and 2).
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| Figure 2.
Examination of polypeptides in cold-acclimated and
nonacclimated apoplastic extracts by SDS-PAGE and immunoblotting. A,
SDS-PAGE (15% acrylamide) of equal volumes of crude cold-acclimated
and nonacclimated apoplastic extracts (lanes 1 and 2, respectively) and
equal amounts (5 µg) of cold-acclimated and nonacclimated apoplastic
proteins (lanes 1 and 3, respectively). Polypeptides were
stained with Coomassie brilliant blue R-250. Low-range prestained
SDS-PAGE molecular-mass standards from Bio-Rad are shown on the left
(lanes M). SDS-PAGE gels with the same sample-loading scheme shown in A
were blotted and probed with anti-GLP antiserum (B), anti-CLP antiserum
(C), and anti-TLP antiserum (D). Positive immunodetection of
polypeptides is indicated by arrows on the right.
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Analysis of the Composition of Apoplastic NPs
The individual proteins shown in Figure 1 were eluted from the
native gels, concentrated by ultrafiltration, and then denatured and
examined by SDS-PAGE. All of the NPs except NP8 and NP9 contained multiple polypeptides when electrophoresed by denaturing SDS-PAGE (Fig.
3A). NP1 was composed of two polypeptides
(12 and 97 kD). NP2 contained two polypeptides that migrated as a
doublet with a molecular mass of 35 kD. NP3 showed four polypeptides,
singlets of 10 and 12 kD and a doublet of 35 kD. NP4 showed seven
polypeptides of 12, 14, 24, 25, 30, 32, and 35 kD. NP5 was composed of
eight polypeptides with molecular masses of 12, 14, 22, 24, 25, 30, 32, and 35 kD. NP6 showed five polypeptides (13, 15, 22, 23, and 25 kD).
NP7 showed four polypeptides (15, 22, 23, and 25 kD). NP8 and NP9
exhibited only one 15-kD polypeptide.
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| Figure 3.
Examination of native apoplastic proteins by
SDS-PAGE and immunoblotting. A, Individual cold-acclimated apoplastic
proteins were eluted from the native gel shown in Figure 1, denatured,
separated by 15% SDS-PAGE, and stained with Coomassie brilliant blue
R-250. Equal amounts of protein (5 µg) were loaded on each lane.
Lanes 1 to 9, NP1 to NP9. Low-range prestained SDS-PAGE molecular-mass
standards from Bio-Rad are shown on the left (lanes M). SDS-PAGE gels
loaded with equal amounts (1 µg per lane) of individual native
apoplastic proteins and with crude cold-acclimated apoplastic extract
in lanes 10 were blotted and probed with anti-GLP antiserum (B),
anti-CLP antiserum (C), and anti-TLP antiserum (D). Positive
immunodetection of polypeptides in apoplastic extracts is indicated by
arrows on the right.
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Analysis of NPs by Immunoblotting and Immunoprecipitation
The composition of each NP was further examined by immunoblotting.
The apoplastic proteins initially separated by native-PAGE were
denatured and separated by SDS-PAGE (Fig. 3A) and were then blotted and
probed with antisera against GLPs (Fig. 3B), CLPs (Fig. 3C), and TLPs
(Fig. 3D). The specificities of these antisera were described
previously (Antikainen et al., 1996). Antiserum raised against the
denatured 32-kD GLP, used in a dilution of 1:2000, recognizes two
polypeptides with molecular masses of 32 and 35 kD, both of which were
identified as GLPs by amino-terminal amino acid sequencing (Hon et al.,
1995). Anti-CLP antiserum recognizes only one 35-kD polypeptide at a
dilution of 1:2000, although the 35- and 28-kD polypeptides were both
identified as CLPs by Hon et al. (1995). The anti-CLP antiserum was
raised against the native 35-kD CLP, which has a chitin-binding domain
that is lacking in the 28-kD CLP. Antiserum raised against the
denatured 25-kD TLP detects only one polypeptide with a molecular mass
of 25 kD at a dilution of 1:10,000. At this dilution, the 16-kD
polypeptide also identified as a TLP by Hon et al. (1995) is not
detected. Each antiserum is specific to one type of AFP and does not
cross-react with other apoplastic proteins. Within each type of AFP,
the antiserum is more reactive with the protein against which it was
raised.
