Plant Cell Physiol. 37(8): 1204-1210(1996)
JSPP © 1996
Alfalfa Nuclei Contain Cold-Responsive Phosphoproteins and Accumulate
Heat-Stable Proteins during Cold Treatment of Seedlings
Wojciech Kawczynski and Rajinder S. Dhindsa
Department of Biology, McGill University, 1205 Avenue Docteur Penfield Montreal, Quebec H3A 1B1, Canada
We have examined whether low temperature, as a pervasive thermodynamic stimulus, is sensed independently in
different parts of the cell by studying low temperature responses of phosphoproteins in isolated nuclei. The isolated
alfalfa (Medicago sativa) nuclei respond to cold by rapid
and reversible changes in phosphorylation level of their proteins. The population of such cold-regulated phosphoproteins and the cold-stimulation of their phosphorylation are
greater in a freezing-tolerant cultivar Apica than in a sensitive cultivar Trek. With a 4-day cold treatment of the seedlings, additional proteins showing cold-stimulated phosphorylation appear in the nucleus of Apica while there is
little change in the case of Trek. Furthermore, nuclei from
cold-treated seedlings of Apica, but not of Trek, show a
large accumulation of heat-stable proteins. These results
support the view that the low temperature sensing and acclimation occur in all vital parts of the cell and that accumulation of heat-stable nuclear proteins may be related to
freezing tolerance.
Key words: Cold acclimation — Freezing tolerance —
Heat-stable proteins — Medicago sativa — Temperature
sensing.
Exposure to low temperature, in the light or dark, induces the expression of cold acclimation-specific genes in
alfalfa cells or seedlings (Mohapatra et al. 1989, Monroy
et al. 1993a, Wolfraim et al. 1993). We have previously
shown that low temperature signal transduction leading to
the expression of these genes is mediated by rapid and reversible changes in protein phosphorylation (Monroy et al.
1993b). Furthermore, cold-triggered influx of cell wall calcium into the cytosol through calcium channels on the plasma membrane has been shown to be required for coldspecific protein phosphorylation, gene expression, and
development of freezing tolerance (Monroy and Dhindsa
1995, Monroy et al. 1993b). Thus we have proposed (Monroy and Dhindsa 1995) that low temperature signal transduction comprises the perception of the signal at the plasma membrane, followed by a cascade of cytosolic events
leading to a communication to the nucleus to activate these
genes.
The present study is based on the reasoning that temperature, being a pervasive thermodynamic factor, should
also be sensed independently at many places in the cell.
Thus in addition to receiving cytosol-mediated signals, organelles may also perceive the low temperature signal directly as their own temperature declines. Furthermore, cold acclimation during which mechanisms to protect against
freezing damage are developed, is likely to occur in all vital
parts of the cell including the nucleus. While the nature of
these protective mechanisms is unclear, many plants synthesize boiling-stable proteins related to the LEA/RAB/dehydrin (Late Embryogenesis Abundant/ Responsive to
Abscisic Acid/ dehydration induced, respectively) family
of proteins in response to dehydrative stresses such as due
to cold, drought and salt. It has been suggested (Baker et
al. 1988, Close et al. 1990) that such proteins protect cellular constituents against dehydration damage—a significant
component of freezing injury (Steponkus 1984). This suggestion has been strengthned by a recent demonstration
that introduction of a Lea gene into rice conferred tolerance to drought and salt stresses (Xu et al. 1996).
In the present study, we have examined direct (in isolated nuclei) and indirect (in vivo) responses of nuclei to low
temperature as indicated by the population pattern of nuclear phosphoproteins. We have also determined the accumulation of heat-stable proteins in the nuclei of cold-treated
seedlings. We have made these determinations on two
cultivars of alfalfa (Medicago sativa) both of which are
tetraploid but have substantially different capacity for cold
acclimation (Mohapatra et al. 1989, Monroy et al. 1993a).
The seedlings of cultivar Trek are frost-sensitive while
those of cultivar Apica are frost-tolerant. Even after a prolonged cold acclimation, the degree of freezing-tolerance
of Trek is only half as much as that of Apica (Monroy et al.
1993a).
The results of our study show that (i) low temperature
modulates the phosphorylation level of proteins in the isolated nuclei; (ii) during prolonged cold treatment of the
seedlings, additional phosphoproteins appear in the nuclei
some of which are specific to the tolerant cv. Apica; and (iii)
heat-stable proteins accumulate in the nucleus during a
prolonged cold-treatment of the seedlings of the freezingtolerant but not the freezing-sensitive cultivar.
