Download Novel plasmodesmata association of dehydrin

Tree Physiology 23, 759–767
© 2003 Heron Publishing—Victoria, Canada
Novel plasmodesmata association of dehydrin-like proteins in coldacclimated red-osier dogwood (Cornus sericea)
DALE T. KARLSON,1,2 TAKESHI FUJINO,3,4 SATOSHI KIMURA,5 KEI’ICHI BABA,3 TAKAO
ITOH3 and EDWARD N. ASHWORTH1,6
1
Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, IN 47907-1165, USA
2
Present address: Department of Low Temperature Sciences, National Agricultural Research Center for Hokkaido Region, Hitsujigaoka, Sapporo
062-8555, Japan
3
Wood Research Institute, Kyoto University, Uji, Kyoto, Japan
4
Present address: Forest Biotechnology Group, Department of Forestry, North Carolina State University, Raleigh, NC 27695, USA
5
Forestry and Forest Products Research Institute, Tsukuba, Ibaraki 305-8687, Japan
6
Author to whom correspondence should be addressed ([email protected])
Received November 6, 2002; accepted January 24, 2003; published online July 1, 2003
Summary Dehydrins are proteins associated with conditions affecting the water status of plant cells, such as drought,
salinity, freezing and seed maturation. Although the function
of dehydrins remains unknown, it is hypothesized that they stabilize membranes and macromolecules during cellular dehydration. Red-osier dogwood (Cornus sericea L.), an extremely
freeze-tolerant woody plant, accumulates dehydrin-like proteins during cold acclimation and the presence of these proteins
is correlated with increased freeze tolerance (Karlson 2001,
Sarnighausen et al. 2002, Karlson et al. 2003). Our objective
was to determine the location of dehydrins in cold-acclimated
C. sericea stems in an effort to provide insight into their potential role in the freeze tolerance of this extremely cold hardy species. Abundant labeling was observed in the nucleus and
cytoplasm of cold-acclimated C. sericea stem cells. In addition, labeling was observed in association with plasmodesmata
of cold-acclimated vascular cambium cells. The unique association of dehydrin-like proteins with plasmodesmata has not
been reported previously.
Keywords: dehydrins, desiccation stress, freeze tolerance,
immunolocalization.
Introduction
Dehydrins are stress responsive proteins that accumulate as
tissue water status declines, such as during seed maturation or
in response to salinity, drought and freezing stress (reviewed
by Close et al. 1993a, 1993b, Close 1996, 1997, Campbell and
Close 1997). Members of this protein family are characterized
by the presence of a lysine-rich consensus sequence (EKKGI
MDKIKEKLPG), a tract of serine residues and typically 1–3
Y motifs (DEYGNP) near the N-terminus (Close et al. 1993b,
Close 1996, 1997, Campbell and Close 1997). Although
linked to desiccation stress, the function of dehydrins is un-
known. Structural features of these proteins have led investigators to speculate on their potential function in stress
tolerance. The lysine-rich consensus sequence is predicted to
form an amphipathic α-helix domain (Close et al. 1993b, Dure
1993, Close 1996) and the Y motif resembles nucleotide binding sites in molecular chaperone proteins (Close 1996). Based
on these features, dehydrins are proposed to bind non-specifically to macromolecules and membranes through hydrophobic interactions with the putative amphipathic α-helix domain
(Close et al. 1993b, Close 1996, 1997). Because of their overall hydrophilic nature, water may associate with dehydrins
(Close 1997) and prevent aggregation of macromolecules during desiccation (Close 1996, 1997).
Several studies have used immunolocalization techniques to
document the presence of dehydrins in the cytoplasm and nucleus of plant cells (Asghar et al. 1994, Godoy et al. 1994,
Houde et al. 1995, Egerton-Warburton et al. 1997, Wisniewski
et al. 1999). Recently, however, Danyluk et al. (1998) and
Rinne et al. (1999) reported that dehydrins were associated
with the plasma membranes of cold-acclimated wheat and
birch, respectively. In addition, Ukaji et al. (2001) identified
an association between a group 3 LEA (late embryogenesis
abundant) protein and the endoplasmic reticulum of cold-acclimated mulberry cortical cells. These latter reports are consistent with the proposed function of these proteins to stabilize
membranes during desiccation stress.
