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Plant Physiol. (1998) 116: 1367-1377
Characterization of a Gene for Spinach CAP160 and Expression of
Two Spinach Cold-Acclimation Proteins in Tobacco1
Claudia Kaye2,
Lisa Neven3,
Andrea Hofig4,
Qin-Bao Li,
Dale Haskell, and
Charles Guy*
Plant Molecular and Cellular Biology Program, Department of
Environmental Horticulture, Institute of Food and Agricultural
Sciences, University of Florida, Gainesville, Florida 32611-0670
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ABSTRACT |
The
cDNA sequence for CAP160, an acidic protein previously linked with cold
acclimation in spinach (Spinacia oleracea L.), was
characterized and found to encode a novel acidic protein of 780 amino
acids having very limited homology to a pair of Arabidopsis thaliana stress-regulated proteins, rd29A and rd29B. The lack of similarity in the structural organization of the spinach and Arabidopsis genes highlights the absence of a high degree of
conservation of this cold-stress gene across taxonomic boundaries. The
protein has several unique motifs that may relate to its function
during cold stress. Expression of the CAP160 mRNA was increased by
low-temperature exposure and water stress in a manner consistent with a
probable function during stresses that involve dehydration. The coding sequences for CAP160 and CAP85, another spinach cold-stress protein, were introduced into tobacco (Nicotiana tabacum) under
the control of the 35S promoter using Agrobacterium
tumefaciens-based transformation. Tobacco plants expressing the
proteins individually or coexpressing both proteins were evaluated for
relative freezing-stress tolerance. The killing temperature for 50% of
the cells of the transgenic plants was not different from that of the
wild-type plants. As determined by a more sensitive time/temperature
kinetic study, plants expressing the spinach proteins had slightly
lower levels of electrolyte leakage than wild-type plants, indicative
of a small reduction of freezing-stress injury. Clearly, the
heterologous expression of two cold-stress proteins had no profound
influence on stress tolerance, a result that is consistent with the
quantitative nature of cold-stress-tolerance traits.
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INTRODUCTION |
Although the concept that cold acclimation in plants involves
altered gene expression during the induction of freezing tolerance is
more than 20 years old, it has only been in recent years that most of
the information concerning the influence of low temperature on gene
expression has become available (Thomashow, 1994 ; Hughes and Dunn,
1996). Similar findings with cold-water fishes (Lin and Gross, 1981),
bacteria (Goldstein et al., 1990), and yeast (Kondo and Inouye, 1991)
parallel the growing body of information on low-temperature-regulated
genes in plants. In plants a significant portion of the genes
demonstrated to be regulated in response to low temperature are, as
yet, without a known function. Some, based on sequence homologies (Hahn
and Walbot, 1989; Gilmour et al., 1992; Guo et al., 1992; Houde et al.,
1992; Sutton et al., 1992; Neven et al., 1993), belong to a superfamily
of developmentally and environmentally regulated proteins linked with
reduced water activities (Dure, 1993; Close, 1996). Others have
properties in common with this superfamily, such as boiling solubility,
regulation by ABA, or responsiveness to water stress (Cattivelli and
Bartels, 1990; Kurkela and Franck, 1990; Lin and Thomashow, 1992; Orr
et al., 1992; Nordin et al., 1993; Yamaguchi-Shinozaki and Shinozaki, 1993a, 1993b).
Freezing stress is a desiccative physical stress (Pearce, 1988), and
during slow equilibrium freezing in plants, unfrozen water is withdrawn
from the inside of the cell by the lower vapor pressure of
extracellular ice (Mazur, 1963). This causes progressive dehydration,
which leads to a drastic loss of cellular volume and an increase in the
concentration of intracellular solutes. The extreme dehydration that
can result from freezing has major effects on cellular components such
as membrane structure (Steponkus, 1984; Steponkus et al., 1988), which
in turn reduces the ability of the cell to survive a freeze/thaw cycle.
Consequently, during the course of evolution plants have acquired
adaptive mechanisms to help ensure their survival in situations in
which freezing occurs (Sakai and Larcher, 1987; Guy et al., 1992). The
idea that many of these proteins might function not only during water
stress but also during freezing stress to afford some manner of
protection is appealing but largely untested. Previous work with
spinach (Spinacia oleracea L.) in our laboratory has
supported a relationship between drought stress and cold-acclimation
responses (Guy et al., 1992; Neven et al., 1993).
The major question yet unanswered is this: By what mechanisms do plants
become freezing tolerant? Undoubtedly, the process is one of great
complexity (Steponkus, 1984; Sakai and Larcher, 1987). The finding that
many cold-stress proteins are related to proteins linked to water
stress or anhydrous conditions is pivotal because it points to a
specific facet of freezing stress that can be more critically examined.
In spinach two cold-stress proteins, CAP85 and CAP160, have been shown
to share features in common with proteins associated with water stress
(Guy et al., 1992; Neven et al., 1993). Both of these proteins exist in
soluble form in spinach cells, and are actively synthesized and
accumulated in leaf tissue exposed to 5°C (Guy and Haskell, 1987,
1988) or subjected to water stress at 25°C (Guy et al., 1992).
