Directory UMM :Data Elmu:jurnal:T:Tree Physiology:vol17.1997:

Tree Physiology 17, 31--37
© 1997 Heron Publishing----Victoria, Canada

Changes in ABA and gene expression in cold-acclimated sugar maple
ANNICK BERTRAND,1 GILLES ROBITAILLE,1 YVES CASTONGUAY,2 PAUL NADEAU2 and
ROBERT BOUTIN1
1

Natural Resources Canada, Canadian Forest Service, Laurentian Forestry Centre, P.O. Box 3800, Sainte-Foy, Québec G1V 4C7, Canada

2

Agriculture and Agri-Food Canada Research Station, 2560 Boul Hochelaga, Sainte-Foy, Québec G1V 2J3, Canada

Received April 25, 1996

Summary To determine if cold acclimation of sugar maple
(Acer saccharum Marsh.) is associated with specific changes
in gene expression under natural hardening conditions, we
compared bud and root translatable mRNAs of potted maple
seedlings after cold acclimation under natural conditions and

following spring dehardening. Cold-hardened roots and buds
were sampled in January when tissues reached their maximum
hardiness. Freezing tolerance, expressed as the lethal temperature for 50% of the tissues (LT50), was estimated at − 17 °C for
roots, and at lower than − 36 °C for buds. Approximately ten
transcripts were specifically synthesized in cold-acclimated
buds, or were more abundant in cold-acclimated buds than in
unhardened buds. Cold hardening was also associated with
changes in translation. At least five translation products were
more abundant in cold-acclimated buds and roots compared
with unhardened tissues. Abscisic acid (ABA) concentration
increased approximately tenfold in the xylem sap following
winter acclimation, and the maximum concentration was
reached just before maximal acclimation. We discuss the potential involvement of ABA in the observed modification of
gene expression during cold hardening.
Keywords: Acer saccharum, cold hardening, freezing tolerance, in vitro translation.

Introduction
Woody species native to the northern temperate zone have an
annual cycle of cold hardening and dehardening. In many
plants, acclimation to cold is associated with major biochemical and physiological changes (Sakai and Larcher 1987), and

altered gene expression (Lee and Chen 1993). However, little
is known about the relationship between the biochemical
changes and the changes in gene expression, particularly in
woody species.
The extent of freezing tolerance of roots of sugar maple
(Acer saccharum Marsh.) has been related to the capacity of
the species to survive harsh winter conditions (Bertrand et al.
1994a). Calmé et al. (1994) found that a portion of the roots
can survive temperatures as low as − 30 °C in December, but
are killed by a − 6 °C frost in August, indicating that major
changes in freezing tolerance occur during the year. Despite
the importance of this phenomenon, little is known about the

cold-hardening process in sugar maple. The identification of
gene products that specifically accumulate in cold-acclimated
tissues could provide a tool to study cold-hardening processes
and to identify cold-tolerant families of trees.
Several studies have shown that changes in abscisic acid
(ABA) concentration are related to the cold-hardening process
in Acer species (Irving 1969, Dumbroff et al. 1979). Recently,

we demonstrated that the concentration of ABA in xylem sap
is a useful indicator of physiological changes occurring in
sugar maple (Bertrand et al. 1994a). Thus, it is possible that
ABA not only plays a direct role in response to cell desiccation
(Hartung and Davies 1991) and as a modulator of growth (Saab
et al. 1990), but is also involved in the control of gene expression during cold hardening (Chandler and Robertson 1994).
The observation of simultaneous changes in endogenous concentrations of ABA and of particular gene products after exposure to a stress, such as cold, further supports the idea that ABA
regulates the expression of certain genes. These observations
led to the hypothesis that low temperatures induce increased
synthesis of ABA in plants, which in turn triggers the expression of genes involved in freezing tolerance.
We analyzed changes in the populations of translatable
mRNAs isolated from sugar maple buds and roots to determine
if cold hardening induces changes in genetic expression. Specifically, we characterized the freezing tolerance of sugar maple buds and roots during a cycle of hardening and dehardening
under natural conditions and compared the patterns of in vitro
translation products in unhardened and cold-hardened tissues.
In parallel, we measured the ABA concentration in the xylem
sap to establish the relationships among acquisition of cold
tolerance, changes in gene expression, and accumulation of
ABA.
Materials and methods