As shown in Figure 3, polypeptides associated with NP2 and some of the
polypeptides associated with NP3 were positively detected by anti-GLP
and anti-CLP antisera. Polypeptides associated with NP4 were detected
by anti-GLP, anti-CLP, and anti-TLP antisera. Polypeptides associated
with NP5 were detected by anti-GLP and anti-TLP antisera, whereas those
associated with NP6 and NP7 were detected only by anti-TLP antiserum.
In addition, NPs separated by nondenaturing gel electrophoresis were
directly transferred to nitrocellulose membranes and probed with
antisera against cold-induced winter rye GLP, CLP, and TLP. The results
of these experiments confirmed the immunoblotting results obtained with
SDS-PAGE (data not shown).
To examine further whether the observed cold-induced GLP, CLP, and TLP
were in fact physically associated, two sets of experiments were
conducted. Because these experiments required the use of higher
antiserum concentrations, the specificity of each of the three antisera
was determined using a dilution of 1:3 in immunoblots of denatured
apoplastic polypeptides from cold-acclimated plants. Under these
conditions, the anti-GLP antiserum detected two polypeptides with
apparent molecular masses of 32 and 35 kD, the anti-CLP antiserum detected two polypeptides at 28 and 35 kD, and the anti-TLP antiserum detected two polypeptides at 16 and 25 kD (Fig.
4). In the first experiments, glucanase
activity in cold-acclimated apoplastic extracts was assayed after
incubating cold-acclimated apoplastic extracts with specific rye
anti-GLP, anti-CLP, or anti-TLP antiserum. As shown in Figure
5, glucanase activity was dramatically
inhibited in the presence of anti-GLP antiserum. Antisera produced
against cold-induced CLP and TLP also inhibited glucanase activity by 93% and 95%, respectively. In other experiments, chitinase activity in the apoplastic extracts was inhibited by adding antiserum against GLP, CLP, or TLP (data not shown). In the second experiments, immunoprecipitation of AFPs by anti-GLP, anti-CLP, and anti-TLP antisera was examined (Fig. 6). The
anti-GLP antiserum precipitated not only 32- and 35-kD GLPs but also a
35-kD CLP and a 25-kD TLP, as evident in the results from SDS-PAGE
(Fig. 6A, lane 1) and immunoblotting (Fig. 6, B-D, lane 1). Different
groups of polypeptides were immunoprecipitated by anti-CLP antiserum
(Fig. 6, lanes 2) and anti-TLP antiserum (Fig. 6, lanes 3), but each of
these antisera also precipitated a GLP, a CLP, and a TLP (Fig. 6,
B-D).
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| Figure 4.
Specificities of anti-GLP, anti-CLP, and anti-TLP
antisera. CA, Cold-acclimated apoplastic proteins (5 µg) were
denatured, separated by 15% SDS-PAGE, and stained with Coomassie
brilliant blue R-250. Similar gels were blotted and probed with
antisera at a dilution of 1:3. Lane GLP was probed with anti-GLP
antiserum, lane CLP was probed with anti-CLP antiserum, and lane TLP
was probed with anti-TLP antiserum. Low-range prestained SDS-PAGE
molecular-mass standards from Bio-Rad (lane M) were used to determine
the molecular masses (kD). The molecular mass of each polypeptide
immunodetected by an antiserum is indicated on the right.
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| Figure 5.
Inhibition of glucanase activity by antisera
produced against AFPs. Glucanase activity was assayed in nonacclimated
(NA) and cold-acclimated (CA) apoplastic extracts and in
cold-acclimated extracts incubated with antisera produced against
cold-induced GLP (G), CLP (C), and TLP (T), or with preimmune serum
(P). The glucanase specific activity was normalized as a percentage of
the specific activity present in the cold-acclimated apoplastic extract
and is shown as the mean ± SE (n = 3).