One approach to determining the role of a stress protein of known or
unknown function is to modify its natural expression (Artus et al.,
1996) or to insert its gene into another organism in which it does not
naturally exist (Georges et al., 1990; Hightower et al., 1991; McKown
and Warren, 1991; Kenward et al., 1993). Here we report the cloning and
characterization of cDNAs and genomic sequences for one of these
proteins, CAP160, and the influence of expression of both CAP160 and
CAP85 proteins on freezing-stress injury in tobacco (Nicotiana
tabacum).
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MATERIALS AND METHODS |
Isolation of CAP160 cDNAs and Genomic Clones
Using a monoclonal antibody (2H8) raised against purified CAP160,
cDNAs were isolated by immunoscreening a Uni-Zap (Stratagene) library
prepared with poly(A+) RNA from spinach
(Spinacia oleracea L.) leaf tissue that had been exposed to
5°C for 2 d (Neven et al., 1993). Sixteen clones were selected
by the immunoscreening. Four different clones were partially sequenced
and all appeared identical. Clone IIa contained the largest insert and
was sequenced in its entirety using the Taq DyeDeoxy
Terminator Cycle Sequencing Kit (Applied Biosystems) on an automated
sequencer (Applied Biosystems). Subclones were prepared by cutting at
internal restriction sites, followed by cloning into pBluescript
(Stratagene). All regions of the cDNA were sequenced several times and,
where necessary, synthetic oligonucleotide primers were used to
complete sequencing. Spinach DNA was partially restricted with
EcoRI and cloned into EMBL4. The library was screened with
CAP160 cDNA clone IIa. Overlapping clones representing the 5 and 3
regions were used to construct the complete gene sequence. Database
searches for homology to CAP160 DNA and protein sequences were
performed with BLAST (Altschul et al., 1990).
Nucleic Acid Isolation and Analyses
RNA was isolated using a phenol-LiCl procedure (Ausubel et al.,
1991), and portions were separated onto a 1.2% agarose gel containing
formaldehyde. Gels were stained with ethidium bromide to visualize rRNA
bands for verification of equal loading, photographed, and then blotted
onto a nylon membrane (Hybond-N, Amersham) and fixed with UV light
(Stratalinker 1800, Stratagene). Blots were hybridized with the
random-primed 32P-labeled cDNA clone IIa, then
washed in 2× SSC, 0.2% SDS at room temperature, 0.5× SSC, 0.2% SDS
at 68°C, and 0.1× SSC, 0.2% SDS at 68°C. Autoradiography was
generally performed for 24 h at  80°C. DNA was isolated by the
method of Gordon-Kamm et al. (1990), cut with restriction enzymes as
indicated, and analyzed by DNA-blot hybridization.
CAP Expression in Escherichia coli
Fusion protein expression in E. coli XL1-Blue cells
harboring pBluescript containing a cDNA insert was induced with IPTG. XL1-Blue cells containing pBluescript without an insert were included in all of the experiments as a control. Induced cells were pelleted by
centrifugation at 1,600g for 15 min in a JA20 rotor
(Beckman), resuspended in lysis buffer (50 mm Tris, pH 8.0, 1 mm EDTA, 1 mm PMSF, 10% Suc, 1 mg/mL
lysozyme), and incubated for 10 min on ice. Triton X-100 was added to
0.1% final concentration and incubated for 10 min on ice. The solution
was centrifuged at 48,000g in a JA20 rotor for 1 h, and
the supernatant was removed and mixed with 2× SDS buffer. Protein
sequestered in inclusion bodies was extracted from the pellet with SDS
buffer. Approximately 5 µg of total protein was fractionated by
SDS-PAGE in 10% acrylamide gels, blotted to PVDF membranes, and probed
with anti-CAP160 or anti-CAP85 antibodies (Guy et al., 1992).
Plant Transformation and Growth Conditions
CAP160 and CAP85 (Neven et al., 1993) cDNA sequences were put
under the control of the cauliflower mosaic virus 35S promoter by
subcloning a 2.7-kb BamHI-XhoI fragment of CAP160
and a 1.8-kb BamHI-XhoI fragment of CAP85 into
the broad-host-range vector pVSTIII (Malik and Wahab, 1993), creating
plasmids pVST-160 and pVST-85, respectively (Fig.
1). These two plasmids were transferred separately into Agrobacterium tumefaciens strain LBA4404 by
direct transformation (Hofgen and Willmitzer, 1988).
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| Figure 1.
Constructs used in the tobacco transformation
experiments (Malik and Wahab, 1993). The right (RB) and left (LB)
borders are indicated by the open boxes at the ends of the
constructs.
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Leaf discs from surface-sterilized young leaves of greenhouse-grown
tobacco (Nicotiana tabacum cv Xanthi) plants were infected with A. tumefaciens harboring pVST-160, pVST85, or pVSTIII.