Plant material
On May 25, 1994, two-year-old sugar maple seedlings (height
50--90 cm and provenance 46°10′ N, 70°37′ W) were planted
in Styroblock containers (15 cavities per container, 750 cm3
per cavity). The substrate was a 2/1/1 (v/v/v) mix of compost/peat moss/sand. Seedlings were grown in the field at the
Centre de recherches acéricoles (Tingwick, Québec) from June

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BERTRAND ET AL.

1 to September 19. Seedlings were watered to saturation
weekly and fertilized on June 28, July 21, and August 15 and
30 with a soluble commercial preparation (CIL, N,P,K,
20,20,20, 3 g l −1). On September 19, the seedlings were transferred to the Laurentian Forestry Centre, Sainte-Foy, Québec,
and kept outdoors during the winter. Substrate temperature
(starting on November 15, at a depth of one-third of the cavity)
and air temperature (starting on November 1, 2.0 m above the
containers) were recorded continuously with copper-constantan thermocouples connected to a data logger (Model CR 10,
Campbell Scientific Inc., Logan, UT), and averaged hourly.

Freezing tolerance determination
Freezing tests were performed on seedlings in a modified
programable (MIC 6000 control, Partlow Corp., New Hartford, NY) cold room (dimensions: 2.7 × 3.9 × 2.1 m, minimum
temperature − 40 °C), as described by Calmé et al. (1994). The
seedlings were placed individually in plastic bags, and the
interior temperatures of the bags were recorded during the test
using copper-constantan thermocouples connected to a data
logger (Model CR21X, Campbell Scientific Inc.). Plants were
equilibrated at 3 °C for 2 h before the temperature was lowered
at a rate of 3 °C h −1, followed by a 1-h plateau. Seedlings were
sampled at ten temperatures at 3 °C intervals; the range of test
temperatures varied according to the physiological state of the
seedlings. Before damage assessment, the seedlings sampled
at each test temperature were placed in a cold room at 3 °C for
2 days, and then in a dark room at 20 °C for 15 days.
Root systems were excised just above the primary lateral
roots and examined using a stereo microscope. Dead and live
roots were separated with dissecting forceps and a razor blade,
dried in a forced-air oven at 60 °C for 48 h, and weighed. The
percentage of damage was determined by dividing the dry

weight of dead root (brown, dark-colored and flaccid) by the
total root dry weight. Two cm of the basal ends of the shoots
was placed in water, and the shoots were kept in a dark room
at 20 °C for 6 days before damage evaluation. Brown discolored phloem and cambium were observed when the shoots
were cut lengthwise with a razor blade. Percentage of damage
was determined by dividing the length of brown phloem and
cambium by the total shoot length. Freezing tolerance was
estimated from the LT50, the freezing temperature at which
50% of the tissues were killed, as determined by the SAS
Probit procedure (SAS Institute Inc., Cary, NC).
Xylem sap extraction and ABA quantification
Root systems were gently washed in tap water to remove
substrate. Stems were cut 12 cm above the primary lateral root
and the basal part was placed immediately in a pressure chamber (PMS Instruments Co., Corvallis, OR). Roots were covered
with a damp paper towel. The pressure in the chamber was
gradually increased, and then maintained at 0.8--1.2 MPa for
approximately 5 min for the collection of 100 ml of xylem sap.
The vials containing the sap were placed on ice and frozen at
− 80 °C within 1 h. After a 30-min incubation with polyvinylpolypyrrolidone (PVPP, 1 mg ml −1), xylem sap was assayed for free ABA concentration by radioimmunoassay