Red-osier dogwood (Cornus sericea L.) is among the most
freeze-tolerant plants, and is capable of surviving extremely
low temperatures (–269 °C) (Guy et al. 1986). Cornus sericea
exhibits a substantial accumulation of a 24-kDa dehydrin-like
protein within xylem tissues during cold acclimation (Karlson
2001, Sarnighausen et al. 2002, Karlson et al. 2003). Biochemical evidence suggests that the 24-kDa protein is associated with the cell walls of wood tissue (Sarnighausen et al.
2002). The purpose of the present study was to clarify the sub-
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cellular distribution of dehydrin-like proteins within C. sericea
stem tissue. In addition, we wanted to compare the location of
dehydrins in an extremely freeze-tolerant plant with that previously reported in less freeze-tolerant plants.
A clonal population of C. sericea (Massachusetts ecotype)
was used in all experiments. Plants were grown in a field plot
adjacent to the Purdue University campus and received supplemental drip irrigation and periodic granular fertilization
(N,P,K (12:12:12)). Stem tips were collected in the field, packaged on ice and carried to the laboratory for chemical fixation
and total protein extraction.
blocking serum (heat inactivated at 65 °C for 5 min). Xylem
tissue was incubated at 4 °C overnight in a 1:250 dilution (in
10 mM PBS, 0.1% BSA pH 7.2) of anti-24-kDa protein immune serum. Samples were washed with gentle agitation with
three changes of PBS (10 min each rinse). Goat anti-chicken
immunogold (5 nm gold particles) secondary antibody
(Aurion, Wageningen, Netherlands) was diluted 1:50 in
10 mM PBS (pH 7.2), 0.1% BSA and added to the samples for
1 h at room temperature. Samples were washed 3 times with
PBS for 5 min and 3 times with ultra-pure water for 3 min. An
IntenSE M silver enhancement kit (Amersham Biosciences,
Piscataway, NJ) was used for the development of the silver deposition reaction onto secondary antibodies. Sections were
viewed with an Olympus Vanox light microscope (Olympus
America, Melville, NY) and photographed with a Spot RT digital camera (Diagnostic Instruments, Sterling Heights, MI).
Preparation of antibodies
Immunolocalization: transmission electron microscopy
The anti-24-kDa protein polyclonal antibody was raised in a
white leghorn hen and purified from eggs as previously described (Sarnighausen et al. 2002). To minimize nonspecific
secondary cell wall labeling, polyclonal chicken anti-24-kDa
protein immune serum preparations were further purified as
described by Karlson et al. (2002).
Ultrathin sections (90 nm) were prepared for TEM immunogold evaluation as previously described (Karlson et al. 2002).
Serial dilutions were performed to identify the anti-24-kDa
protein immune serum concentration that consistently produced maximum labeling with minimal nonspecific background. Anti-24-kDa immune protein serum was diluted 1:15
and pre-immune serum was diluted 1:3.75 in 10 mM PBS,
0.1% BSA (pH 7.2). Five nm (prepared by Debra Sherman,
Purdue University, West Lafayette) or 10 nm (British BioCell
International, Cardiff, U.K.) colloidal gold-conjugated rabbit
anti-chicken secondary antibodies were used for immunolabeling of 24-kDa protein-like antigens at a 1:50 dilution. A
monoclonal antibody directed against (1-3)-β-D-glucan (BioSupplies, Parkville, Australia) was diluted 1:100 to locate
callose deposition. Identical incubation and washing procedures were followed, as previously described (Karlson et al.
2002), except for the use of 10 nm gold rabbit anti-mouse secondary antibodies diluted 1:50 for callose localization (Sigma
Immuno Chemicals).
All micrographs were scanned at 600 dpi for high resolution
printing as previously described (Kolosova et al. 2001). In Figures 3A–3H, the gold particle size was doubled with Adobe
Photoshop 5.5 software to allow easier viewing of gold particles at low magnifications. Digital enlargement of gold particles was performed as previously described (Kolosova et al.
2001).