Successful transformants were selected on regeneration medium
containing 4.3 g/L MS salts (Murashige and Skoog, 1962), 1 mL/L 1000×
B5 vitamin mixture, 0.3 mg/L IAA, 10 mg/L dimethylallylaminopurine, 30 g/L Suc, 100 mg/L kanamycin, and 250 mg/L cefotaxime or
carbenicillin. Young shoots were transferred to this medium with no
hormones and no antibiotic for rooting. Rooted plants were transferred to soil and maintained either in a growth chamber at 25°C or in the
greenhouse. Seeds were collected from all primary transformants and
sown on solid medium containing MS salts and 200 mg/L kanamycin. Plantlets that remained green were transferred to soil and grown under greenhouse conditions.
Protein Analysis
Leaf material was frozen in liquid nitrogen and then powdered
using a mortar and pestle. The frozen powder was transferred directly
to SDS buffer (Neven et al., 1993) and boiled for 5 min. Insoluble
proteins were removed by spinning the samples for 10 min in a
microcentrifuge. The supernatant was transferred to fresh tubes and
stored at 20°C. Total protein content was determined by the
Bradford method (1976). The presence of CAP160 and CAP85 were
determined by protein-blot analysis of crude protein extracts using
monoclonal antibodies 2H8 and 5A10, respectively. Equal loading of
protein-blot lanes was verified by Coomassie brilliant blue staining of
a duplicate gel.
Partial sequencing of CAP160 purified from spinach tissue was
accomplished after cleavage with cyanogen bromide as previously described (Neven et al., 1993).
Tissue prints were made from flower, stem, and pedicel tissue from
cold-acclimated and nonacclimated transformed tobacco and control,
mock-transformed tobacco (Cassab and Varner, 1987). The resulting
membranes were probed with CAP85 monoclonal antibody 5A10 (Neven et
al., 1993), CAP160 monoclonal antibody 2H8 (Haskell et al., 1993), or
dehydrin antibody (Close et al., 1993).
Freeze Tests
Leaves were harvested from either greenhouse- or
growth-chamber-grown plants at the temperatures indicated. Leaf discs
(three per tube) were frozen at a cooling rate of 0.5 or 1°C/h, as
indicated. All samples were equilibrated for 1 h at 0°C, and ice
nucleation was induced at 1°C by placing a small ice chip in
contact with the tissue. Frozen samples were thawed at 4°C in a
refrigerator overnight. The leaf discs were placed in 5 mL of
double-distilled water and incubated for 2 h. Electrolyte loss was
measured with a conductivity meter. After the initial conductivity
measurement, the relative electrolyte loss was determined by refreezing
the tissue at 80°C, thawing, and measuring the conductivity a
second time.
Drought Tolerance
Young plants at the three-leaf stage were transferred to
vermiculite and watered every other day with 5% PEG in one-tenth MS
salts. Control plants received one-tenth MS salts alone. After 2 weeks,
the concentration of PEG was raised to 10%. Six weeks later the plants
were evaluated visually and fresh weights were determined.
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RESULTS |
The CAP160 protein is known to be actively synthesized and/or
accumulated in leaf tissue exposed to 5°C (Guy and Haskell, 1987,
1988) or during water stress at 25°C (Guy et al., 1992). Therefore,
any cDNAs for this protein selected by the antibody screening should
hybridize with mRNA that was up-regulated during low-temperature
exposure or water stress. Several of the clones hybridized strongly to
a 2.7-kb mRNA that was up-regulated during exposure to 5°C and was
down-regulated upon return to 25°C (Fig. 2A). Similarly, the cDNAs hybridized to a
2.7-kb mRNA that was abundant in water-stressed leaf tissue, but was
low or absent in nonstressed tissue (Fig. 2A). The mRNA for CAP160
reached a maximum abundance in leaf tissue within 24 h of 5°C
exposure and remained high for up to 7 d of low-temperature
exposure. The transcript abundance was slightly increased at 2 h
of 5°C and by 6 h had accumulated to a high level (Fig. 2B).
Expression of CAP160 was moderately stimulated by a heat-shock
treatment of 40°C for 2 h, but not by higher or lower heat-shock
temperatures (Fig. 2C).
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| Figure 2.
RNA-blot analyses of the influence of cold
acclimation, deacclimation, water stress, and heat shock on the CAP160
steady-state mRNA abundance. A, Acclimation, deacclimation, and
water-stress responses. B, Short-term response to low temperature. C,
Response to heat shock. The cDNA hybridizes to a major 2.7-kb mRNA.
Each lane was loaded with 5 µg of total RNA, and equal loading was verified by ethidium-bromide staining before blotting. Similar results
were observed in several hybridization experiments. Details regarding
the physiologic parameters for the water-stressed samples have been
published elsewhere (Guy et al., 1992; Neven et al., 1993).
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Sequence analysis of several cDNAs indicated an open reading frame
encoding an acidic protein of 780 amino acids (Fig.