Materials and Methods—Seeds of alfalfa (Medicago
1204
Responses of alfalfa nuclei to cold
saliva L. cv. Apica and Trek) were surface sterilized and
planted under sterile conditions between two 3MM Whatman paper sheets laid on water-saturated vermiculite. Seedlings were grown in the dark for a week at 25°C. One-week
old seedlings were used for cold treatment which was carried out in the dark at 4°C. Seedlings were grown in the
dark to facilitate nuclear isolation. It should be noted that
a 4-day cold treatment causes the accumulation of cold
acclimation-specific mRNAs in the dark as well as light
(Mohapatra et al. 1989, Monroy et al. 1993a, b, Wolfraim
et al. 1993), indicating that all stages of low temperature
signal transduction leading to the expression of cold acclimation-specific genes are light-independent.
Nuclei were isolated according to established procedures (Green et al. 1989, Liu and Whittier 1994). Briefly,
seedlings (5 to 10 g fresh weight) were ground to a powder
in liquid nitrogen and mixed with homogenization buffer
(10 ml (g fresh weight)" 1 ) consisting of 1 M 2-methyl-2,4pentanediol (Hexylene Glycol), 10 mM P I P E S / K O H pH
7 . 5 % (v/v) Triton X-100, 5 mM 0-mercaptoethanol. The
results of initial experiments using a combination of protease inhibitors showed that protein patterns with or without these inhibitors were the same. The mixture was passed
sequentially through four layers of cheesecloth, one layer
of Miracloth, 60 fiva nylon mesh and 40 pm nylon mesh.
The resulting filtrate was centrifuged at 3,000 x g for 10
min and the supernatant was discarded. The pellet was resuspended in Buffer A (1/30 of the volume of the homogenization buffer) consisting of 0.5 M Hexylene Glycol, 10
mM P I P E S / K O H p H 7.0, 0.5% (v/v) Triton X-100, and 5
mM /?-mercaptoethanol. The suspension was centrifuged at
3 , 0 0 0 x g for 10min and the supernatant was discarded.
The pellet was resuspended in a volume of Buffer B (Buffer
A without Triton X-100) equal to 1/2 of the volume of Buffer A used in the previous step. The nuclear preparation
was verified by staining with DNA-specific stain (4',6-diamidino-2-phenylindole) coupled with light microscopy
and by extraction of nucleic acids and staining the latter
with ethidium bromide. This procedure has been previously used to isolate nuclei for the rapid preparation of
megabase plant DNA (Liu and Whittier 1994). The prepared samples of nuclei were either used in experiments or
they were mixed with 1/3 volume of 80% glycerol, aliquoted and stored at — 85°C. Nuclei stored in this manner
yielded similar phosphoprotein patterns and labeling intensity as the freshly prepared nuclei.
For labeling the nuclei in organello, samples were
thawed, diluted with an equal volume of Buffer B, and
nuclei were pelleted by centrifugation at 3,000xg for 10
min and resuspended in Buffer C (50 mM Tris-HCl pH 8.0,
10 mM MgCl 2 , 10 mM Hexylene Glycol, 10 mM yS-mercaptoethanol) to a final protein concentration of 1 /ig/A~l.
Samples to be labeled at 25°C were equilibrated to their
labeling temperature for 15 min prior to labeling. No
1205
changes in proteins were detected during the equilibration
period. Samples to be labeled at 4°C were kept on ice. Proteins were labeled in organello with [y- 32 P]ATP (HOTBq
mmol" 1 , used at 20.35 kBq (jig protein)" 1 (Amersham,
Oakville, Ontario) for 10 min. Labeling was terminated by
an addition of 3 volumes of Buffer C. Nuclei were pelleted
by centrifugation at 3,000 x g for 10 min, and were lysed in
the appropriate loading buffer.
Nuclear phosphoproteins were analyzed by SDSPAGE. Thawed samples of nuclei were diluted with an
equal volume of Buffer B and the nuclei were pelleted by
centrifugation at 3 , 0 0 0 x g for 10min. To extract nuclear
proteins, nuclei were lysed in either SDS loading buffer (for
1-D SDS-PAGE) or in isoelectric focusing (IEF) loading
buffer (for 2-D SDS-PAGE). No detectable degradation of
proteins occurred during extraction with these detergentcontaining buffers since the inclusion of protease inhibitors
did not affect the protein pattern on the gel. One-D SDSPAGE was performed using 1 3 % polyacrylamide gels according to Laemmli (1970). Standard IEF followed by 2-D
SDS-PAGE was performed according to O'Farell (1975).
