Download Seasonal regulation of a 24-kDa protein from red

Tree Physiology 22, 423–430
© 2002 Heron Publishing—Victoria, Canada
Seasonal regulation of a 24-kDa protein from red-osier dogwood
(Cornus sericea) xylem
ERIC SARNIGHAUSEN,1,2 DALE KARLSON1,3 and EDWARD ASHWORTH1,4
1
Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, IN 47907-1165, USA
2
Present address: Institut für Biologie II, Albert-Ludwigs Universität Freiburg, D-79104 Freiburg, Germany
3
Present address: Department of Low Temperature Sciences, National Agricultural Research Center, Hitsujigaoka, Sapporo 0628555, Japan
4
Author to whom correspondence should be addressed ([email protected])
Received July 9, 2001; accepted October 26, 2001; published online March 1, 2002
Summary Previously, we showed that an apparent cell wall–
plasma membrane interaction in xylem ray parenchyma differed between cold acclimated and non-acclimated red-osier
dogwood (Cornus sericea L.) (Ristic and Ashworth 1994). For
the present study, a calcium chloride extraction method was
used to identify cell-wall-associated xylem proteins that accumulated during periods of cold acclimation. A 24-kDa protein
represented the predominant protein in both total protein and
CaCl2 extracts during cold acclimation of field-grown plants.
Two-dimensional gel electrophoresis separated the 24-kDa
protein into four basic isoforms. The most abundant and basic
isoform had a high glycine content. In-gel digestion of this basic 24-kDa isoform generated three partial peptide fragments,
of which one exhibited homology to the dehydrin protein family. An anti-dehydrin polyclonal antibody cross-reacted with
the 24-kDa protein, providing further evidence that this protein
is related to dehydrins. The 24-kDa protein began to accumulate in late August, reached a maximum in midwinter, declined
during the spring months and was absent in early summer.
Keywords: cell wall, cold acclimation, dehydrin, seasonal
proteins, woody plants.
Introduction
Woody plant survival in temperate zones is dependent on the
coordinated timing of physiological processes with seasonal
oscillations in environmental conditions (Hummel et al.
1982). Temperate woody plants perceive environmental cues,
such as decreasing photoperiod and temperature in the fall,
and subsequently acclimate and develop increased tolerance to
freezing temperatures (Weiser 1970, Burke et al. 1976). Numerous changes occur during cold acclimation (CA), including modifications in cellular ultrastructure (e.g., Pomeroy and
Siminovitch 1971, Wisniewski and Ashworth 1986, Sagisaka
et al. 1990, Kuroda and Sagisaka 1993), altered gene expression and protein synthesis (e.g., Guy 1990, Pearce 1999,
Thomashow 1999), increases in soluble sugars and compatible
solutes (e.g., Ashworth et al. 1993, Nomura et al. 1995, Allard
et al. 1998) and changes in lipid composition and plasma
membrane properties (e.g., Steponkus 1984).
The composition and properties of plant cell walls also
change during CA in some species. Alterations in amounts of
lignin and suberin (Griffith and Brown 1982), extracellular
soluble and pectic polysaccharides, callose (Wallner et al.
1986) and cell-wall-associated proteins (e.g., Jian et al. 1987,
Weiser et al. 1990, Kozbial et al. 1998) have been observed
during CA. Changes also occur in the physical properties of
the cell wall (reviewed by Tao and Li 1993) and include
changes in cell wall thickness (Huner et al. 1981), cell wall
tensile strength and pore size (Huner et al. 1981, Rajashekar
and Lafta 1996).
It is not surprising that the composition and properties of
cell walls change during CA. Cell walls are at the interface
between extracellular ice and the cell protoplasm and, in conjunction with the plasma membrane, form a barrier to the penetration of extracellular ice crystals. Several investigators have
identified components within the apoplast that influence ice
crystal growth. For instance, mucilaginous polymers (Olien
1978), glycoproteins (Jian et al. 1987), ice nucleating proteins
(Brush et al. 1994) and anti-freeze proteins (Griffith et al.
1992, Marentes et al. 1993, Hon et al. 1994, Anitkainen et al.
1996, Pihakaski-Maunsbach et al. 1996, Griffith et al. 1997,
Chun et al. 1998, Worrall et al. 1998) have been documented to
accumulate extracellularly during CA and to affect extracellular ice growth. In addition, Goodwin et al. (1996) identified a cold-regulated linker protein that was hypothesized to
maintain a close association between the cell wall and plasma
membrane during freezing stress. Other alterations of cell wall
characteristics such as porosity, strength and thickness may
also influence cellular responses during freezing events. Enhanced wall strength may provide resistance to freeze-induced
cellular dehydration and limit cell collapse and deformation
(Burke et al. 1983, Rajashekar and Burke 1996, Rajashekar
and Lafta 1996). Alterations of cell wall porosity may affect
the efficacy of the cell wall as a barrier to extracellular ice
(Rajashekar and Lafta 1996) and influence freezing behavior
in woody plants (Wisniewski et al. 1991).
424
SARNIGHAUSEN, KARLSON AND ASHWORTH
Evidence that cell walls and their associated proteins undergo changes during CA led us to investigate whether seasonal regulation of cell-wall-associated proteins occurs in the
woody shrub Cornus sericea L. We chose to study C. sericea
because it is among the most freeze-tolerant plants (Guy et al.