3). The position of the first ATG in the
sequence has a similarity to a translation initiation site consensus
(Joshi, 1987), and the open reading frame is broken by an in-frame stop
codon 20 bp upstream from the first ATG, suggesting that the cDNA
contains the entire coding sequence. The deduced protein has a
molecular mass of 83.9 kD and a pI of 4.6. Whereas the calculated pI is in good agreement with the pI of 4.5 estimated from two-dimensional gels for spinach CAP160, the molecular mass of the deduced protein is
little more than one-half of that estimated by SDS-PAGE (Guy and
Haskell, 1987).
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| Figure 3.
Nucleotide and deduced amino acid sequence of
CAP160 clone IIa. Amino acids in boldface and underlined correspond to
cyanogen bromide-generated peptide sequences from purified CAP160. The dark-shaded box denotes a motif rich in negatively charged residues, and the lighter-shaded box denotes a repeat sequence. An imperfectly tandem repeated sequence is double underlined, and a repeating four-residue motif of P(I/S)(T/K)(G/W) is underlined.
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Protein-blot analyses of E. coli possessing pBluescript
without an insert showed no immunoreactivity with anti-CAP160
antibodies, whereas cells containing the CAP160 cDNA expressed
immunoreactive proteins with or without induction with IPTG (Fig.
4A). Expression was higher with IPTG, and
the level of CAP160 accumulation in E. coli appeared to be
similar to that found in cold-acclimated spinach leaves (Guy et al.,
1992). Two major bands (120 and 140 kD) and a number of minor bands
were present, each of which migrated faster than leaf CAP160 (Fig. 4A,
upper band in lanes 7 and 16). The lower doublet bands in the leaf
extracts of 115 and 110 kD are thought to be proteolytic breakdown
products of CAP160 because they generally increase while the 160-kD
band decreases with time after extraction. Induced bacterial cells
contained a minor band at 160 kD (Fig. 4A, lanes 2, 4, 12, and 14) that
comigrated with leaf CAP160 (Fig. 4A, lanes 7 and 16). Because an
in-frame stop codon is present before the first ATG, it is probable
that the immunoreactive recombinant protein of 160 kD in induced cells is not a  -galactosidase fusion protein, but an authentic-size CAP160
protein resulting from the reinitiation of translation at the first ATG
site. The proteins of < 160 kD may be proteolytic products or may
result from alternative reinitiation of translation at ATG sites
farther downstream. Nevertheless, a small percentage of the protein
produced appeared to be full length. This was not unexpected because
eukaryotic proteins are often found to be unstable when expressed in
E. coli (Ausubel et al., 1991).
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| Figure 4.
CAP160 and CAP85 protein expression in E. coli XL1-Blue cells. A, Lane 1, pBluescript without an insert;
lane 2, CAP160 clone IIa; lane 3, clone IIb; lane 4, clone VIII; lane
5, clone VII; lane 6, clone IX; lane 7, spinach leaf CAP160; lane 8, soluble fraction of pBluescript without an insert minus IPTG; lane
9, insoluble fraction of pBluescript without an insert minus IPTG; lane
10, soluble fraction of pBluescript without an insert plus IPTG; lane
11, insoluble fraction of pBluescript without an insert plus IPTG;
lane 12, soluble fraction of CAP160 IIa minus IPTG; lane 13, insoluble
fraction of CAP160 IIa minus IPTG; lane 14, soluble fraction of CAP160
IIa plus IPTG; lane 15, insoluble fraction of CAP160 IIa plus IPTG; and
lane 16, spinach leaf CAP160. B, Lane 1, Spinach leaf CAP85; lanes 2 and 3, CAP85 out-of-frame clone S42a plus IPTG; and lanes 4 and 5, CAP85 in-frame clone S42aE plus IPTG.
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Whether the truncated products retained biological activity remains
unknown. CAP160 protein products were present in both soluble and
insoluble forms, and it appeared that a significant portion of the
expressed protein was not sequestered in inclusion bodies. The 160-kD
form was observed only in the soluble fraction (Fig. 4A, lanes 12 and
14). The reason for the low mobility of CAP160 in SDS-PAGE is unknown,
but might result from the high content of Ser, Ala, and negatively
charged residues (Kondo and Inouye, 1991). The CAP85 cDNA, in which the
deduced protein sequence is smaller than the apparent size estimated by
SDS-PAGE (Neven et al., 1993), also produced a protein that comigrated
with CAP85 from leaf tissue (Fig. 4B).
Additional evidence linking the cDNA of clone IIa with CAP160 is
provided by protein sequence information. Two amino acid sequences from
peptide fragments prepared by cyanogen bromide cleavage of purified
CAP160 closely matched the deduced sequences at residues 83 to 94 and
174 to 192 (Fig. 3).
CAP160 has several unusual motifs that may have a functional
significance. It has an acidic region toward the N terminus in which 18 of 26 residues are either aspartic acid or Glu, including a central
core where five aspartic acids are followed by four Glu residues (Fig.