For the first dimension, 2 % ampholytes consisting of a 3 :
2 mixture of Bio-Lyte 5-7 and Bio-Lyte 3-10 (Bio-Rad,
Richmond, California) were used. For phosphoprotein
analysis, samples containing equal amounts of trichloroacetic acid-precipitable radioactivity (245,000 cpm per gel)
were loaded on each first dimension gel. The labeled phosphoproteins were visualized by autoradiography. Autoradiograms were obtained by exposing Fuji RX X-Ray
films (Fuji Photo Film Co., Ltd, Japan) to dried acrylamide gels for 62 h at — 85°C.
To obtain heat-stable protein fraction, the nuclei from
the thawed samples were pelleted as above and resuspended
in Buffer C (50 mM Tris-HCl p H 8.0, 10 mM MgCl 2) 10
mM Hexylene Glycol, 10 mM 2-mercaptoethanol) and
tubes containing the nuclei were placed in boiling water for
20 min. Coagulated proteins were pelleted by centrifugation. Proteins from the supernatant (heat-stable fraction)
were precipitated with acetone and then resuspended in 1-D
loading buffer. Total or heat-stable proteins were separated
by 1-D SDS-PAGE and visulized by staining with Coomassie Brilliant Blue R-250.
Protein concentrations of samples were determined by
the dye-binding method (Bradford 1976) using the Bio-Rad
(Richmond, California) Protein Assay Kit according to
manufacturer's instructions.
The relationship of the heat-stable proteins to dehydrins was determined by immunoblotting. The immunoblots were prepared essentially as described by Winston et
al. (1992). Proteins separated by 1-D SDS-PAGE were electrophoretically transferred to P V D F membranes (ICN,
Costa Mesa, California) using Trans-Blot Cell (Bio-Rad,
Richmond, California). Membranes were blocked in 1%
(v/v) instant non-fat milk in Phosphate Buffered Saline
1206
Responses of alfalfa nuclei to cold
(PBS) (137 mM NaCl, 2.7 mM KC1, 4.3 mM Na 2 HPO 4)
1.4 mM KH 2 PO 4 , pH 7.3) and incubated with rabbit antibodies raised against a synthetic peptide containing the consensus KIKEKLPG amino acid sequence found near the
carboxy terminus of dehydrins (Close et al. 1993). The
membranes were washed in PBS and then incubated with
secondary goat anti-rabbit horseradish peroxidase conjugate antibodies (Bio-Rad). Secondary antibodies were detected using Diaminobenzidine as a substrate.
Results—To determine whether ambient temperature
directly modulates the phosphorylation status of nuclear
proteins, nuclei were isolated from control or cold-treated
seedlings of the two alfalfa cultivars and their proteins
were labeled in organello for lOmin with [y-32P]ATP at
either 4°or 25°C. Proteins were analyzed by 2-D SDSPAGE coupled with autoradiography. Patterns of nuclear
phosphoproteins from untreated (A, B) or cold-treated (C,
D) seedlings of cultivar Apica, radiolabeled at either 25 °C
(A, C) or 4°C (B, D) are shown in Figure 1. In the nuclei
from control seedlings (Fig. 1 A), there are several phosphoproteins which are phosphorylated at 25°C, and those numbered 6, 10 and 11 are the most prominently labeled. When
similar nuclei are labeled at 4°C (Fig. IB), the phosphorylation level of the majority of proteins increases considerably. For example, phosphoproteins 6, 7, 10 and 14
show a large increase in their phosphorylation at 4°C.
There are other phosphoproteins which are almost undetectable when labeled at 25°C but are clearly seen when la-
kDa
97.2
I I
50
3
J
35.1
12
29.7
21.9
16
15
kDa
97.2
21.9
15'
Fig. 1 Temperature-sensing by isolated nuclei of a freezing-tolerant alfalfa cultivar Apica. Nuclear phosphoproteins of cultivar Apica
labeled in organello. Proteins from control (A, B) or cold-treated (C, D) seedlings were labeled for 10 min at either 25°C (A, C) or 4°C
(B, D). Labeled proteins were separated by IEF followed by SDS-PAGE and the phosphoproteins were visualized by autoradiography.