1986) and may contain unique proteins that are important in
establishing a high degree of freeze-tolerance. A second reason for studying C. sericea is that microscopic evaluation of
C. sericea xylem ray parenchyma during exposure to freezestress revealed that the interaction between the plasma membrane and the cell wall differed between cold-acclimated and
non-acclimated specimens (Ristic and Ashworth 1994). We
postulate that this interaction is protein-mediated and that an
investigation of cell wall proteins in C. sericea may provide
insight into specific molecular characteristics unique to extremely freeze-tolerant organisms.
subjected to repeated washings including: one wash with
40 ml of 2 mM Na2S2O5, 0.5% (w/v) Nonidet P-40; seven
washes with 2 mM Na2S2O5; one wash with 20 mM sodium acetate, pH 5.5, 20 mM NaCl; and an additional wash with 40 ml
of purified water. All steps were performed at 4 °C. Putative
cell-wall-associated proteins were extracted from filtered material with 50 ml of 0.4 M CaCl2, 5 mM DTT and 0.5 mM
PMSF for 24 h. After centrifugation for 45 min at 12,000 g
(4 °C), the supernatant was dialyzed against 4 l of 20 mM
Tris-HCl, pH 7.5 with 0.02% sodium azide for 20 h (Spectra/
Por 3 dialysis tubings, MWCO 3500, Spectrum Laboratories,
Rancho Dominguez, CA) and concentrated to a final volume
of 5 ml with 200 ml of 30% (w/v) PEG-8000 in dialysis buffer.
Proteins were precipitated with a fivefold volume of acetone
and stored at –20 °C overnight. Protein concentrations were
subsequently determined as described by Bradford (1976).
Electrophoretic separation of proteins
Materials and methods
Plant material
Stems from current-season growth were harvested periodically from red-osier dogwood (C. sericea) growing in the vicinity of the Purdue University campus, West Lafayette, IN.
Bark tissue was scraped from twigs with a scalpel. Bark and
internodal xylem segments were immediately frozen in liquid
nitrogen. Leaves were sampled in June and immediately
frozen in liquid nitrogen. All tissues were subsequently
lyophilized and stored at –80 °C until used for protein extractions.
Protein extractions
Lyophilized tissue was ground to a fine powder in a ball mill.
Extraction of total proteins was performed according to Sauter
et al. (1989) with slight modifications. Dried samples
(300 mg) were homogenized on ice in extraction buffer
(62.5 mM Tris-HCl, pH 6.8, 2% (w/v) lauryl sulfate (lithium
salt), 5% (v/v) 2-mercaptoethanol, 1 mM PMSF) and boiled
for 5 min. After cooling to room temperature, the extracts were
centrifuged at 12,000 g (4 °C) for 45 min. Total proteins were
precipitated from the supernatant with a fivefold volume of acetone and maintained at –20 °C overnight. Soluble proteins
were extracted as described by Perras and Sarhan (1989).
Dried wood powder (320 mg) was extracted on ice with extraction buffer (100 mM Tris-HCl, pH 8.0, 100 mM NaCl,
1 mM EDTA, 1 mM EGTA, 50 mM DTT, 1 mM PMSF and
0.5% (w/v) PPVP). The slurry was filtered through cotton
wool and centrifuged at 16,500 g for 90 min at 4 °C. Proteins
were precipitated from the supernatant with a fivefold volume
of acetone and maintained at –20 °C overnight.
To isolate putative cell-wall-associated proteins from xylem
tissue, we used a modified calcium chloride extraction procedure (Bao et al. 1992). Ground xylem tissue (2.5 g) was vacuum infiltrated with 40 ml of extraction buffer (0.1% (w/v)
potassium acetate, pH 5.0, 4 mM Na2S2O5, 30 mM ascorbic
acid) for 10 min and subsequently filtered through two layers
of Miracloth in a Buchner funnel. The filtered material was
One-dimensional sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) was performed according to
Laemmli (1970). Two-dimensional electrophoresis (non-equilibrium pH gradient electrophoresis (NEpHGE) and SDSPAGE) was performed according to O’Farrell et al. (1977)
with crystal violet as a tracking dye for NEpHGE (Nakayasu
1995). Protein gels were subsequently Coomassie stained or
transferred to nitrocellulose for immunoblotting.
In-gel digestion and peptide sequencing
In-gel digestion of proteins was performed as described by
Ferrara et al. (1993) with either trypsin or Lys C. Proteolytic
fragments in 1% TFA were separated on a C18 reverse phase
HPLC column with a gradient of increasing concentrations of
acetonitrile. Collected peptide fragments were subjected to
Edman degradation (Edman 1950) and the amino acid sequence determined with a Procise Protein Sequencing System
(Model 491, Applied Biosystems, Foster City, CA).
Amino acid composition analysis
Following two-dimensional electrophoresis, proteins were
transferred to PVDF membranes using 10 mM 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS), pH 11.0 as a transfer
buffer (Matsudaira 1987) and stained with Ponceau S, and
spots of interest were excised. Following acid hydrolysis
(110 °C for 22 h), amino acids were derivatized with AccQFluor reagent (Waters Corporation, Milford, MA) and subsequently separated by AccQ-Tag (Waters Corporation) reverse
phase column chromatography following the manufacturer’s
instructions. Chromatographic separation of samples was conducted with a Beckman System Gold HPLC (Beckman Coulter, Fullerton, CA). Individual peaks were identified by means
of authentic standards.