3). Two imperfect repeats of 29 residues each are found in the
N-terminal half of the protein. They are separated from each other by
133 residues. These two repeats are rich in Thr (24%), Gly (14%), and
Asp (14%). Another two large, imperfect repeats of 58 and 56 amino
acids, separated by 1 residue, are found beginning at residue 460. Thr
(approximately 13%) is the single most abundant residue in these
repeats, followed by Val (10.5%). Immediately following the second
large repeat is a series of seven 4-residue repeats with the consensus
of P(S/I)TG. Preceding the first large repeat is one copy of the
4-residue repeat PSTG. Given the presence of the Pro and Gly in this
repeating sequence, this region might be a loop or a solvent-exposed
region of the protein. The Ser and Thr residues both bearing hydroxyls
might be potential phosphorylation sites on the protein. All of these characteristic features of CAP160 were individually used to do BLAST
searches of the databases for matches that could provide insight into
its function. In addition, the CAP160 sequence was searched for known
protein motifs found in the Prosite library (release 12.2, 3/95) of the
Genetics Computer Group (Madison, WI) sequence analysis software
package, but no convincing matches were found that would give a clue to
its function or identity.
A search of the GenBank database revealed no compelling sequence
matches for the entire protein. Although it was suspected that CAP160
was likely the homolog of a pair of Arabidopsis thaliana proteins with several names (cor78, lti140,
rd29A, rd29B, and lti65), the total
protein sequence similarity was less than 20%. Nevertheless, four
discrete regions within the CAP160 sequence did have high homology with
the Arabidopsis proteins (Fig. 5), leading us to believe that CAP160 is a probable homolog to these proteins. The two Arabidopsis proteins also have an acidic region (EEDDDDDELEPE or DVEDDDDEYDEQDPE) with similarities to the acidic region of CAP160, which could have some importance to a common function.
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| Figure 5.
Alignment of CAP160 protein sequence with the
Arabidopsis rd29A and rd29B proteins. Identical residues are indicated
by dots.
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Replicated blots of genomic DNA using cDNA probes showed some
blot-to-blot variation, but overall produced the predicted banding pattern based on the DNA sequence from genomic clones for CAP160 (Fig.
6). The banding pattern was relatively
simple even when stringency was reduced, indicating that CAP160 is not
a member of a large, high-homology gene family.
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| Figure 6.
Genomic DNA blot for CAP160. Results of three
different experiments are shown. Clone IIa was used to make the
hybridization probe.
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The isolation of genomic clones for CAP160 permitted the
characterization of the organization of the gene. The CAP160 protein is
encoded on seven exons extending over 8 kb. The exons range from 71 to
1254 bp in length, whereas the introns tend to be larger than the
exons, ranging from a minimum of 440 bp to a maximum of 1437 bp (Fig.
7). Characterization of genomic clones
with differences in restriction fragment length revealed the existence
of a 352-bp deletion in intron 1 (Fig. 7). Aside from this deletion,
the nucleotide sequences in both intron and exon regions of all
isolated clones were virtually identical, suggesting the possibility
that if it does not represent a second gene sequence, this clone
represents an allelic sequence.
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| Figure 7.
Structure of the CAP160 gene. Flanking sequences
are indicated by dark shading, noncoding and intron sequences are
lightly shaded, and exons are shown as unshaded boxes. The hatched area in intron 1 shows the position of a large deletion present in some
sequences.
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Subcellular-localization studies were conducted with leaf and hypocotyl
tissue to determine where in the cell CAP160 is found. These studies
were performed at the same time as studies of another spinach CAP, the
results of which have been previously reported (Neven et al., 1993).
This previous report includes the marker protein analysis for relative
fraction contamination. The major organellar fractions were prepared,
and these extracts were probed with 2H8, a monoclonal antibody specific
for CAP160, using protein-blot methods (Fig.
8). A major portion of CAP160 was present
in a freely soluble fraction, which would be consistent with a protein
present in the cytosol and not tightly associated with any
macromolecular structure that would be pelleted at relatively low
g forces. However, an equally large proportion was found in
the mitochondrial fraction. Whether the protein was inside or outside
of the mitochondria was not determined. Protein blots using antibodies
for marker proteins for all of the fractions shown in Figure 8
indicated that the presence of the strong CAP160 reactivity in
the mitochondrial fraction was well in excess of the documented
cross-contamination (Neven et al., 1993), and thus was likely to
reflect an actual cofractionation.
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| Figure 8.
Subcellular localization of CAP160. The presence
of CAP160 was detected by protein blot using a monoclonal antibody,
2H8, which is specific for CAP160. For marker protein analysis and relative contamination for each subcellular fraction, see Neven et al.
(1993).