Arrows indicate phosphoproteins specific to the cold-treated sample.
Responses of alfalfa nuclei to cold
beled at 4°C (proteins 3, 8, 9, 12 and 15). When nuclei are
taken from seedlings exposed to cold for 4 d, they have considerably more complex phosphoprotein profiles (Fig. 1C,
D) than those from control seedlings (Fig. 1A, B). Some
phosphoproteins are specific to the nuclei of cold-treated
seedlings (indicated by arrows in Fig. ID) and also show
greater phosphorylation at 4°C (Fig. ID) than at 25°C
(Fig. 1C). Thus it may be concluded that in the case of
cultivar Apica, nuclei have some cold-regulated phosphoproteins constitutively and several additional ones appear
during a 4-d cold treatment of the seedlings.
Profiles of nuclear phosphoproteins from control and
cold-treated seedlings of cultivar Trek, labeled in organello
at 4° or 25 °C are shown in Figure 2A-D. It can be seen that
the Trek nuclei contain fewer phosphoproteins as com-
1207
pared to the Apica nuclei (Fig. 1). There is little difference
in the phosphoprotein profiles between nuclei labeled at
25°C and those labeled at 4°C. Furthermore, the phosphoprotein patterns of nuclei from untreated and cold-treated
seedlings are quite similar. Thus in the case of cultivar
Trek, cold treatment of the seedlings causes little change in
the population of nuclear phosphoproteins.
Patterns of total and heat-stable nuclear proteins from
untreated (NT) and cold-treated (CT) seedlings of Apica
and Trek cultivars, separated by 1-D SDS-PAGE and
visualized by staining with Coomassie Blue, are shown in
Figure 3. It can be seen from the pattern of total nuclear
proteins (Fig. 3A) that many proteins are common to the
two cultivars. However, there are several proteins, particularly in the high molecular mass range, which are
-•IEF
kDa
97.2
I I
e t
50
35.1
c d
g
h
29.7
21.9
kDa
97.2
50 -
35.1 29.7-
21.9-
Fig. 2 Temperature-sensing by isolated nuclei of a freezing-sensitive alfalfa cultivar Trek. Nuclear phosphoproteins of cultivar Trek labeled in organello. Proteins from control (A, B) or cold-treated (C, D) seedlings were labeled for 10 min at either 25°C (A, C) or 4°C (B,
D). Labeled proteins were separated by IEF followed by SDS-PAGE and the phosphoproteins were visualized by autoradiography.
Responses of alfalfa nuclei to cold
1208
present in nuclei from Apica but not in those from Trek.
The level of proteins (indicated by arrows in Fig. 3A) with
approximate molecular masses of 48, 34.5, and 32.5 kDa in
cultivar Apica increase with cold treatment. Proteins in
cultivar Trek show little change with cold treatment.
The varietal and cold-induced differences in protein
populations are more dramatic in the case of heat-stable nuclear proteins (Fig. 3B). In cultivar Apica, exposure to cold
for 4 d caused major changes in the pattern of heat-stable
nuclear proteins. Thus proteins with approximate molecular masses of 50, 45, 37, and 23.5 kDa (indicated by arrows
in Fig. 3B) are undetectable in the untreated seedlings of
Apica but show a large accumulation with cold treatment.
In cultivar Trek, the 4-d cold treatment of seedlings
does not cause accumulation of any heat-stable protein. It
causes a decline in the level of at least one protein, with a
molecular mass of about 23.5 kDa, which shows increased
accumulation in the case of Apica nuclei.
Because dehydrins are also heat-stable, and since some
of the cold acclimation-specific genes of alfalfa encode pro-
Apiea
CT
Trek
NT
Discussion—The present study was undertaken based
Pre-Immune Serum
Heat-Stable
Total
Heat-Stable
Total
NT
teins which show homology to dehydrins (Wolfraim et al.