Preparation of antibodies
Individual white leghorn hens were screened to identify an individual in which preimmune IgY preparations did not crossreact with C. sericea protein extracts. The 24-kDa protein
band was cut from a one-dimensional protein gel, homoge-
TREE PHYSIOLOGY VOLUME 22, 2002
SEASONAL REGULATION OF PROTEINS IN C. SERICEA
425
nized in 1 ml of 3.6% (w/v) saline solution and mixed with
750 µl of complete Freund’s adjuvant for the primary injection. Two hundred µg of protein were injected into a white leghorn hen. Two more injections were performed at 2-week
intervals with 100 µg of protein mixed with incomplete
adjuvant. Eggs were collected immediately after the second
booster. The IgY was purified from chicken egg yolks as described by Song et al. (1985).
Immunoblotting
After electrophoresis, proteins were electro-transferred to
nitrocellulose membranes with 25 mM 3-(cyclohexylamino)2-hydroxy-1-propanesulfonic acid (CAPSO)-NaOH transfer
buffer, pH 10.0 (Szewczyk and Kozloff 1985). Membranes
were blocked with 3% BSA and incubated overnight with a
1:5000 dilution of anti-24-kDa protein polyclonal antibody (or
1:1000 anti-dehydrin, with 3% gelatin blocking solution) and
subsequently washed with 50 mM Tris-HCl, 200 mM NaCl,
pH 7.5 (TBS) and TBS with 0.05% (w/v) Tween-20 (TBST)
and twice more with TBS for 10 min per wash. Membranes
were incubated with an alkaline phosphatase conjugated antichicken secondary antibody (1:100,000 dilution; Sigma
Immuno-Chemicals) for 2 h and subsequently washed with
TBS/TBST/TBS. Color reactions were developed with nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate
as described by de Maio (1994). The anti-dehydrin polyclonal
antibody (Close et al. 1993) was a generous gift from Dr. Timothy Close (University of California, Riverside, CA). Goat
anti-rabbit secondary antibody conjugated with horseradish
peroxidase was used in these studies.
Figure 1. (A) SDS-PAGE and (B) immunoblot analysis of total protein extracts from C. sericea xylem. Individual lanes represent seasonal extractions from: (1) January, (2) March, (3) April, (4) May, (5)
June, (6) August, (7) early September, (8) late September, (9) October, (10) November and (11) December. Each lane was loaded with
15 µg of protein. Arrows designate the 24-kDa protein.
close homology to any known protein, similarity to dehydrins
from wheat (Triticum aestivum L.), barley (Hordeum vulgare L.) and rice (Oryza sativa L.) was detected (not shown).
Partial peptide sequence analysis of the most basic 24-kDa
isoform
Results
Seasonal analysis of total and CaCl2-extractable protein
fractions from C. sericea wood
Total and CaCl2-extractable proteins were isolated from
C. sericea wood throughout the year and analyzed by SDSPAGE (Figures 1 and 2). Among total protein extracts, the accumulation of a 24-kDa protein correlated with periods of cold
acclimation. The 24-kDa protein was abundant in January and
March, low in April, and was undetectable in May and June
(Figure 1). The protein was detected again in late August and
continued to accumulate until the end of the year. Four protein
bands of 37, 22, 19 and 17 kDa were reduced in May and June,
whereas two protein bands (84 and 57 kDa) increased during
this time of the year (Figure 1). The 24-kDa protein band was
enriched in the CaCl2-extractable protein fraction of the wood
and constituted a major portion of this protein fraction in winter samples (Figure 2). The 24-kDa band was resolved into at
least four basic proteins by NEpHGE/SDS-PAGE two-dimensional electrophoresis (Figure 3).
Amino acid composition analysis of the most basic 24-kDa
isoform
The most basic 24-kDa isoform had a high glycine content
(Table 1). Although a SWISS-PROT search did not reveal
In-gel digestion of the most basic 24-kDa isoform with trypsin
and Lys C generated multiple peptide fragments. The partial
amino acid sequences of three fragments were determined
Figure 2. SDS-PAGE of CaCl2-extractable proteins from C. sericea
xylem. Lanes represent extracts from wood collected in (1) January,
(2) March, (3) April, (4) May, (5) June, (6) August, (7) early September, (8) late September, (9) October, (10) November and (11) December. Each lane was loaded with 15 µg of protein. An arrow indicates
the 24-kDa protein band. Note the seasonal fluctuation of the predominant 24-kDa protein.
TREE PHYSIOLOGY ONLINE at http://heronpublishing.com
426
SARNIGHAUSEN, KARLSON AND ASHWORTH
Table 2. Partial amino acid sequence of the most basic 24-kDa protein
isoform. Three peptide fragments were isolated following in-gel digestion and partially sequenced. The third fragment had significant
homology to a potato dehydrin (DHN1) (Baudo et al. 1996) and the
symbols *, : and • indicate amino acid residues that were identical,
highly conserved and conserved, respectively.
Figure 3. Two-dimensional (NEpHGE/SDS-PAGE) electrophoresis
of cold-acclimated C. sericea xylem CaCl2 extracts (70 µg protein).
Arrows indicate the four basic isoforms of the 24-kDa protein.
(Table 2). Analysis of protein sequence databases with Clustal
X software indicated homology of Fragment 3 with DHN1
(Baudo et al. 1996), a cold acclimation-related dehydrin from
potato (Solanum tuberosum L.).