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Expression of CAP160 and CAP85 in Transgenic Tobacco
Constructs using the cDNAs for CAP160 and CAP85 (Neven et al.,
1993) were placed under the control of the 35S promoter and used in
A. tumefaciens-based transformation experiments. Putative transformants were assayed for expression of CAP160 and CAP85 by
protein-blot analyses using highly specific monoclonal antibodies. In
these experiments protein was isolated from plants grown under either
growth-chamber conditions (25°C) or greenhouse conditions. Some of
the leaves (as indicated) were taken from plants that were grown under
sterile conditions in sealed containers with a very high humidity
level. Several transformants were found to express the mRNA for each
introduced gene (Fig. 9). No
cross-reacting protein was found in either wild-type tobacco (not
shown) or in tobacco transformed with the vector alone. CAP85 was
resolved as a doublet consisting of an intense 85-kD band and a smaller and less-intense 80- to 83-kD band in spinach (Neven et al., 1993), and
its expression also resulted in a doublet in transgenic tobacco. R0 CAP85 plants (Nt25 and
Nt35) were crossed with R0 CAP160
plants (Nt40), and the progeny were evaluated for expression
of the two proteins. A large number of the plants expressed both
proteins, a few expressed only CAP160, but no plants were found that
expressed only CAP85 or neither of the two proteins (Fig.
10).
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| Figure 9.
Analysis of spinach CAP160 RNA and protein
expression in transgenic lines of tobacco. Spinach RNA and protein are
shown for reference. Nt10 was transformed with the
vector only. Hybridizations were done with radiolabeled cDNA clone IIa,
and protein detection was with monoclonal antibody 2H8. RNA blots were
loaded with 5 µg of total RNA per lane. Protein blots were loaded
with 20 µg of total buffer-soluble protein per lane.
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| Figure 10.
Analysis of spinach CAP85 expression in tobacco
(A) and coexpression of CAP85 and CAP160 in progeny from a cross of
lines expressing individually either CAP85 or CAP160 (B).
Cross-reacting proteins were detected with a mixture of monoclonal
antibody 2H8 for CAP160 and monoclonal antibody 5A10 for CAP85.
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Tissue prints were done on nitrocellulose with cold-acclimated or
nonacclimated stem, pedicel, and flower tissue taken from transformed
tobacco plants to assess expression patterns at the tissue level. The
resulting membranes were exposed to CAP85, CAP160, or both antibodies.
Both proteins were expressed in all tissues examined, and no difference
was seen between cold-acclimated and nonacclimated tissue (not shown).
When the same tissues were analyzed by tissue printing and probed with
a dehydrin antibody (Close et al., 1993), greater staining was seen in
the cold-acclimated tissue than in the nonacclimated tissue. Tissue
prints from control, mock-transformed tobacco did not react with
either of the two monoclonal antibodies for the spinach proteins, but
did react with the anti-dehydrin antibody.
Seeds were collected from all of the R0 plants,
regardless of whether they expressed the introduced CAP genes. Seeds
were sown on kanamycin and the number of green plants (kanamycin
resistant) versus white plants was evaluated. The number of white
plants versus green plants was somewhat lower than the expected
Mendelian ratio of 1:3 (1:6.6 for CAP160, 1:5 for CAP85), indicating
the possibility that more than one, but probably only a few, copies of
the transferred genes integrated into the genome. Selected green plants
were evaluated by protein-blot analysis, and all expressed either the
expected CAP160 or CAP85. Green, 1-month-old plantlets were transferred
to soil, and at the three-leaf stage they were transferred into either
cold (3°C) or warm (20°C) conditions. After 14 d, protein was
isolated and analyzed by protein blot. In all cases the quantities of
CAP85 and CAP160 protein were lower in the plants grown in the cold
(data not shown).
Freezing Tests
The first test for increased hardiness was an electrolyte-leakage
test performed on leaf discs of transformed and mock-transformed plants. Initial tests were conducted on R0 plants
grown at 25°C. Only one transformant, CAP160 Nt40,
exhibited any evidence of a decrease in electrolyte leakage, but even
that was not statistically significant (Table
I). A sample from each plant was taken
each time the electrolyte-leakage test was performed to assay for
CAP160 or CAP85 expression.
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Table I.
Freezing tolerance of an Ro
transgenic tobacco line expressing the spinach CAP160 protein
The data are presented as the arcsin-transformed percentage of the
relative electrolyte leakage. Two different mock-transformed lines were
used and the data were combined. The experiment was repeated five
times.
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The second set of experiments was performed on the
R1 generation. In this case, two sets of
experiments were run. The first was a determination of
LT50 as described above (Fig.
11). A slight improvement was seen in
the hardiness of plants expressing the spinach protein only at 1°C,
but overall the LT50 values were not different. The
second test, a "kinetics-of-freezing" analysis, was designed to
reveal more subtle differences between the plants. In this test,
samples were placed at the approximated LT50 of 2°C and removed at various times. At all times, the plants
expressing the spinach proteins exhibited less electrolyte leakage than
the untransformed control plants. Because of the large variability in
the mock-transformed plants, no significant difference was found
between it and either the untransformed plants or those expressing the
spinach proteins (Fig. 12).
View larger version (42K):
[in this window]
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| Figure 11.