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| Figure 6.
Immunoprecipitation of native AFPs by antisera
produced against specific AFPs. Equal amounts (5 µg per lane) of
proteins immunoprecipitated by anti-GLP (lanes 1), anti-CLP (lanes 2),
or anti-TLP (lanes 3) antiserum, or by preimmune serum (lanes 4), were
denatured and separated by 15% SDS-PAGE. Cold-acclimated apoplastic
extract is shown as a positive control in lanes 5. A, Gel stained with
Coomassie brilliant blue R-250. Gels were blotted and probed with
anti-GLP antiserum (B), anti-CLP antiserum (C), and anti-TLP antiserum
(D). Low-range prestained SDS-PAGE molecular-mass standards from
Bio-Rad (lanes M) were used to determine the molecular masses (kD).
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Purification and Characterization of NP3
The separation of native and denatured proteins presented in
Figures 1 and 3 could result from the comigration of proteins similar
to GLPs, CLPs, and/or TLPs. To test this possibility, we isolated one
complex from apoplastic extracts of cold-acclimated rye leaves by
chitin-affinity chromatography (Huynh et al., 1992; Hon et al., 1994).
With this procedure, only chitinases or lectins with a chitin-binding
domain specifically bind to the colloidal chitin used to pack the
affinity column. By changing the pH of the washing buffer from 8.0 to
4.5, the nonspecifically bound proteins were washed off the column. The
specifically bound chitinases were then washed off the column using 20 mM acetic acid, pH 3.0. In our experiments, only one
protein was bound specifically to the chitin-affinity column. When
separated by native-PAGE, this protein had an RF
value similar to that of NP3 in the crude cold-acclimated apoplastic
extract (Fig. 7A). When denatured and
separated by SDS-PAGE (Fig. 7B), this purified protein was composed of
four polypeptides with molecular masses of 10, 12, 35, and 35 kD. The two 35-kD polypeptides were positively detected by antisera produced against cold-induced winter rye GLP and CLP (Fig. 7C).
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| Figure 7.
Purification and identification of NP3.
Cold-acclimated (CA) rye apoplastic extract and purified NP3 were
examined by native-PAGE (A), SDS-PAGE (B), and immunoblotting (C). NP3
was purified from rye apoplastic extract by chitin-affinity
chromatography. Low-range prestained SDS-PAGE molecular-mass standards
from Bio-Rad (lanes M) were used to determine the molecular masses
(kD). The gels in A and B were stained with Coomassie brilliant blue
R-250. Anti-GLP and anti-CLP antisera were used to detect GLP and CLP,
respectively.
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Antifreeze Activities of NPs
The antifreeze activities of individual NPs eluted from native
gels were compared with NP3 purified by chitin-affinity chromatography, crude apoplastic extract from nonacclimated leaves, and crude apoplastic extract from cold-acclimated leaves. The protein
concentration of each sample was adjusted to about 0.5 mg
mL1. As summarized in Table
I, the highest antifreeze activity was found in the cold-acclimated crude apoplastic extract, which was composed of a mixture of all AFPs (rated 5). The next highest antifreeze activity was found in NP4, which contained GLP, CLP, and TLP
(rated 4). This was followed by NP2 and NP3, which contained CLP and
GLP (rate 3); NP5, which contained GLP and TLP (rated 3); and NP6 and
NP7, which contained TLP (rated 2). NP1, NP8, and NP9 and crude
apoplastic extract from nonacclimated leaves did not have any
antifreeze activity (rated 0).
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Table I.