(RIA), as described by Bertrand et al. (1994a). Antibodies
specific to free (+) cis-trans ABA were prepared according to
Weiler (1980). The antibody showed a linear response from 0.1
to 10.0 pmol under RIA conditions. The antiserum was highly
specific with a cross-reactivity of 53% for (±) cis-trans ABA,
32% for (±) racemic ABA and 7% for methylated (±) ABA.
The affinity constant for the antiserum was 2.47 × 10 −9 l mol −1.
All standards and samples were assayed in triplicate. Depending on their initial concentration, samples were diluted 1/20 or
1/200 in phosphate buffered saline (Weiler 1980).
Extraction of RNA
Buds were collected for RNA extraction at their maximum
hardiness on January 18, 1995 (cold-hardened) and when they
were swollen, on May 15, 1995 (unhardened). Roots were
collected on January 8, 1995 (cold-hardened) and on June 4,
1995 (unhardened).
The lateral and terminal buds of approximately ten seedlings
and the fine roots of three seedlings were excised, frozen
immediately in liquid nitrogen and kept at − 80 °C until extraction. Six grams of buds or roots were ground to a fine powder
with a pestle in a mortar containing liquid nitrogen, and total
RNA was extracted as described by De Vries et al. (1988).

Twelve milliliters of a preheated (90 °C) 1/1 mixture of RNA
extraction buffer (100 mM Tris-NaOH (pH 9.0), 100 mM LiCl,
10 mM EDTA and 1% (w/v) SDS) and phenol equilibrated
with TLE (200 mM Tris-HCl (pH 8.0), 100 mM LiCl, 5 mM
EDTA) containing 0.1% (w/v) 8-hydroxyquinoline, were
added to each sample. After mixing at room temperature for
5 min on a rotary shaker at 300 rpm, chloroform (1 ml g −1 fresh
weight of sample) was added. The mixture was shaken at
300 rpm for 20 min, and then centrifuged at 20,000 g for
30 min. The aqueous phase was recovered and the chloroform
extraction repeated. The final aqueous phase was adjusted to
2 M LiCl and allowed to precipitate for 16 h at 4.0 °C. Samples
were then centrifuged at 12,000 g for 30 min. The resulting
pellet was washed once with 2 M LiCl, twice with 80% ethanol
and vacuum dried. The final pellet was dissolved in 0.5 ml
Tris-EDTA (10 mM Tris-HCl and 1 mM EDTA, pH 7.4). The
color of the samples indicated the presence of phenolic compounds. Further chromatographic purification was performed
by two separations on Sephadex G-50 exclusion matrix (20%
(w/v) Sephadex G-50 equilibrated with Tris-EDTA (10 mM
Tris-HCl, 1 mM EDTA and 100 mM NaCl, pH 8.0)). Two ml

of the Sephadex G-50 preparation was placed in a Poly-Prep
column (Bio-Rad, Hercules, CA) and washed twice with H2O
treated with 0.1% (v/v) diethylpyrocarbonate (DEPC). TrisHCl EDTA was added to bring sample volumes to 0.2 ml, and
the entire volume was loaded on a column and centrifuged at
1500 g for 4 min. Purified samples were precipitated for 2 h at
− 20 °C in an ice-cold ethanol mix (2.5 ml cold ethanol, 0.1 ml
sample buffer (10 mM TrisHCl, 1 mM EDTA, 3.0 M NaCl,
pH 7.4) and 0.01 ml glycogen) (Pharmacia mRNA purification
kit), centrifuged at 12,000 g for 15 min, washed with ethanol,
vacuum dried and stored at − 80 °C.
Four mg of nucleic acids was purified by oligo (dT) cellulose chromatography (mRNA purification kit; Pharmacia,

GENE EXPRESSION IN COLD-HARDENED SUGAR MAPLE

33

Baie d’Urfé, Québec, Canada). Total and poly A-tailed RNA
were quantified by spectrophotometry at 260 nm, and the
integrity of the RNA was verified by size distribution analysis
on denaturating 1% agarose formaldehyde gels (Fourney et al.