The 24-kDa protein: seasonal accumulation, tissue
specificity, and immunological relatedness to dehydrin
proteins
A polyclonal antibody raised against the 24-kDa protein was
used to confirm the seasonal accumulation of the 24-kDa protein and its distribution among tissues. Western blot analysis
of total protein extracts from wood using the anti-24-kDa protein polyclonal antibody detected 24-kDa protein accumula-
Fragment
Deduced amino acid sequence
1
2
3
DHN1
LPGGRKPEQT SQYPAATATRP
QGGVLQR
SNAHGNP–QQTGGNTGGYGAT
–DE YGNP I QQTGGTMGEYGTT
: : * ** * * * * * • • * • * * •*
tion in August and progressive accumulation through the winter months (Figure 1b). The 24-kDa protein was not detected
with this antibody during May and June (Figure 1b). When
CaCl2 extracts were probed with the anti-24-kDa protein
polyclonal antibody, additional proteins that cross-reacted
with this antibody were identified. These cross-reacting protein bands were regulated in a manner similar to the 24-kDa
protein, being more abundant in late fall and winter and at low
or undetectable amounts in summer (Figure 4). Tissue-specific
total protein extractions revealed that the 24-kDa protein was
more prevalent in wood tissue than in bark, and absent in
leaves (Figure 5). Although detectable in the soluble protein
fraction, the 24-kDa protein was more prevalent in the total
protein fraction and CaCl2-extractable fraction.
A polyclonal antibody that was raised against the synthetic
peptide fragment CTGEKKGIMDKIKEKLPGQH, which defines the consensus lysine-rich region of dehydrins (Close et
al. 1993), was used to probe extracts from cold-acclimated
C. sericea wood. The 24-kDa protein band was recognized by
Table 1. Amino acid composition of the most basic 24-kDa protein
isoform.
Amino acid
Symbol
%
Ala
Arg
Asx
Glx
Gly
His
Ile
Leu
Lys
Met
Phe
Pro
Ser
Thr
Tyr
Val
A
R
N/D
Q/E
G
H
I
L
K
M
F
P
S
T
Y
V
8.1
5.0
7.2
13.3
23.4
2.0
1.7
4.1
6.6
1.6
0.0
6.5
5.4
10.3
2.4
2.5
Figure 4. Immunoblot analysis of CaCl2-extractable proteins from
C. sericea xylem. The blot was probed with a 1:5000 dilution of anti24-kDa protein polyclonal antibody. Individual lanes represent extractions from: (1) March, (2) April, (3) May, (4) August, (5) early
September, (6) late September, (7) October, (8) November, (9) December and (10) January. Seasonal regulation of the 24-kDa protein
and additional proteins were detected with protein blot analysis.
TREE PHYSIOLOGY VOLUME 22, 2002
SEASONAL REGULATION OF PROTEINS IN C. SERICEA
Figure 5. Immunoblot analysis from C. sericea leaf, xylem and bark
protein extracts. The blot was probed with a 1:5000 dilution of the
anti-24-kDa protein polyclonal antibody. (1) Total leaf proteins from
June, (2) soluble xylem proteins from January, (3) total xylem proteins from January and (4) total bark proteins from December were
extracted from field-harvested samples. Note absence of 24-kDa antigen in the leaf sample, and reduced amounts in the soluble wood and
bark extracts.
this anti-dehydrin antibody, indicating similarity between the
24-kDa protein and the dehydrin protein family (Figure 6).
Discussion
We report the seasonal accumulation of a 24-kDa protein in
wood of red-osier dogwood. The 24-kDa protein was most
prominent during winter months and disappeared in late
spring, thus correlating with the plant’s state of cold acclimation (Figures 1 and 2). The 24-kDa protein was enriched in the
CaCl2-extractable protein fraction of the wood (Figure 2),
which indirectly supports the notion that the 24-kDa protein is
associated with the cell wall. Proteins may bind to the cell wall
in a non-covalent manner, and the basic amino acid residues
are thought to interact with the block polyanion regions of pectin. Inclusion of CaCl2 in the extraction medium releases these
proteins from the cell wall (Cassab and Varner 1988). Bao et
al. (1992) used this CaCl2 technique to isolate an extensin-like
protein from the cell walls of loblolly pine (Pinus taeda L.)
woody cells.
Figure 6. Immunoblot analysis with anti-24-kDa protein polyclonal
antibody and an anti-dehydrin antibody detected similar proteins in
cold-acclimated C. sericea xylem. Membranes were incubated with
(1) a 1:5000 dilution of anti-24-kDa protein polyclonal antibody and
(2) a 1:1000 dilution of an anti-dehydrin antibody. Note similar detection of the 24-kDa protein and less abundant proteins in both preparations.
427
Accumulation of cell wall proteins, or an induction of their
transcripts during cold acclimation, has been reported. In
Arabidopsis thaliana (L.) Heynh., expression of genes encoding cell wall modifying enzymes is up-regulated following
cold treatment (Xu et al. 1996). Proposed functions of cell wall
proteins that accumulate during cold acclimation include mechanical protection of the wall itself (Weiser et al. 1990), stabilization of the linkage between the cell wall and the plasma
membrane during cold stress (Goodwin et al. 1996), and antifreeze (Sabala et al. 1996) or disease resistance activity (Antikainen and Griffith 1997, Griffith et al. 1997). In addition,
Livingston and Henson (1998) reported increased activities of
fructan exohydrolase and invertase in the apoplastic fluid extracted from winter oat (Avena sativa L.) crown tissue during
cold hardening. It is important to note, however, that none of
these cold-inducible cell wall proteins were similar to the
24-kDa protein identified from C. sericea wood.