Materials and methods
Plant material
Protein extraction and blot analysis
Total proteins were extracted from lyophilized C. sericea xylem as previously described (Karlson et al. 2003). Separated
proteins were electro-transferred to nitrocellulose membranes
in a 25 mM CAPSO-NaOH transfer buffer, pH 10.0 (3cyclohexylamino-2-hydroxy-1-propanesulfonic acid) (Szewczyk and Kozloff 1985). Protein blot analysis was performed
as previously described (Sarnighausen et al. 2002). A dot blot
analysis using serial dilutions of immune and pre-immune
chicken IgY serum, with alkaline phosphatase-conjugated
secondary rabbit anti-chicken antibodies (Sigma Immuno
Chemicals, St. Louis, MO), determined that the immune serum preparation was four times more concentrated (not
shown). Therefore, pre-immune serum was used at a 1:1250
dilution and immune serum was used at a 1:5000 dilution.
Preparation of tissue for light and TEM microscopy
Stem tips from current year’s growth were examined exclusively in these experiments. One-mm-thick cross sections
from the second internode were quartered, fixed and embedded for transmission electron microscopy (TEM) and
immunolocalization as previously described (Karlson et al.
2002).
Results
Immunolocalization: silver enhancement
Immunodetection of 24-kDa protein with protein blot
analysis
Distribution of dehydrin-like proteins in stem cross sections
was observed by silver enhanced light microscopy. Specimens
were sectioned (0.5 µm) with a Diatome Histo knife (Diatome,
Fort Washington, PA) and transferred to clean glass slides.
Samples were heated at 55 °C for 24 h and subsequently
blocked for 1 h in 10 mM phosphate buffered saline (PBS,
pH 7.2) with 0.1% bovine serum albumin (BSA) and 5% goat
In cold-acclimated C. sericea xylem total protein extracts,
anti-24-kDa protein immune serum recognized the 24-kDa
protein and several less prominent proteins (Figure 1). Blot-affinity purification of anti-24-kDa protein immune serum to the
24-kDa antigen was attempted and failed to minimize cross reaction with less prominent proteins in cold-acclimated xylem
total and CaCl2 protein extracts (data not shown). As a result,
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immunolocalization data represents in situ labeling of several
24-kDa-like antigens within cold-acclimated tissues. Blot
analyses of total protein extracts from two negative controls,
non-acclimated C. sericea and cold-acclimated Cornus florida L.
xylem, confirmed the absence of 24-kDa protein antigen
within these tissues (Figure 1). In addition, pre-immune serum
did not recognize the 24-kDa antigen within total protein extracts from cold-acclimated C. sericea xylem (Figure 1).
Tissue localization of the 24-kDa protein using light
microscopy
Silver enhancement was used to visualize the general distribution of 24-kDa protein-like antigens in semi-thin stem cross
sections of cold-acclimated C. sericea. Both brightfield and
darkfield illumination were used to observe tissues and visualize deposition of silver grains. Abundant labeling was observed within the pith, axial and ray parenchyma cells of
cold-acclimated stems of C. sericea (Figures 2A and 2B). The
Figure 1. Protein blot analysis demonstrating the specificity of
polyclonal anti-24-kDa protein immune serum. Ten µg of total xylem
protein extracts from non-acclimated and cold-acclimated C. sericea,
and cold-acclimated C. florida were probed with a 1:5000 dilution of
anti-24-kDa protein immune serum. Anti-24-kDa pre-immune serum
was used at a 1:1250 dilution and incubated with cold-acclimated
C. sericea xylem total protein extracts. Note the recognition of a
24-kDa protein in cold-acclimated C. sericea extracts and the absence
in extracts from non-acclimated C. sericea. Additional cross-reactive
bands were detected within cold- and non-acclimated C. sericea xylem and likely represent related members of a small C. sericea dehydrin-like protein family (Karlson 2001). Note limited cross-reactivity
against extracts from cold-acclimated C. florida and absence of
cross-reactivity with pre-immune serum.
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vacuoles within these cells were unlabeled, as were the lumens
of xylem vessel elements and fibers. Cambial cells, phloem
companion cells and bark cortical cells were labeled with silver grains, whereas phloem fiber cells and enucleate sieve elements were not (Figures 2A and 2B). Non-acclimated stem
sections of C. sericea were not labeled (Figures 2C and 2D).