Comparison of antifreeze activity of individual
native proteins with the activity of crude apoplastic extracts from
cold-acclimated and nonacclimated plants
The individual proteins were separated by native-PAGE (Fig. 1), eluted,
and assayed for antifreeze activity. Antifreeze activity of each sample
was rated from 0 to 5 based on the shape of ice crystals grown in
solution (see ``Materials and Methods''), with 5 representing the
highest activity and 0 representing no activity. The protein
concentration of each sample was determined using the Bradford (1976)
method, as modified by Bio-Rad, and is presented as the mean ± SE (n = 3).
|
|
At this time, we cannot explain why nonacclimated apoplastic extracts
lack antifreeze activity after they have been concentrated, because
they contain NP2, NP4, NP5, and NP6 (Fig. 1), all of which exhibit
antifreeze activity when isolated from cold-acclimated extracts (Table
I). It may be that different isozymes of GLPs, CLPs, and/or TLPs
accumulate at 20°C or that the proteins produced at 5°C are
posttranslationally modified in some way to acquire antifreeze
activity. Either of these possibilities could explain the differences
in polypeptide composition between cold-acclimated and nonacclimated
extracts observed on SDS-PAGE (Fig. 2).
| |
DISCUSSION |
Association of Winter Rye AFPs in Vivo
Our biochemical and immunological evidence shows that apoplastic
extracts from cold-acclimated winter rye leaves contain nine proteins
(Fig. 1). Seven of these are composed of multiple polypeptides when
denatured and separated by SDS-PAGE (Fig. 3A), and, surprisingly, GLPs,
CLPs, and TLPs are associated with six of them (Fig. 3, B-D). The
association of glucanase with other proteins was observed previously
(Ballance and Manners, 1978; Ji and Kuc, 1995). For example,
native -1,3-glucanase purified from germinated rye by ion-exchange
chromatography on DEAE- and CM-cellulose followed by gel filtration on
Bio-Gel P-60 was associated with three unidentified proteins (Ballance
and Manners, 1978). Moreover, an acidic -1,3-glucanase extracted
from cucumber leaves infected with tobacco necrosis virus was
associated with a class III chitinase because both enzymes migrated
together on native-PAGE (Ji and Kuc, 1995).
It is possible that, by chance, the different proteins have the same
mobility on a native gel, so they form a single band on the gel but
migrate as several polypeptides on SDS-PAGE. To distinguish between
these possibilities, NP3 (Fig. 7) was isolated by chitin-affinity
chromatography and electrophoresed using the native-gel system in the
absence of other apoplastic proteins. Only one NP was visible (Fig.
7A), and it contained both GLP and CLP, as determined by immunoanalysis
(Fig. 7C). The binding between GLP and CLP appeared to be strong
because GLP and CLP remained associated when other proteins were washed
off the chitin-affinity column using solutions with pHs ranging from
8.0 to 4.8. The physical association among GLP, CLP, and TLP was also
evident in the results of immunoinhibition experiments in which
-1,3-glucanase activity in cold-acclimated crude apoplastic extracts
was dramatically inhibited by the presence of anti-GLP, anti-CLP, or
anti-TLP antiserum, but not by preimmune serum (Fig. 5), and in
immunoprecipitation experiments in which anti-GLP, anti-CLP, or
anti-TLP antiserum precipitated all three AFPs (GLPs, CLPs, and TLPs)
from cold-acclimated apoplastic extracts (Fig. 6). We interpret these
results to indicate that all of these polypeptides are exposed on the
surface of the NP complex. Alternatively, if the complexes are small,
then the binding of any one of the antisera may hinder access of the
polymeric substrate to the active site of the glucanase.
Why are there so many different NPs in the apoplastic extract? We
hypothesize that the individual NPs are produced by different cell
types. For example, immunolocalization studies of AFPs in winter rye
revealed that all three classes of AFPs are localized in the epidermis
of cold-acclimated leaves (Antikainen et al., 1996). Thus, NP4, NP5,
NP6, and/or NP7 may be produced by epidermal cells. In contrast, just
two classes of AFPs, GLPs and CLPs, accumulate in cell walls
surrounding the intercellular spaces in the mesophyll (Antikainen et
al., 1996; Pihakaski-Maunsbach et al., 1996). We would expect NP2 and
NP3 to be secreted by mesophyll cells. Therefore, each NP identified in
Figure 1 may have a different, tissue-specific location within the
apoplast of a rye leaf.