Two-dimensional electrophoresis (NepHGE/SDS-PAGE)
resolved the C. sericea 24-kDa band into at least four basic
proteins (Figure 3). The most abundant and basic isoform was
partially sequenced and the amino acid composition determined (Tables 1 and 2) and found to be similar to the dehydrin
proteins. Based on total amino acid composition, the closest
similarity was to rab15, an ABA-inducible protein found in
water-stressed wheat roots (King et al. 1992). Sequence analysis from one partial peptide fragment revealed similarity to
DHN1 (Baudo et al. 1996), a low-temperature-inducible and
ABA-responsive dehydrin in potato. In addition, protein blot
analysis confirmed immunological similarity of the 24-kDa
protein to dehydrin proteins (Figure 6).
Dehydrins are characteristically hydrophilic and heat stable,
and may be induced by multiple desiccation stimuli such as
drought, salinity and freezing (Close et al. 1989, Close 1996,
1997, Campbell and Close 1997). Numerous investigations in
woody plants have documented the presence of dehydrin-like
proteins and their accumulation during cold acclimation
(Arora et al. 1992, 1996, Arora and Wisniewski 1996, Salzman et al. 1996, Wisniewski et al. 1996, Rinne et al. 1998,
Sauter et al. 1999). Despite extensive research, the precise
function of dehydrins remains obscure. It is hypothesized that
dehydrins stabilize macromolecules during desiccation stress
(e.g., Close 1996, 1997). Wisniewski et al. (1999) demonstrated that a dehydrin in peach (Prunus persica (L.) Batsch)
exhibited both cryoprotective and anti-freeze activity.
Several studies showed that dehydrin-like proteins were localized in the nucleus and cytoplasm (Asghar et al. 1994,
Godoy et al. 1994, Egerton-Warburton et al. 1997, Wisniewski
et al. 1999). However, two recent studies have described dehydrin-like protein accumulation in the vicinity of the plasma
membrane and the cell wall in cold-acclimated tissues (Danyluk et al. 1998, Rinne et al. 1999). Danyluk et al. (1998) reported accumulation of the dehydrin-like protein WCOR410
in association with the plasma membrane in root, crown, stem
and leaf tissues of winter wheat during cold acclimation. In addition, immunogold labeling was also detected within intercellular spaces and over the fibrillar network at cell wall junctions. However, WCOR410 does not contain a signal sequence
TREE PHYSIOLOGY ONLINE at http://heronpublishing.com
428
SARNIGHAUSEN, KARLSON AND ASHWORTH
that would target the protein for extracellular transport. In contrast to the basic 24-kDa protein accumulated in C. sericea
wood, WCOR410 is an acidic protein with a pI of 5.1. Micrographs from Rinne et al. (1999) showed sparse immunogold
labeling in the vicinity of the plasma membrane in cold-acclimated birch (Betula pubescens Ehrh.) apices.
Cornus sericea displays extreme freeze-tolerance. In the
cold hardened state, stems can survive gradual immersion in
liquid helium (Guy et al. 1986). The 24-kDa protein represents
the major component of the CaCl2-extractable fraction of the
wood proteins (Figure 2) and represents only a minor component of the Tris-extractable soluble protein fraction (Figure 5),
which indirectly supports a putative cell wall association.
However, it cannot be ruled out that the close association of the
24-kDa protein with the cell wall occurs only during extraction. The majority of immunolocalization studies of dehydrin-like proteins have indicated a cytosolic and nuclear distribution (Asghar et al. 1994, Godoy et al. 1994, Close 1997,
Egerton-Warburton et al. 1997, Wisniewski et al. 1999).
In summary, we have demonstrated the seasonal regulation
of a predominant 24-kDa protein that accumulates during periods of cold acclimation in one of the most freeze-tolerant species known. The 24-kDa protein is enriched in the CaCl2extractable fraction, which indirectly supports a putative cell
wall localization. The 24-kDa protein is immunologically related to dehydrins; however, our finding that it is associated
with the cell wall contrasts with most previous studies that indicate a cytosolic and nuclear localization of this protein family. Therefore, the possible association of a dehydrin-like protein to the cell wall of C. sericea is perplexing. To resolve this
conflict, we plan to use the anti-24 kDa protein polyclonal antibody in immunolocalization experiments. These investigations should clarify subcellular localizations and may provide
further insight into the possible function of the 24-kDa protein
during cold acclimation of Cornus sericea.
Acknowledgments
The authors thank Dr. Timothy Close, University of California, Riverside, for his kind donation of the anti-dehydrin polyclonal antibody.
The authors also thank Vicki Stirm for her technical assistance
throughout the project. This research was supported by the United
States Department of Agriculture through the National Research Initiative Competitive Grants Program under Agreement 94-371000689.
References
Allard, F., M. Houde, M. Krol, A. Iranov, N.P.A. Huner and F. Sarhan.
1998. Betaine improves freezing tolerance in wheat. Plant Cell
Physiol. 39:1194–1202.
Antikainen, M. and M. Griffith. 1997. Antifreeze protein accumulation in freezing-resistant cereals. Physiol. Plant. 99:423–432.