1993), we have examined if alfalfa nuclei contain dehydrinrelated proteins. The immunoblots of total and heat-stable
proteins separated by SDS-PAGE and probed with anti-dehydrin anti-serum are shown in Figure 4. The reaction of
the preimmune serum with total (A) and heat-stable (B) nuclear proteins is shown in the upper panel. It can be seen
that preimmune serum shows a weak reaction to alfalfa proteins (see Discussion below). The immunoblot probed with
the anti-dehydrin anti-serum is shown in the lower panel
for total (C) and heat-stable (D) proteins. It should be
noted that none of the bands among total proteins reacts
with the anti-dehydrin antibody (see discussion). However,
at least two dehydrin-related proteins are detectable with
the anti-dehydrin antibody in the heat-stable protein fraction obtained from nuclei isolated from both untreated and
cold-treated Apica seedlings. The reaction of anti-dehydrin
anti-serum with nuclear proteins of Trek seedlings is much
weaker.
CT
Apica
NT CT
Trek
NT
Apica
CT
Trek
N T C T N T C T
Apiea
Trek
N T C T N T C T
kDa
112
84
34.9-
f
53.2
Anti-Dehydrin Antibody
Total
Heat-Stable
34.9
Apica
NT
CT
Trek
NT
CT
Apica
NT
CT
Trek
NT
CT
kDa
53.2-
28.7
20.5
34.9-
A
B
Fig. 3 Nuclei of the freezing-tolerant cultivar Apica accumulate
heat-stable proteins during cold treatment of the seedlings. Total
(A) and heat-stable (B) nuclear proteins of alfalfa cultivars Apica
and Trek. Thirty fig of total protein were loaded per lane in (A).
Each sample of heat-stable protein used in (B) was fractionated
from 800fig of total proteins. Proteins were separated by SDS
PAGE using 13% polyacrylamide, and stained with Coomassie
Brilliant Blue R-250. Arrows indicate proteins the level of which
increases with exposure to cold. NT, untreated control; CT, coldtreated.
Fig. 4 Dehydrin-related proteins of alfalfa nuclei. Western blot
analysis of total (A, C) and heat-stable (B, D) nuclear proteins of
alfalfa cultivars Apica and Trek. Proteins transferred to membranes were probed with either preimmune serum (A, B) or with
anti-dehydrin antibodies (C, D). Each sample of total protein contained 30 fig proteins, and each sample of heat-stable proteins
(12.5 fig) was fractionated from 400 fig of total proteins. Arrows
indicate protein bands reacting with anti-dehydrin anti-serum but
not with pre-immune serum. NT, untreated control; CT, coldtreated.
Responses of alfalfa nuclei to cold
on the reasoning that temperature, being a pervasive
thermodynamic factor, is expected to be sensed and responded to in all vital parts of the cell. We have tested the
validity of this reasoning by examining the responses of isolated alfalfa nuclei to low temperature. We have used temperature-modulated protein phosphorylation as a marker
for temperature sensing because altered protein phosphorylation is known to mediate plant responses to a number
of environmental stimuli, including cold (Daminov et al.
1992, Datta and Cashmore 1989, Fallon et al. 1993, Felix et
al. 1991, Monroy et al. 1993b, Trewavas and Gilroy 1991).
The results of our study show that isolated alfalfa nuclei
can sense temperature directly through registering changes
in phosphorylation of their pre-existing proteins; and, indirectly in vivo (mediated by cytosolic events) through importing new phosphoproteins during prolonged cold treatment of the seedlings.
The results of the present study show that the two
alfalfa cultivars which differ in their capacity for cold acclimation (Mohapatra et al. 1989, Monroy et al. 1993a)
also differ in responses of their nuclei to low temperature.
The large number of phosphoproteins showing cold-regulated phosphorylation level probably reflects the complexity of the low temperature signal transduction process and
the multigenic nature of freezing tolerance. The two cultivars differ in three important aspects, (i) In the isolated
nuclei, the level of cold-stimulated phosphorylation of their
phosphoproteins is much greater in Apica than in Trek; (ii)
the number of cold-regulated phosphoproteins in the isolated nucleus is greater in Apica than in Trek; and (iii) during
cold treatment of the seedlings, the nuclei of Apica, but not
of Trek, import additional phosphoproteins which also
show cold-stimulated phosphorylation. It is tempting to
suggest that these differences between the two cultivars are
related to their observed differential gene expression and development of freezing tolerance during cold acclimation.
The rapid and reversible nature of the temperature-induced
changes in protein phosphorylation observed in the present
study may explain why low temperature is continuously
needed to maintain the cold acclimated state (Levitt 1980).