Other investigators have also reported tissue-specific locations of
glucanase and chitinase. In tomato plants, pathogen-induced -1,3-glucanase and chitinase were both found in the abaxial
epidermal layer near the stomata (Wubben et al., 1993).
Ethylene-induced chitinase and -1,3-glucanase have also been
localized together in abaxial epidermal cells and in parenchymal cells
adjacent to vascular strands in bean leaves (Mauch et al.,
1992). Additional evidence for the physical association between
-1,3-glucanase and chitinase was obtained by immunolocalization of
-1,3-glucanase and chitinase in the large, electron-dense aggregates
located in the vacuoles of lower epidermal cells of ethylene-treated
bean leaves (Mauch et al., 1992). The common compartmental location of
glucanase and chitinase is consistent with our hypothesis that the two
enzymes are physically associated with each other.
The arrangement of specific proteins and enzymes as part of functional
complexes has been found in many systems. For example, the cellulose
(mainly -1,4-glucanase) activity of many cellulolytic bacteria
occurs in discrete, multifunctional, multienzyme complexes called
cellulosomes. These organized complexes account for the efficient
solubilization of insoluble cellulose (Bayer et al., 1994). A second
example of a multienzyme complex is acetyl-CoA carboxylase, which
consists of biotin carboxylase and a biotin-carboxyl carrier protein
and is a regulatory enzyme of fatty acid synthesis (Roesler et al.,
1996).
Although -1,3-glucanase and chitinase are encoded by two different
small gene families (Linthorst, 1991), the expression of the two genes
is often coordinately regulated upon pathogen infection and exposure to
other environmental stresses such as wounding, drying, and flooding
(Ohashi and Ohshima, 1992; Stintzi et al., 1993). Glucanase and
chitinase enzymatic activities increase concomitantly in many plants
not only in response to pathogen attack but also in response to
pathogen-derived elicitors and the plant hormone ethylene (Mauch et
al., 1992). Moreover, it has been reported that -1,3-glucanase or
chitinase purified from pea cannot inhibit the growth in culture of
most of the fungi tested when used individually. However, a combination
of these enzymes effectively inhibits the growth of most fungi tested
(Mauch et al., 1988), which suggests that the two enzymes act
synergistically in plant defense.
It is possible that GLPs, CLPs, and TLPs play a synergistic role in
improving the winter survival of winter rye plants. First, -1,3-glucanase and chitinase activities are both high in apoplastic extracts from cold-acclimated winter rye leaves; therefore, they may
act together to provide resistance to pathogens (Hon et al., 1995).
Second, GLPs, CLPs, and TLPs may work in concert to modify the growth
of intercellular ice as part of the mechanism of freezing tolerance.
These proteins are all present in the apoplast of rye leaves after cold
acclimation (Hon et al., 1995), and they are located in the epidermis
and in the cell walls lining the intercellular spaces of
cold-acclimated leaves, where they may interact with ice (Antikainen et
al., 1996; Pihakaski-Maunsbach et al., 1996). The GLPs and CLPs appear
to be physically associated with each other (Fig. 7), which is
significant because the antifreeze activity of complexes that contain
both GLPs and CLPs is higher than that of complexes that lack these
proteins (Table I).
Possible Roles of AFP Complexes
The general mechanism of action of AFPs can be explained by the
adsorption-inhibition theory described in detail by Raymond and DeVries
(1977). An ice crystal normally grows as a broad front with a
low radius of curvature. However, when ice crystals are grown in a
solution containing AFPs, the AFPs interact with ice in two unique
ways. First, AFPs adsorb onto the nonbasal planes of ice at the
ice-water interface (Raymond et al., 1989) and exert a
concentration-dependent effect on ice-crystal growth features (DeVries,
1986). Second, AFPs adsorbing onto the ice surface block the binding of
additional water molecules, which creates an ice-crystal surface with
many highly curved fronts and a high surface free energy. Consequently,
the growth of these fronts is halted because it is less energetically
favorable for water molecules to bind to this surface. The temperature
must be lowered further to decrease free energy before crystal growth
proceeds. As a result, the freezing point of the solution is depressed.