Influence of CAP85 or CAP160 expression or
coexpression of both proteins on the severity of injury after freezing.
Error bars represent the sd of two to four experiments.
|
|
View larger version (38K):
[in this window]
[in a new window]
| Figure 12.
Differential sensitivity of tobacco expressing
spinach cold-stress proteins in response to a time-course freeze/thaw
regime. Error bars represent the sd of five experiments.
|
|
Whole plants expressing CAP160 or CAP85, mock-transformed (pVSTIII,
vector only) plants, or control cv Xanthi plants (nontransformed) were
transferred at approximately the three-leaf stage into a cold room.
These plants were incubated for 1 month at 5°C. Visual inspection
revealed no obvious difference in either the amount of leaf damage or
plant size. The temperature was reduced to approximately 0°C, and
although all of the plants suffered minor leaf damage, all survived
(data not shown). Protein analyses indicated no differences in the
levels of either spinach protein over that of similar plants kept at
20°C.
| |
DISCUSSION |
The function of CAP160 in spinach and its role in plant cold
stress remains obscure. Homology searches indicate that CAP160, in its
entirety, is a novel protein, the biological function of which cannot
be identified by shared homology with proteins of known function.
CAP160 does have limited similarity to the Arabidopsis lti78, or COR78 protein, a low-temperature-induced (Horvath
et al., 1993; Nordin et al., 1993) and desiccation-responsive protein, also known as rd29A and rd29B (Yamaguchi-Shinozaki, 1993a, 1993b). The
shared homology is enough for us to consider it a spinach homolog to
these proteins. The primary structure of CAP160 shows no similarity to
antifreeze or ice-nucleation proteins, the function of which is to
alter the freezing of water (Green and Warren, 1985; Davies and Hew,
1990). However, several noteworthy features of the primary structure of
CAP160 (Fig. 3) could provide helpful clues to its functional role in
low-temperature-stress response mechanisms. These include a highly
negatively charged motif at residues 61 to 86, which includes a run of
nine consecutive aspartates and glutamates and several repeat motifs, a
large tandem imperfect repeat, and a four-residue imperfect-repeating
motif present in eight copies. All of these features were used to
search the databases for matching sequences that might provide a clue
to CAP160's function. Only the acidic residue tract 61 to 86 yielded a
noteworthy similarity.
A variety of proteins possess extensive tracts of negatively charged
residues, including nucleolins (Lapeyre et al., 1987), some
transcription factors (Hisatake et al., 1991), Na/Ca,K exchanger (Reilander et al., 1992), spermine-binding protein (Chang et al., 1987), calsequestrin (Yazaki et al., 1990), and ubiquinol-Cyt c reductase protein (Van Loon et al., 1984). Of these, the
relationship to that of nucleolin seems most notable. Nucleolin is a
major nucleolar phosphoprotein in eukaryotic cells (Fang and Yeh, 1993) that has sequence-specific RNA-binding activity that regulates, or
participates in, rRNA processing (Hanakahi et al., 1997). CAP160 lacks
many of the structural features of plant nucleolin, such as
nuclear-localization sequences (Bogre et al., 1996), and we do not
consider it to be a nucleolin. However, it has some unusual similarities, including the fact that it is a phosphoprotein (Guy and
Haskell, 1989) and is extremely sensitive to proteolytic cleavage even
in purified form (D. Haskell and C. Guy, unpublished data). Nucleolin
has a self-cleaving activity that resides in the C-terminal domain and
that apparently recognizes repeated motifs within the molecule (Fang
and Yeh, 1993). CAP160 has repeats, although not as many as are found
in nucleolins.
Because one of the major effects of low-temperature exposure is a
significant reduction in translation rate, perhaps CAP160's function
is to participate as an accessory protein in the assembly of ribosomes
or to help accelerate translation in some way. Evidence exists to
suggest that the capacity of the translation apparatus is enhanced
during cold acclimation (Guy et al., 1985) during a time when CAP160 is
accumulating. CAP160's subcellular localization appears to be
predominately cytosolic, which would be consistent with such a
function. However, given its apparent cofractionation with
mitochondria, alternative functions are also a possibility.
Analysis of CAP160 gene structure revealed further differences from
that of the Arabidopsis cold- and drought-responsive rd29 genes. CAP160 has more and much larger introns than either of the two
Arabidopsis genes, six versus three each. In addition, the two
Arabidopsis genes are within 2 kb of each other, but we could find no
evidence from genomic clones that such a close tandem arrangement
exists for CAP160 in spinach.
The coding sequences from cDNA clones for two genes that are
up-regulated during cold acclimation in spinach were placed under the
control of the nearly constitutive cauliflower mosaic virus promoter
and transformed separately into tobacco, a freezing-sensitive plant.