Subcellular localization of the 24-kDa protein and
(1-3)-β-D-glucan (callose)
The subcellular location of the 24-kDa protein in cold-acclimated C. sericea stem tissues was determined by immunogold
labeling and TEM. The nucleus and cytoplasm of xylem parenchyma and cortical cells were uniformly labeled with
immunogold particles (Figures 3A and 3E). Exposure of similar tissues to pre-immune serum produced only background
labeling (compare Figures 3A and 3E to 3B and 3F). The nucleus and cytoplasm of non-acclimated C. sericea cells (Figures 3C and 3G) and cold-acclimated C. florida cells (Figures
3D and 3H) did not contain significant label. In each instance,
immunolocalization data were consistent with protein blot
analyses, demonstrating the recognition of 24-kDa antigen in
cold-acclimated C. sericea xylem tissue and the absence of
24-kDa antigen in both non-acclimated C. sericea and coldacclimated C. florida (compare Figures 1 and 3).
Abundant gold labeling was found in association with the
plasmodesmata of cold-acclimated xylem cells in C. sericea.
Areas of the cell wall containing plasmodesmata were densely
labeled (Figures 3I–J, 4A–B, 5A–C), with labeling most pronounced in xylem parenchyma cells adjacent to the cambial
region. Such labeling was largely absent in non-acclimated
cells (Figure 3K) and in cold-acclimated specimens that were
incubated with pre-immune serum (Figures 3L and 5D). Gold
particles were predominantly associated with the neck and
collar regions of plasmodesmata (Figures 4A, 4B and 5A–C)
as opposed to the interior or desmotubule region. Although the
exterior portions of the plasmodesmata were more heavily labeled than the interior regions, the latter areas contained the
anti-24-kDa antigen as noted in Figure 4B. There appeared to
be no difference in the polarity of labeling between adjacent
cells with the collar and neck regions associated with adjacent
cells equally labeled. The pattern of labeling with anti-24-kDa
antiserum was strikingly similar to the pattern of labeling observed when using a monoclonal immune serum specific for
(1-3)-β-D-glucan (callose) (Figures 5E and 5F).
Discussion
We have previously described the seasonal regulation of a predominant 24-kDa dehydrin-like protein within the CaCl2
extractable fraction from C. sericea xylem (Sarnighausen et
al. 2002). Cell wall proteins were preferentially isolated by extraction with CaCl2 to disrupt ionic associations, thereby extracting proteins with cell-wall specificity (Cassab and Varner
1988). Bao et al. (1992) previously isolated a structural
extensin-like protein from loblolly pine cell walls by this
method. Thus, chemical evidence supported a putative associ-
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Figure 2. Distribution of 24-kDalike protein within C. sericea stem
tissue. Stem cross sections (0.5 µm)
were cut from cold-acclimated
(A, B) and non-acclimated (C, D)
tissues and incubated with anti24-kDa protein immune serum
(1:250) and goat anti-chicken
immunogold labeled secondary antibody (1:50). Light (A, C) and
dark field (B, D) microscopy images are presented. A silver enhancement reaction and dark field
illumination were used to facilitate
immunolocalization. Positive labeling appears as a dense white signal
in dark field micrographs. In coldacclimated sections (A, B), xylem
parenchyma (XP), vascular cambium (C) and cortical cells within
xylem (CX) and phloem (CB) exhibited substantial labeling. Note
the absence of gold labeling within
vacuoles (V) of pith parenchyma
(PP) and xylem parenchyma (XP).
Also note the lack of label within
phloem fibers (PF), enucleate sieve
elements (SE) and the presence of
labeling within adjacent companion
cells (CC). Note the lack of similar
labeling patterns within non-acclimated (C, D) specimens. Bar =
6 µm.
ation of the 24-kDa protein with the cell walls of cold-acclimated C. sericea xylem tissue (Sarnighausen et al. 2002).
Amino acid sequence analysis of internal peptide fragments
indicated that the 24-kDa protein had limited homology to
dehydrin proteins (Ashworth et al. 1998, Sarnighausen et al.
2002), a protein family that is typically found associated with
the cell nucleus and cytoplasm and not associated with cell
walls (Asghar et al. 1994, Godoy et al. 1994, Houde et al.
1995, Egerton-Warburton et al. 1997, Wisniewski et al. 1999).