Theoretically, one way to increase the effectiveness of an AFP is to
increase the size of the protein so that it blocks a greater area on
the ice-crystal surface. Wu et al. (1991a) demonstrated that this size
effect does occur. They showed that an insect AFP conjugated with
rabbit anti-AFP IgG, which has no antifreeze activity by itself, plus
goat anti-rabbit IgG, which also has no antifreeze activity by itself,
exhibits greater antifreeze activity than the insect AFP alone.
Furthermore, they showed that a second protein isolated from insect
hemolymph can bind to the insect AFP and enhance its activity (Wu et
al., 1991b). Thus, the high level of antifreeze activity observed in
crude hemolymph extracts obtained from overwintering larvae probably
requires the interaction of the insect AFP (12-22 kD) and its
activator protein (70 kD).
The AFPs in winter rye may function in a fashion similar to the insect
AFP plus its activator protein. By forming complexes composed of GLPs,
CLPs, and/or TLPs, the AFPs may block a larger area of the ice surface,
thus making it more difficult for the ice to overgrow the complex(es).
As a result, the growth of ice crystals is inhibited to a greater
extent and antifreeze activity increases. The fact that the apoplastic
extract, which is a mixture of AFPs, has the highest antifreeze
activity, followed by the complex containing GLP, CLP, and TLP, and
then complexes consisting of GLP and TLP or GLP and CLP (Table I),
provides indirect evidence to support our hypothesis.
Physical damage caused by ice can occur in frozen tissues when small
ice crystals condense into larger ones, a process known as
recrystallization (Knight and Duman, 1986). Although recrystallization occurs slowly during prolonged freezing at very low temperatures, it
can happen very quickly at temperatures near the melting point of ice.
In nature, the inhibition of ice recrystallization may be the primary
role of AFPs in freezing-tolerant organisms (Knight and Duman, 1986).
Winter rye leaves form extracellular ice during winter (Pearce, 1988)
and are exposed to fluctuating subzero temperatures that promote the
recrystallization of ice. Winter rye AFPs inhibit ice recrystallization
effectively at very low concentrations (25 µg protein
L1) (Griffith and Antikainen, 1996). The
ability of rye AFPs to inhibit ice recrystallization at low protein
concentrations may be related to both the size of proteins and the
presence of multiple ice-binding sites on each protein. For
example, it is possible that each AFP complex can interact with more
than one ice crystal, because each of the components of the complex is
able to interact with ice. In summary, we conclude that winter rye AFPs
form oligomeric complexes in the apoplast that enhance the ability of
these proteins to inhibit the growth and recrystallization of ice.
| |
FOOTNOTES |
1
This work was supported by a research grant from
the Natural Sciences and Engineering Research Council of Canada to M.G.
*
Corresponding author; e-mail griffith{at}uwaterloo.ca; fax
1-519-746-0614.
Received August 17, 1998;
accepted January 8, 1999.
| |
ABBREVIATIONS |
Abbreviations:
AFP, antifreeze protein.
CLP, chitinase-like
protein.
GLP, -1,3-glucanase-like protein.
NP, native protein.
TLP, thaumatin-like protein.
| |
ACKNOWLEDGMENTS |
We thank Dr. G. McLeod (Agriculture Canada) for the cv Musketeer
rye seeds and J. Krupp, A. McElligott, C. Lumb, and V. Jackson for
growing the plants. We also thank Drs. B.R. Glick, B.A. Moffatt, and
R.W. Johnson for technical assistance and helpful discussions.
| |
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Abstract
Introduction