1988).

Institute Inc.) after the confirmation of homogeneity of variances by residual analysis.

In vitro translation

Freezing tolerance

In vitro translation was performed with 1 mg poly A-tailed
RNA using a wheat germ extract system (Promega Co., Madison, WI). The standard reaction mixture had a final volume of
50 µl and contained 25 µl of wheat germ extract, 1 µl (40 units
µl −1) of placental ribonuclease inhibitor (RNasin, Promega),
4 µl of 1 mM amino acid mixture (lacking methionine), 3.75 µl
of 1 M potassium acetate and 1 µg of poly A-tailed RNA
heated at 65°C for 10 min. Forty µCi of 35S-methionine (1,000
Ci mmol −1; Amersham, Oakville, ON, Canada) was added to
each reaction, and DEPC-treated water was added to attain
final volume. After a 60-min incubation at 25 °C, in vitro
translation reactions were stopped by placing the reaction
tubes on ice. Five µl of the translation mixture was precipitated

with trichloroacetic acid (TCA) to determine the extent of label
incorporation. The remaining 45 µl was thoroughly mixed
with 55 µl of O’Farrell’s lysis buffer (O’Farrell et al. 1977)
(9.5 M urea, 5% (v/v) 2-mercaptoethanol, 2% (v/v) Nonidet
P-40, 2% (w/v) LKB ampholines (pH 3.5--10; LKB, Baie
d’Urfé, Québec, Canada)), and briefly centrifuged at 13,000 g
before analysis of translation products by two-dimensional
polyacrylamide gel electrophoresis (PAGE).

Seedlings became cold hardened under fall and winter conditions. The lowest air temperature of − 32 °C was reached on
January 10 (Figure 1). The lowest soil temperatures were
reached in November and December, before a sufficient snow
cover stabilized the temperature slightly below the freezing
point. Freezing tolerance (LT50) of the roots and shoots differed
significantly between sampling dates (P < 0.0001) (Figure 2).
Freezing tolerance of the roots increased markedly from August to mid-January (LT50 = − 3.6 °C and − 17.0 °C, respectively). Root freezing tolerance then decreased, and the tissues
reached an LT50 of − 11.4 °C on May 15. Shoot LT50 decreased
rapidly from − 5 °C on August 11 to − 36 °C on November 16.
Between November 16 and March 22, the shoot LT50 was
lower than − 36 °C, but could not be precisely evaluated because the lowest temperature reached in the cold room was
− 40 °C.

Electrophoresis
In vitro translation products were analyzed by two-dimensional PAGE as described by Bertrand et al. (1994b). Gels were
prepared for fluorography according to Bonner and Laskey
(1974), dried and exposed to Kodak X-Omat AR5 X-ray film
at − 80 °C. To give equivalent overall intensities on the fluorograms, exposure times were normalized according to the total
dpm of 35S-methionine incorporated during in vitro translation.
Exposure times varied from 3 to 30 days. Fluorograms were
scanned with a Bio-Rad Video Densitometer Model 620, and
the images were processed, analyzed and compared using the
Bio-Rad 2D-Analyst II computer program.

Results

Xylem sap ABA concentration
The ABA concentration in the xylem sap varied significantly
(P < 0.0001) during the experiment (Figure 3). It increased
from 200 pmol ml −1 of xylem sap on September 23 to reach a
maximum concentration of 1600 pmol ml −1 on December 14.
The highest concentration of ABA, 500 pmol ml −1, preceded
the maximum hardened state of roots, which was attained on
January 18. From March 22 to May 15, ABA remained at a
stable and low concentration of approximately 200 pmol ml −1,
comparable to the concentration measured in August of the
preceding summer.