Analysis of four full-length cDNAs isolated from an expression library made from cold-acclimated C. sericea xylem and
immuno-screened with the anti-24-kDa protein immune serum identified a novel YnSKn class of dehydrin-like proteins
(Karlson 2001, Sarnighausen et al. 2003). The deduced amino
acid sequences from these cDNAs contained no extracellular
signal peptides, as would be expected for cell wall proteins,
and instead were predicted to reside in the nucleus and cyto-
plasm (Karlson 2001). Considering these apparent contradictions and the little that is known about dehydrins in extremely
freeze-tolerant organisms, it was of interest to determine the
subcellular location of the 24-kDa protein with TEM and
immunogold labeling.
Protein blot analysis of total protein extracts demonstrated
multi-specificity of the polyclonal anti-24-kDa protein immune serum (Figure 1). Specificity could not be improved
with an affinity purified dehydrin-specific antibody (Sarnighausen et al. 2002). Blot-affinity purification of the 24-kDa
protein band failed to eliminate the cross-reactivity of the
anti-24-kDa protein immune serum with other protein bands
(data not shown). These results were not surprising because of
the high sequence similarity and the occurrence of up to 34
conserved repeats within each of the four C. sericea dehydrinlike cDNAs (Sarnighausen et al. 2003). However, since the
anti-24-kDa protein immune serum cross-reacts with other
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Figure 3. Subcellular localization of 24-kDa-like proteins within C. sericea and C. florida xylem parenchyma cells. Ultrathin (90 nm) cross sections were treated with dilutions of anti-24-kDa protein immune serum (1:15) and pre-immune serum (1:3.75). Samples were subsequently
treated with colloidal gold-conjugated rabbit anti-chicken secondary antibodies and examined using TEM. Cold-acclimated C. sericea xylem ray
parenchyma nuclei (A, B) and cytosol (E, F) were labeled with anti-24-kDa protein immune serum (A, E) and pre-immune serum (B, F), respectively. Note the limited labeling of non-acclimated C. sericea xylem ray parenchyma nuclei (C) and cytoplasm (G) treated with anti-24-kDa protein immune serum. Also note absence of label in the nucleus (D) and cytoplasm (H) in xylem parenchyma cells from cold-acclimated C. florida.
Low magnification of cold-acclimated C. sericea xylem cells (I) adjacent to vascular cambium. Arrow in panel I indicates region magnified in
panel J. Note the abundant labeling in simple pit and association with plasmodesmata (J). Note the absence of significant labeling in simple pits
from a cold-acclimated C. sericea stem cross section incubated with pre-immune serum (K) and in non-acclimated C. sericea incubated with
anti-24-kDa protein immune serum (L). Arrows indicate the location of plasmodesmata in panels (I–L). The gold particle size was doubled using
Adobe Photoshop 5.5 in micrographs (A–H) for easier viewing. Abbreviations: CY = cytosol; MV = microvacuoles; NU = nuclei; NC = nucleoli;
PL = plastid; SW = secondary cell walls; and V = vacuoles. Bar = 500 nm (exception, panel E = 200 nm and panel I = 4 µm.)
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Figure 4. Association of 24-kDa-like proteins with plasmodesmata in cold-acclimated C. sericea. (A, B) Ultrathin (90 nm) longitudinal serial sections from within the vascular cambium of cold-acclimated C. sericea stem tips were incubated with dilutions of anti-24-kDa protein immune serum (1:15) and subsequently treated with colloidal gold-conjugated rabbit anti-chicken secondary antibody (1:50). A and B represent serial
sections of the region highlighted in C. Arrows indicate plasmodesmal regions within simple pits. Note the evident labeling of plasmodesmal neck
regions as indicated by the right and middle arrows in panel A and the middle arrow in panel B. Note the presence of gold labeling within the cell
wall. Abbreviations: CW = cell walls; MV = microvacuoles; and CY = cytosol. Bar in A, B = 500 nm. Bar in C = 5 µm.
proteins in cold-acclimated tissue and a small dehydrin-like
protein family exists within C. sericea, interpretation of immunogold labeling data must account for the possibility that
multiple 24-kDa-protein-like antigens may be recognized in
situ.
The dense labeling observed in cold-acclimated C. sericea
nuclei and cytosol is consistent with predictions of protein location based on PSORT analysis of the four cDNAs isolated
from cold-acclimated C. sericea xylem tissue (Karlson 2001).