We noticed that irrespective of the source of the nuclei,
phosphorylation of almost every protein was found to be
higher at 4°C than at 25°C. This may be due to a greater
sensitivity of protein phosphatases to low temperature as
compared to that of protein kinases. We have previously
reported the upregulation by cold of a member of calciumdependent protein kinase gene family (Monroy and Dhindsa 1995). We have recently found that action of protein kinase(s) and inactivation of protein phosphatase type 2A are
required for low temperature signal transduction cascade
leading to the expression of cas genes (Monroy et al. unpublished). A report on these data is being submitted separately.
The mechanism(s) by which cold exerts its effects on
1209
protein phosphorylation level is presently unclear. When
[35S]thio-ATP, which, on incorporation, is resistant to protein phosphatase activity, was used instead of [ 32 P]ATP,
cold-induced phosphoprotein pattern did not change (data
not shown). This suggests that cold activation of protein kinase may be involved. Up-regulation of calcium-dependent
protein kinase transcripts (Monroy and Dhindsa 1995),
cold activation of a protein kinase in blotto (Labbe et al.
unpublished) and cold inactivation of protein phosphatase
2A (Monroy et al. unpublished) have been observed. Furthermore, temperature-dependent conformational changes
in macromolecular complexes have been recently demonstrated (Powers and Noller 1995). Thus the observed coldinduced changes in nuclear protein phosphorylation may
be mediated by conformational changes in the phosphoproteins and/or in the protein kinases/phosphatases involved.
Presence of protein kinases (Suzuki and Verma 1989,
Li et al. 1991) and phosphatases (Huber et al. 1994,
MacKintosh et al. 1991) in the nucleus has been documented. The present study suggests that cold-regulation of
such enzymes also occurs in the isolated nucleus. Cold
regulation of protein kinases/phosphatases is expected to
enable the plant to distinguish between cold and other
stresses all of which cause an increase in cytosolic calcium.
A shared event of increased cytosolic calcium gets coupled
with specifically cold-regulated enzyme activities leading to
cold acclimation-specific signal transduction pathways.
The cold-induced changes which confer freezing tolerance are expected to occur in the nucleus as well. While the
nature of these changes is unclear, cold-induced expression
of genes encoding highly hydrophillic proteins, related to
the LEA/RAB/dehydrin family of heat-stable proteins, is
commonly observed (Lin et al. 1990, Houde et al. 1992,
Wolfraim et al. 1993). A protective role of these proteins
against dehydration due to cold, drought and salt has been
suggested (Baker et al. 1988, Close and Chandler 1990) and
recently demonstrated (Xu et al. 1996). It is expected that
such protective mechanisms developed during exposure to
low, non-freezing temperatures must also operate in the
nucleus. The results of our study show that a cold treatment of the seedlings induces the accumulation of heatstable proteins in the nucleus of the tolerant but not the sensitive cultivar of alfalfa.
Localization of dehydrins to the nucleus has been demonstrated in plants subjected to dehydration stress (Asghar
et al. 1994, Goday et al. 1994). Anti-dehydrin antibodies
used in the present study were raised in rabbits. The preimmune serum also shows weak reaction with alfalfa proteins
probably because the commercial food for rabbits generally contains alfalfa. At least two heat-stable nuclear proteins constitutively present in Apica appear to react with
the anti-dehydrin anti-serum. Although the heat-stable nuclear proteins constitute a subset of total nuclear proteins,
no dehydrin-related protein was detected among the total
1210
Responses of alfalfa nuclei to cold
nuclear proteins of Apica. In alfalfa, heat-stable nuclear
proteins constitute about 3% of the total nuclear proteins.
Furthermore, as the results of this study show, not all heatstable proteins are immunologically related to dehydrins.
Thus the most prominently cold-induced heat-stable nuclear proteins in Apica, with molecular masses of about 42
and 50 kDa (Fig. 3B), do not react with anti-dehydrin antiserum (Fig. 4D). LEA proteins have been classified into several families depending on the presence of amino acid
sequence domains (Baker et al. 1988) and dehydrins correspond only to the Dl 1 family. Thus the heat-stable nuclear
proteins which do not react with the anti-dehydrin antibodies may be homologous to LEA proteins other than the
members of Dl 1 family.
We thank Dr. T. J. Close (University of California, Riverside)
for the generous supply of the preimmune serum and the anti-dehydrin anti-serum. This research was supported by Grant A2724
from the Natural Sciences and Engineering Research Council of
Canada to R.S.D.
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(Received October 14, 1996; Accepted November 11, 1996)