Experimental design and statistical analysis
The experimental design was a randomized complete block
with sampling dates as the main effect, completely randomized
within each of the three blocks. The experiment included a
total of 396 seedlings: 3 blocks × 11 sampling dates × 12
seedlings. One seedling was sampled for ABA quantification
for each block and date, and one seedling was sampled for each
of the 10 temperatures tested during the freezing test. Buds and
roots of the remaining seedlings were sampled twice for RNA
extraction (January 18 and May 15, 1995 for buds and January
18 and June 4, 1995 for roots). Sampling dates for LT50 and
ABA determination were August 11, September 23, October
14, November 2, November 16, December 14, January 18,
March 22, April 12, April 26 and May 15. Data were evaluated
by analysis of variance using the SAS software package (SAS

Figure 1. Minimum daily air and soil temperatures at the outdoor site.

34

BERTRAND ET AL.

groups of transcripts encoding low molecular weight proteins
was lower in cold-hardened than in unhardened buds (Figure 4), in vitro translation of mRNA from cold-hardened buds
showed that ten transcripts or groups of transcripts were newly
synthesized or were present in higher amounts in cold-hardened than in unhardened buds (Figure 4).
Many transcripts or groups of transcripts encoding polypeptides of different molecular weight or charge were more abundant in unhardened than in cold-hardened roots (Figure 4);
however, cold hardening increased the abundance of twelve
transcripts or groups of transcripts in the roots (Figure 4).
Among the transcripts associated with the cold-hardened state,
five were common to both buds and roots (Figure 4).

Discussion
Figure 2. Temperature causing 50% death (LT50) of root (j) and shoot
(h) tissues. Mean ± SE for n = 3. The shoot LT50 was lower than
− 36 °C between November 16, 1994 and March 22, 1995, but could
not be precisely evaluated because the lowest temperature reached by
the cold room was −40 °C.

Figure 3. Concentration of ABA in the xylem sap of sugar maple
seedlings during cold hardening and dehardening. Mean ± SE for
n = 3.

Changes in the populations of translatable mRNAs in buds
and roots
Analysis of the in vitro translation products by two-dimensional PAGE revealed between 150 and 200 polypeptides,
depending on the tissue and the state of hardening. Comparison of in vitro translation products from RNA extracted from
cold-hardened and from unhardened buds revealed major
modifications in translatable mRNA populations during cold
acclimation. Although the abundance of nine transcripts or

Freezing tolerance of sugar maple buds and roots increased
markedly following fall acclimation and was associated with
significant modifications in gene expression. Some in vitro
translation products were down-regulated by natural cold
hardening, whereas the relative abundance of approximately
ten transcripts increased in both bud and root tissues. Among
these, five transcripts or groups of transcripts were more abundant in both cold-hardened buds and roots of sugar maple. Our
observation of the down- and up-regulation of certain genes of
sugar maple buds is similar to the observations of Hance and
Bevington (1992), who reported an increase in protein synthesis in sugar maple embryos after a 20-day acclimation at 4 °C
and a concomitant repression of protein synthesis in cotyledons. These authors concluded that, during stratification, the
growth potential of the embryonic axes increases because of
the greater capacity for translation of mRNA, whereas the
inhibitory effect that the cotyledons exert on the growth of the
embryo is removed by repression of the expression of specific
cotyledon genes. There seems to be an interesting parallel
between these observations and our results, because most of
the down-regulated transcripts encoding low molecular weight
proteins that we observed were specific to buds, but were not
found in roots of sugar maple. Conversely, a majority of the
up-regulated transcription products were common to buds and
roots and may be part of the general acclimation response of
seedlings to cold.
Other reports of changes in gene expression that increase
cold hardiness in woody species are mainly quantitative and do
not provide details on the nature of the modifications
(Clapham et al. 1994, Lloyd et al. 1996). However, changes in
population of in vitro translation products in response to cold
have been documented for many other plants that show cold
hardening (Sitbon et al. 1993). In alfalfa, the abundance of
some in vitro translation products is related to the degree of
hardinesss of cultivars, indicating that the genes encoding
these polypeptides may constitute genetic markers for cold
tolerance (Mohapatra et al. 1989, Newton et al. 1991, Castonguay et al. 1993). The cold-induced in vitro translation products could play structural, regulatory or protective roles in the
cellular modifications that occur during winter.