These two genes were expressed in the transformed tobacco as determined
by protein-blot analysis, whereas no cross-reacting proteins to the
monoclonal antibodies were found in nontransformed tobacco. However,
both of the proteins were expressed in tobacco at an apparently lower
level than under low-temperature-induction conditions in spinach. Tests
designed to determine whether their expression conferred increased
tolerance to low-temperature stress indicated little overall benefit to
tobacco. Freezing tolerance is considered to be a quantitative trait
(Hayes et al., 1993); therefore, it is not surprising that the
heterologous expression of one or two genes induced during cold
acclimation would significantly alter the LT50 of
freezing-sensitive tobacco. This result is similar to that when a
cold-regulated, osmotin-like protein was overexpressed in potato and
had no effect on freezing tolerance (Zhu et al., 1996), but it is in
contrast to the constitutive overexpression of the COR15a protein in
Arabidopsis (Artus et al., 1996).
In the work with Arabidopsis, overall cell survival was only subtly
enhanced after a freeze/thaw cycle for overexpressing cells, but a more
dramatic, positive benefit of overexpression of COR15a was the
preservation of chloroplast electron-transport function by this
chloroplast stromal protein. Other examples of this approach include
several attempts to enhance yeast and plant freezing tolerance through
the introduction of cold-water fish antifreeze proteins (Georges et
al., 1990; Hightower et al., 1991; McKown and Warren, 1991; Kenward et
al., 1993). Expression of fish antifreeze proteins has not
significantly altered freezing of plant tissues (Hightower et al.,
1991). The results of the expression of spinach cold-stress proteins in
tobacco do not rule out the possibility that major enhancements in the
tolerance of freezing-sensitive plants might be achieved through the
expression of one or a few stress proteins, but instead further support
the view of the quantitative nature of plant freezing-stress tolerance (Hayes et al., 1993).
CAP85 and CAP160 are continuously and highly expressed during cold
acclimation and under continuing cold treatment, making them unlikely
candidates for regulatory genes, which in all likelihood would be rare
messages expressed very early during cold acclimation. A large number
of genes are induced in the process of cold acclimation (Kaye and Guy,
1995). Although many of these genes fall into one of a few multigene
families, particularly the LEA/dehydrin family, their function remains
unknown. Most are probably involved in the metabolic adjustment of the
plant during cold acclimation. The two clones used in this study, CAP85
and CAP160, are both members of the LEA/dehydrin superfamily. LEA and
dehydrin proteins are known to accumulate during the later stages of
seed development or during water stress and are thought to play a role
during seed dry-down (Close et al., 1993). Recent evidence suggests
that at least one of the spinach proteins may have cryoprotective
properties (Kazuoka and Oeda, 1992). Purified CAP85 was shown to
protect lactate dehydrogenase in vitro from a freeze/thaw inactivation (Kazuoka and Oeda, 1992). This opens the door to the possibility that
many low-temperature-regulated genes may have cryoprotective properties
(Artus et al., 1996). More likely, CAP160's role, given its
subcellular localization and its induction during drought stress, will
be stabilization of supramolecular structures such as membranes,
ribosomes, or cytoskeletal elements; acting as that of a proteinaceous
compatible solute that reduces the toxic effects of cellular solutes at
a high concentration during dehydration; or that of an enzyme that
synthesizes a product with compatible solute properties. Regardless of
its mode of action, CAP160 represents a new class of intracellular,
low-temperature-stress proteins with a function correlated with
enhanced freezing tolerance only in spinach.
| |
FOOTNOTES |
1
This research was supported in part by the U.S.
Department of Agriculture/National Research Initiative Competitive
Grants Program (grant no. 93-37100-8906). This is Florida Agricultural Experiment Station journal series no. R-06081.
2
Present address: Centre de Coopération
Internationale en Recherche Agronomique pour le Développement, 2477 ave du Val de Montferrand, 34032 Montpellier, cedex 1, France.
3
Present address: U.S. Department of
Agriculture-Agricultural Research Service, Yakima Agricultural Research
Laboratory, 5230 Konnowac Pass Road, Wapato, WA 98951.
4
Present address: Department of Oral Biology,
University of Florida, Gainesville, FL 32610.
*
Corresponding author; e-mail clg{at}gnv.ifas.ufl.edu; fax
1-352-392-3870.
Received August 21, 1997;
accepted December 9, 1997.
The accession numbers for the sequences described in this article are
AF035535 (cDNA) and AF035536 (genomic clone).
| |
ABBREVIATIONS |
Abbreviations:
CAP, cold-acclimation protein.
IPTG, isopropyl-1-thio--galactoside.
LEA, late embryogenesis abundant.
LT50, killing temperature for 50% of the cells.
MS, Murashige and Skoog.
| |
ACKNOWLEDGMENTS |
We are grateful to J. Anderson, C. Zhang, D. Sung, and an
anonymous reviewer for their constructive comments during the
preparation and review of the manuscript, and T. Close for the gift of
the anti-dehydrin antibody. Facilities and assistance for protein and
DNA sequencing, antibody production, and computer services were
provided by the core laboratories of the Interdisciplinary Center for
Biotechnology Research. We also thank the Florida High Tech Council for
support.
| |
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Abstract
Introduction