Anitkainen, M., M. Griffith, J. Zhang, W. Hon, S.C. Yang and
K. Pihakaski-Maunsbach. 1996. Immunolocalization of antifreeze
proteins in winter rye leaves, crowns, and roots by tissue printing.
Plant Physiol. 110:845–857.
Arora, R. and M. Wisniewski. 1996. Accumulation of a 60-kD
dehydrin protein in peach xylem tissues and its relationship to cold
acclimation. HortScience 31:923–925.
Arora, R., M.E. Wisniewski and R. Scorza. 1992. Cold acclimation in
genetically related (sibling) deciduous and evergreen peach (Prunus persica [L.] Batsch). I. Seasonal changes in cold hardiness and
polypeptides of bark and xylem tissues. Plant Physiol. 99:
1562–1568.
Arora, R., M. Wisniewski and L.J. Rowland. 1996. Cold acclimation
and alterations in dehydrin-like and bark storage proteins in the
leaves of sibling deciduous and evergreen peach. J. Am. Soc.
Hortic. Sci. 121:915–919.
Asghar, R., R.D. Fenton, D.A. DeMason and T.J. Close. 1994. Nuclear and cytoplasmic localization of maize embryo ad aleurone
dehydrin. Protoplasma 177:87–94.
Ashworth, E.N., V.E. Stirm and J.J. Volenec. 1993. Seasonal variations in soluble sugars and starch within woody stems of Cornus
sericea L. Tree Physiol. 13:379–388.
Bao, W., D.M. O’Malley and R.R. Sederoff. 1992. Wood contains a
cell-wall structural protein. Proc. Natl. Acad. Sci. 89:6604–6608.
Baudo, M.M., L.A. MezaZepeda, E.T. Palva and P. Heino. 1996. Induction of homologous low temperature and ABA-responsive
genes in frost resistant (Solanum commersonii) and frost-sensitive
(Solanum tuberosum cv Bintje) potato species. Plant Mol. Biol.
30:331–336.
Bradford, M.M. 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of
protein-dye binding. Anal. Biochem. 72:248–254.
Brush, R.A., M. Griffith and A. Mlynarz. 1994. Characterization and
quantification of intrinsic ice nucleators in winter rye (Secale
cereale) leaves. Plant Physiol. 104:725–736.
Burke, M.J., L.V. Gusta, H.A. Quamme, C.J. Weiser and P.H. Li.
1976. Freezing and injury in plants. Annu. Rev. Plant Physiol. 27:
507–528.
Burke, M.J., C.B. Rajashekar and M.F. George. 1983. Freezing of
plant tissue and evidence of large negative pressure potentials.
Plant Physiol. 72:44.
Campbell, S.A. and T.J. Close. 1997. Dehydrins: genes, proteins, and
associations with phenotypic traits. New Phytol. 137:61–74.
Cassab, G.I. and J.E. Varner. 1988. Cell wall proteins. Annu. Rev.
Plant Physiol. Plant Mol. Biol. 39:321–353.
Chun, J.U., X.M. Yu and M. Griffith. 1998. Genetic studies of antifreeze proteins and their correlation with winter survival in wheat.
Euphytica 102:219–226.
Close, T.J. 1996. Dehydrins: emergence of a biochemical role of a
family of plant dehydration proteins. Physiol. Plant. 97:795–803.
Close, T.J. 1997. Dehydrins: a commonality in the response of plants
to dehydration and low temperature. Physiol. Plant. 100:291–296.
Close, T.J., A.A. Kortt and P.M. Chandler. 1989. A cDNA-based comparison of dehydration-induced proteins (dehydrins) in barley and
corn. Plant Mol. Biol. 13:95–108.
Close, T.J., R.D. Fenton and F. Moonan. 1993. A view of plant dehydrins using antibodies specific to the carboxy terminal peptide.
Plant Mol. Biol. 23:279–286.
Danyluk, J., A. Perron, M. Houde, A. Limin, B. Fowler, N. Benhamou
and F. Sarhan. 1998. Accumulation of an acidic dehydrin in the vicinity of the plasma membrane during cold acclimation of wheat.
Plant Cell 10:623–638.
de Maio, A. 1994. Protein blotting and immunoblotting using nitrocellulose membranes. In Protein Blotting: A Practical Approach.
Ed. B.S. Dunbar. IRL Press, Oxford, pp 11–32.
Edman, P. 1950. Method for determination of the amino acid sequence in peptides. Acta Chem. Scand. 4:283–293.
Egerton-Warburton, L.M., R.A. Balsamo and T.J. Close. 1997. Temporal accumulation and ultrastructural localization of dehydrins in
Zea mays. Physiol. Plant 101:545–555.
TREE PHYSIOLOGY VOLUME 22, 2002
SEASONAL REGULATION OF PROTEINS IN C. SERICEA
Ferrara, P., J. Rosenfeld, J.C. Guillemot and J. Capdevielle. 1993. Internal peptide sequence of proteins digested in-gel after one- or
two-dimensional gel electrophoresis. In Techniques in Protein
Chemistry. Vol. 4. Ed. R. Hogue-Angeletti. Academic Press, New
York, pp 379–387.
Godoy, J.A., R. Lunar, S. Torres-Schumann, J. Moreno, R.M. Rodrigo
and J.A. Pintor-Toro. 1994. Expression, tissue distribution and
subcellular localization of dehydrin TAS14 in salt-stressed tomato
plants. Plant Mol. Biol. 26:1921–1934.