GENE EXPRESSION IN COLD-HARDENED SUGAR MAPLE

35

Figure 4. Two-dimensional PAGE analysis of 35S-methionine-labeled in vitro translation products from unhardened and cold-hardened buds and
roots of sugar maple. Triangles identify peptides or groups of peptides that were less abundant in cold-hardened tissues. Squares and rectangles
identify peptides or groups of peptides that were newly-induced or more abundant in cold-hardened tissues. Mr = Molecular weight.

Freezing has a major effect on cellular water relations (Guy
1990). Tolerance to cold-induced dehydration is a key survival
stategy in most cold-hardy plant species (Lee and Chen 1993).
A characteristic of cold hardening in sugar maple is a modification of the concentration of ABA in the xylem sap. We
observed a marked increase in ABA concentration in mid-November, at the time when air and soil temperatures were below
freezing. The ABA concentration increased tenfold and
reached a maximum before maximum hardiness was reached
in buds and roots. This could be a direct effect of cold-induced
dehydration because desiccation results in an accumulation of
ABA (Hartung and Davies 1991). The increased apoplastic
ABA concentration may be involved in the modulation of
sugar maple gene expression during cold acclimation. Exogenous application of ABA to many species, including Acer
negundo L., increases freezing tolerance in the absence of low
temperature exposure, and also induces changes in gene ex-

pression that are, in some cases, similar to those elicited by low
temperatures (Thomashow 1990). Our finding that the highest
apoplastic ABA concentration preceded the state of maximum
cold hardiness of the tissues is in accordance with the proposition that ABA is involved in the cold-hardening process
through its effect on gene expression (Zeevart and Creelman
1988).
In both cold-hardened buds and roots, we have identified an
increase in the abundance of a group of transcripts encoding
proteins with an apparent molecular weight of approximately
40 kDa. There may be a relationship between our findings and
the previous reports of the accumulation of a 42 kDa protein
that accumulated in both ABA- and low-temperature-treated
cells of alfalfa (Robertson and Gusta 1986), plantlets of Solanum commersonii Dun (Tseng and Li 1991) and other plants
(Close et al. 1993). This protein could be one of the dehydrins,
a family of proteins that is synthesized in response to any

36

BERTRAND ET AL.

environmental stress that has a dehydration component, or in
response to ABA (Close et al. 1993). Based on their amino acid
sequence and cellular localization, it has been suggested that
dehydrins play a structural role in membrane stabilization.
Genetic changes induced by ABA are generally similar but not
identical to changes induced by cold. Some polypeptides are
common to both treatments, but others are specific (Robertson
and Gusta 1986, Tseng and Li 1991). Furthermore, low temperature tolerance in ABA-treated potato cells is transient
when cells are not exposed to cold, but cells remain cold-tolerant if they are kept at low temperatures (Lee et al. 1992).
Amzallag and Lerner (1995) proposed that the stressing factor
must be present in order for the adaptive response to a stress to
be maintained. In sugar maple, the tenfold increase in ABA
concentration in the xylem sap might be required to trigger the
initial acclimation response involving the synthesis of certain
stress proteins; however, continuous exposure to low temperatures, as occurs under natural hardening conditions, may be
essential to maintain cold hardiness.
The identification of genes expressed during cold acclimation could provide an important basis for future genetic improvement, however few studies have related cold acclimation
to the accumulation of specific transcripts in woody plants. We
have now identified gene products that specifically accumulate
in cold-acclimated sugar maple tissues. If kinetic studies link
the accumulation of these proteins to increasing cold tolerance,
they could be used as indicators of the physiological state of
the tree. In addition, identification of the genes encoding these
proteins would allow for a description of cold-tolerant genotypes of sugar maple.
Acknowledgments
We thank Mr. Pierre Lechasseur, Ms. Lucette Chouinard and Mr.
Pascal Aubé for their technical assistance, and Mr. Jacques St.-Cyr for
the preparation of plates.

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