This observation is also consistent with the majority of previous reports on the subcellular location of dehydrins (Asghar et
al. 1994, Godoy et al. 1994, Houde et al. 1995, Egerton-Warburton et al. 1997, Wisniewski et al. 1999). Our present data
add credence to the notion that the accumulation of dehydrins
in the nucleus and cytoplasm is a common feature among diverse plant genera exposed to desiccation stress.
The dense immunogold labeling of the nucleus and cytoplasm appeared inconsistent with our earlier report that the
24-kDa protein was extracted in the CaCl2 extractable cell wall
fraction (Sarnighausen et al. 2002). However, on close examination of specimens by TEM, we observed an association of
24-kDa-protein-like antigens with the cell wall. Dense labeling was observed in association with the plasmodesmata linking adjacent cambial cells (Figures 3I–K, 4 and 5A–C).
Protocols to isolate plasmodesmata-associated proteins generally employ rigorous methods to purify cell wall extracts enriched with plasmodesmata (Kotlizky et al. 1992, Epel et al.
1995). That plasmodesmata are the only membranous structures that remain associated with cell walls following tissue
pulverization, multiple extractions/washes and nitrogen bomb
disruption has been confirmed by TEM. All other plasma
membrane and cytoplasmic inclusions were removed by these
fractionation techniques (Kotlizky et al. 1992, Epel et al.
1995). Several plasmodesmata-associated proteins have been
identified by cell wall extraction techniques similar to our
method of isolating the 24-kDa protein from C. sericea wood.
These studies subsequently used TEM to confirm association
of this protein with the plasmodesmata (Yahalom et al. 1991,
Epel et al. 1996, Blackman and Overall 1998, Blackman et al.
1998). Thus, it is possible that the enrichment of 24-kDa protein in the CaCl2 extractable fraction resulted not from a general association of this protein with the cell wall but from its
association with plasmodesmata.
The pattern of immunogold labeling in specimens incubated
with either anti-24-kDa immune serum or monoclonal anticallose serum was similar, with most labeling associated with
the plasmodesmata neck region (compare Figures 5A–C to
5E–F). Prior studies have reported that callose is predominantly deposited within plasmodesmata neck regions
(Radford et al. 1998, Blackman et al. 1999), an observation
that is consistent with our results. Whether this co-localization
is physiologically relevant is unknown. Immunogold labeling
with the anti-24-kDa immune serum was not restricted to plasmodesmata neck regions and was seen within trans-wall re-
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Figure 5. Association of 24-kDalike proteins and callose with
plasmodesmata in C. sericea. Ultrathin (90 nm) longitudinal stem
sections of the vascular cambium
within cold-acclimated C. sericea
stem tips were incubated with either anti-24-kDa protein immune
serum (A–C), pre-immune serum
(D) or monoclonal immune serum
raised against (1-3)-β-D-glucan
(E, F). Specimens were subsequently labeled with either colloidal gold conjugated rabbit
anti-chicken secondary antibody
or colloidal gold conjugated rabbit
anti-mouse secondary antibody
(E, F). Note the similar pattern of
labeling to the neck region of
plasmodesmata observed with
both the anti-24-kDa protein immune serum (A–C) and
anti-callose monoclonal serum (E,
F). Also note the absence of significant labeling in the specimen
incubated with anti-24-kDa protein pre-immune serum (D). Abbreviations: CW = cell walls;
MV = microvacuoles; and CY =
cytosol. Bar in panel D = 250 nm;
all others = 500 nm.
gions of plasmodesmata in serial sections of cold-acclimated
vascular cambium cells (Figure 5). It is unclear whether the
more abundant labeling of the plasmodesmata neck region reflects an uneven distribution of the antigen within the plasmodesmata or the uneven diameter of plasmodesmata. In the
latter case, the smaller diameter (~40 nm, Epel et al. 1996) of
the trans-wall plasmodesmata regions might remain “embedded” within portions of our 90-nm ultrathin sections. Because
accessibility of 24-kDa epitopes to the anti-24-kDa protein
immune serum would be limited to cut surfaces, the larger diameter of the neck region would more likely be exposed during sectioning. This may explain why some regions of
plasmodesmata appear unlabeled, even though electron dense
trans-wall desmotubules are visible within the sections.