Goodwin, W., J.A. Pallas and G.I. Jenkins. 1996. Transcripts of a gene
encoding a putative cell wall–plasma membrane linker protein are
specifically cold-induced in Brassica napus. Plant Mol. Biol. 31:
771–781.
Griffith, M. and G.M. Brown. 1982. Cell wall deposits in winter rye
Secale cereale L. ‘Puma’ during cold acclimation. Bot. Gaz. 143:
486–490.
Griffith, M., P. Ala, D.S.C. Wang, W. Hon and B.A. Moffatt. 1992.
Antifreeze protein produced endogenously in winter rye leaves.
Plant Physiol. 100:593–596.
Griffith, M., M. Antikainen, W.-C. Hon, K. Pihakaski-Maunsbach,
X.-M. Yu, J.U. Chun and D.S.C. Yang. 1997. Antifreeze proteins in
winter rye. Physiol. Plant. 100:327–332.
Guy, C. 1990. Cold acclimation and freezing stress tolerance: Role of
protein metabolism. Annu. Rev. Plant Physiol. Plant Mol. Biol.
41:187–223.
Guy, C.L., K.J. Niemi, A. Fennel and J.V. Carter. 1986. Survival of
Cornus sericea L. stem cortical cells following immersion in liquid
helium. Plant Cell Environ. 9:447–450.
Hon, W., M. Griffith, P. Chong and D.S.C. Yang. 1994. Extraction and
isolation of antifreeze proteins from winter rye (Secale cereale L.)
leaves. Plant Physiol. 104:971–980.
Hummel, R.L., P.D. Ascher and H.M. Pellet. 1982. Inheritance of the
photoperiodically induced cold acclimation response in Cornus
sericea L., red-osier dogwood. Theor. Appl. Genet. 62:385–394.
Huner, N.P.A., J.P. Palta, P.H. Li and J.V. Carter. 1981. Anatomical
changes in leaves of puma rye in response to growth at cold-hardening temperatures. Bot. Gaz. 142:55–62.
Jian, L.C., L.H. Sun and D.L. Sun. 1987. Glycoproteins at the cell surface in cold hardy and cold tender wheat (Triticum aestivum). In
Plant Cold Hardiness. Ed. P.H. Li. Alan R. Liss, New York, pp
59–66.
King, S.W., C.P. Joshi and H.T. Nguyen. 1992. DNA sequence of an
ABA-responsive gene (rab 15) from water-stressed wheat roots.
Plant Mol. Biol. 18:119–121.
Kozbial, P.Z., A. Jerzmanowski, A.H. Shirsat and A. Kacperska.
1998. Transient freezing regulates expression of extensin-type
genes in winter oilseed rape. Physiol. Plant. 103:264–270.
Kuroda, H. and S. Sagisaki. 1993. Ultrastructural changes in cortical
tissues of apple (Malus pumila Mill.) associated with cold hardiness. Plant Cell Physiol. 34:357–365.
Laemmli, U.K. 1970. Cleavage of structural proteins during assembly
of their head of bacteriophage T4. Nature 227:680–685.
Livingston, D.P., III and C.A. Henson. 1998. Apoplastic sugars,
fructan exohydrolase, and invertase in winter oat: Responses to
second-stage cold hardening. Plant Physiol. 116:403–408.
Marentes, E., M. Griffith, A. Mlynarz and R.A. Brush. 1993. Proteins
accumulate in the apoplast of winter rye leaves during cold acclimation. Physiol. Plant. 87:499–507.
Matsudaira, P. 1987. Sequence from picomole quantities of proteins
electroblotted onto polyvinylidene difluoride membranes. J. Biol.
Chem. 262:10,035–10,038.
429
Nakayasu, H. 1995. Crystal violet as an indicator dye for nonequilibrium pH gradient electrophoresis (NEpHGE). Anal. Biochem. 229:259–262.
Nomura, M., Y. Muramoto, S. Yasuda, T. Takabe and S. Kishitani.
1995. The accumulation of glycinebetaine during cold acclimation
in early and late cultivars of barley. Euphytica 83:247–250.
O’Farrell, P.Z., H.M. Goodman and P.H. O’Farrell. 1977. High resolution two dimensional electrophoresis of basic as well as acidic
proteins. Cell 12:1133–1142.
Olien, C.R. 1978. Analyses of freezing stress and plant response. In
Plant Cold Hardiness and Freezing Stress. Eds. P.H. Li and A.
Sakai. Academic Press, New York, pp 37–38.
Pearce, R.S. 1999. Molecular analysis of acclimation to cold. Plant
Growth Regul. 29:47–76.
Perras, M. and F. Sarhan. 1989. Synthesis of freezing tolerance proteins in leaves, crown and roots during cold acclimation of wheat.
Plant Physiol. 89:577–585.
Pihakaski-Maunsbach, K., M. Griffith, M. Antikainen and A.B.
Maunsbach. 1996. Immunogold localization of glucanase-like antifreeze protein in cold acclimated winter rye. Protoplasma 191:
115–125.
Pomeroy, M.K. and D. Siminovitch. 1971. Seasonal cytological
changes in secondary phloem parenchyma cells in Robinia pseudoacacia in relation to cold hardiness. Can. J. Bot. 49:787–795.
Rajashekar, C. and M. Burke. 1996. Freezing characteristics of rigid
plant tissues—development of cell tension during extracellular
freezing. Plant Physiol. 111:597–603.