Plasmodesmata are passageways for symplasmic transport
and create a cytoplasmic continuum throughout living plant
tissues. Transport through plasmodesmata is more complex
than simple diffusion (Crawford and Zambryski 1999a) and
associated plant proteins are likely involved (XoconostleCazares et al. 1999). Therefore, one could speculate that the
role of dehydrins in plasmodesmata of cold-acclimated
C. sericea is to stabilize macromolecules associated with
intercellular transport during freeze-induced desiccation
stress. Although this hypothesis seems attractive, the maximum accumulation of 24-kDa protein correlates to periods of
dormancy within C. sericea (Karlson et al. 2003). In poplar
and birch apical meristems, the symplasmic continuum “shuts
down” during dormancy, with concomitant growth cessation
and callose accumulation. As a result, cytoplasmic continuity
is disrupted and individual cells are isolated (Rinne and van
der Schoot 1998). Our experiments using monoclonal immune
serum specific for (1-3)-β-D-glucan confirmed the presence of
callose in association with plasmodesmata within cold-acclimated C. sericea xylem ray parenchyma. Therefore, a potential role of dehydrin-like proteins for maintaining intercellular
communication during cold acclimation in C. sericea xylem
seems unlikely.
It has been proposed that dehydrin-like proteins interact
non-specifically with membrane surfaces during desiccation.
Perhaps dehydrins provide structural protection for plasmodesmata during freeze-induced dehydration. Two different
mechanisms of injury to plasmodesmata during desiccation
can be envisioned. In one scenario, contraction of the protoplasm during desiccation creates tension between the retracting plasma membrane and the cell wall. As cells become
plasmolyzed, membranes remain attached to the cell wall
through trans-wall plasmodesmata continuums (Oparka 1994,
Blackman and Overall 1998). Ristic and Ashworth (1994) observed a retraction of the plasma membrane from the cell walls
and a reduction of protoplast volume during freezing stress in
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C. sericea xylem parenchyma cells. During such events, localized tension would be exerted on plasmodesmata, which may
compromise intercellular continuity. Injury to plasmodesmata
could also occur by direct effects on membranes. Plasmodesmata contain two types of membranes, an outer plasma
membrane and an inner core of modified endoplasmic reticulum (Crawford and Zambryski 1999b). Because of the cylindrical nature of the structure of plasmodesmata, they have a
large membrane surface area relative to their volume and these
membranes would be in close proximity, a feature that may
render them more susceptible to desiccation-induced injury.
Steponkus and coworkers (1993) suggested that membrane
surfaces are brought into close contact during desiccation
stress and lamellar-to-hexagonal II phase transitions are initiated (reviewed by Fujikawa et al. 1999). Recent microscopic
studies have demonstrated the association of dehydrin- like
proteins with the plasma membrane (Danyluk et al. 1998,
Rinne et al. 1999) and have determined that hydrophilic proteins may have a structural function, minimizing membrane
damage during freezing stress (Steponkus et al. 1998). Thus, it
is possible that the association of dehydrins with plasmodesmata could play a role in averting such injury.
The apparent association of a dehydrin-like protein with
plasmodesmata is a unique observation. Unfortunately, our
present study cannot unequivocally link the 24-kDa dehydrinlike protein to the plasmodesmata of cold-acclimated cells,
because our anti-24-kDa immune serum cross-reacts with less
abundant proteins in cold-acclimated tissues. Therefore, although it seems likely that the labeling associated with the
plasmodesmata reflects the presence of the highly abundant
24-kDa dehydrin-like protein, this labeling could result from
the presence of immunologically related proteins found in
cold-acclimated red-osier dogwood. Regardless, the possibility that a protein accumulates in association with plasmodesmata in cold-acclimated cells is intriguing and warrants
further investigation. To our knowledge, this is the first report
that the protein complement of plasmodesmata changes in response to environmental factors.
Acknowledgments
The authors thank Debra Sherman from the Electron Microscopy
Center, Purdue University for generously providing technical advice
and for assistance in digital processing of light and electronic micrographs. We also thank Barbara Damsz, Purdue University, for guidance throughout the silver enhancement localization study and for
assessment of TEM micrographs. This work was supported in part by
a National Science Foundation Dissertation Enhancement Grant No.
INT-9908427 to DTK.
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