Rajashekar, C.B. and A. Lafta. 1996. Cell-wall changes and cell tensions in response to cold acclimation and exogenous abscisic acid
in leaves and cell cultures. Plant Physiol. 111:605–612.
Rinne, P.L.H., A. Welling and P. Kaikuranta. 1998. Onset of freezing
tolerance in birch (Betula pubescens Ehrh.) involves LEA proteins
and osmoregulation and is impaired in an ABA-deficient genotype.
Plant Cell Environ. 21:601–611.
Rinne, P.L.H., P.L.M. Kaikuranta, L.H.W. van der Plas and C. van der
Schoot. 1999. Dehydrins in cold-acclimated apices of birch (Betula
pubescens Ehrh.): production, localization and potential role in rescuing enzyme function during dehydration. Planta 209:377–388.
Ristic, Z. and E.N. Ashworth. 1994. Response of xylem ray parenchyma cells of red-osier dogwood (Cornus sericea L.) to freezing
stress: microscopic evidence of protoplasm concentration. Plant
Physiol. 104:737–746.
Sabala, I., U. Egertsdotter, H. von Firks and S. von Arnold. 1996.
Abscisic acid induced secretion of an antifreeze-like protein in embryogenic cell lines of Picea abies. J. Plant Physiol. 149:163–170.
Sagisaka, S., M. Asada and Y.H. Ann. 1990. Ultrastructure of poplar
cortical cells during the transition from growing to wintering stages
and vice versa. Trees 4:120–127.
Salzman, R.A., R.A. Bressan, P.M. Hasegawa, E.N. Ashworth and
B.P. Bordelon. 1996. Programmed accumulation of LEA-like proteins during desiccation and cold acclimation of overwintering
grape buds. Plant Cell Environ. 19:713–720.
Sauter, J.J., B. van Cleve and S. Wellenkamp. 1989. Ultrastructural
and biochemical results on the localization and distribution of storage proteins in a poplar tree and in twigs of other tree species. Holzforschung 43:1–6.
Sauter, J.J., S. Westphal and M. Wisniewski. 1999. Immunological
identification of dehydrin-related proteins in the wood of five species of Populus and in Salix caprea L. J. Plant Physiol. 154:
781–788.
Song, C., J. Yu, D.H. Bai, P.Y. Hester and K. Kim. 1985. Antibodies to
the α-subunit of insulin receptor from eggs of immunized hens. J.
Immunol. 135:3354–3359.
TREE PHYSIOLOGY ONLINE at http://heronpublishing.com
430
SARNIGHAUSEN, KARLSON AND ASHWORTH
Steponkus, P.L. 1984. Role of the plasma membrane in freezing injury and cold acclimation. Annu. Rev. Plant Physiol. 35:543–584.
Szewczyk, B. and L.M. Kozloff. 1985. A method for efficient blotting
of strongly basic proteins from sodium dodecyl sulfate-polyacrylamide gels to nitrocellulose. Anal. Biochem. 150:403–407.
Tao, D. and P.H. Li. 1993. Cell wall and freezing injury: A mini-review. In Advances in Plant Cold Hardiness. Eds. P.H. Li and L.
Christersson. CRC Press, Boca Raton, pp 195–201.
Thomashow, M.F. 1999. Plant cold acclimation: Freezing tolerance
genes and regulatory mechanisms. Annu. Rev. Plant Physiol. Plant
Mol. Biol. 50:571–599.
Wallner, S.J., M. Wu and S.J. Anderson-Krengel. 1986. Changes in
extracellular polysaccharides during cold acclimation of cultured
pear cells. J. Am. Soc. Hortic. Sci. 111:769–773.
Weiser, C.J. 1970. Cold resistance and injury in woody plants. Science 169:1269–1278.
Weiser, R.L., S.J. Wallner and J.W. Weddel. 1990. Cell wall extensin
mRNA changes during cold acclimation of pea seedlings. Plant
Physiol. 93:1021–1026.
Wisniewski, M.E. and E.N. Ashworth. 1986. A comparison of seasonal ultrastructural changes in stem tissues of peach [Prunus
persica (L.) batsch.] that exhibit contrasting mechanisms of cold
hardiness. Bot. Gaz. 147:407–417.
Wisniewski, M., G. Davis and K. Schaffer. 1991. Mediation of deep
supercooling of peach and dogwood by enzymatic modifications in
cell-wall structure. Planta 184:254–260.
Wisniewski, M., T.J. Close, T. Artlip and R. Arora. 1996. Seasonal
patterns of dehydrins and 70-kDa heat-shock proteins in bark tissues of eight species of woody plants. Physiol. Plant. 96:496–505.
Wisniewski, M., R. Webb, R. Balsamo, T.J. Close, X-M. Yu and M.
Griffith. 1999. Purification, immunolocalization, cryoprotective,
and antifreeze activity of PCA60: A dehydrin from peach (Prunus
persica). Physiol. Plant. 105:600–608.
Worrall, D., L. Elias, D. Ashford, M. Smallwood, C. Sidebottom,
P. Lillford, J. Telford, C. Holt and D. Bowles. 1998. A carrot
leucine-rich-repeat protein that inhibits ice recrystallization. Science 282:115–117.
Xu, W., P. Campbell, A.K. Vargheese and J. Braam. 1996. The
Arabidopsis XET-related gene family: environmental and hormonal regulation of expression. Plant J. 9:879–889.
TREE PHYSIOLOGY VOLUME 22, 2002