Download Arabidopsis thaliana avoids freezing by

Journal of Experimental Botany, Vol. 57, No. 14, pp. 3687–3696, 2006
doi:10.1093/jxb/erl125 Advance Access publication 21 September, 2006
RESEARCH PAPER
Arabidopsis thaliana avoids freezing by supercooling
Marjorie Reyes-Dı́az1, Nancy Ulloa3, Alejandra Zúñiga-Feest3, Ana Gutiérrez2, Manuel Gidekel2,
Miren Alberdi3, Luis J. Corcuera1 and León A. Bravo1,*
1
Departamento de Botánica, Facultad de Ciencias Naturales y Oceanográficas, Universidad de Concepción,
Casilla 160-C, Concepción, Chile
2
Departamento de Agroindustrias, Universidad de La Frontera, Temuco
3
Instituto de Botánica, Facultad de Ciencias, Universidad Austral de Chile, Valdivia, Chile
Received 15 May 2006; Accepted 11 July 2006
Abstract
Arabidopsis thaliana (L.) Heynh. has been described
as a freezing-tolerant species based on freezingresistance assays. Nonetheless, this type of experiment
does not discriminate between freezing-tolerance and
freezing-avoidance mechanisms. The purpose of this
paper was to determine which of these two freezingresistance mechanisms is responsible for freezing resistance in A. thaliana. This was achieved by comparing
the thermal properties (ice-nucleation temperature
and the freezing temperature) of leaves and the lethal
temperature to 10, 50 and 90% of the plants (LT10, LT50,
and LT90, respectively). Two wild-type genotypes were
used (Columbia and Ler) and their mutants (esk-1
and frs-1, respectively), which differ in their freezing
resistance. This study’s results indicated that the
mutant esk-1, described as a freezing-tolerant species
showed freezing tolerance only after a cold-acclimation
period. The mutant frs-1, described as freezing sensitive,
presented freezing avoidance. Both wild genotypes presented LT50 similar to or higher than the ice-nucleation
temperature. Thus, the main freezing-resistance mechanism for A. thaliana is avoidance of freezing by supercooling. No injury of the photosynthetic apparatus was
shown by measuring the maximal photochemical efficiency (Fv/Fm) and pigments (chlorophyll and carotenoid) during cold acclimation in all genotypes. During cold
acclimation, Columbia and esk-1 increased total soluble
carbohydrates in leaves. esk-1 was the only genotype
that presented freezing tolerance after cold acclimation.
This feature could be related to an increase in sugar
accumulation in the apoplast.
Key words: Apoplastic fluid, Arabidopsis thaliana, chlorophyll
fluorescence, cold acclimation, esk-1, freezing avoidance,
freezing tolerance, frs-1, sucrose, supercooling, total soluble
sugars.
Introduction
Freezing temperature is a major limiting factor of growth
and distribution of plants in many areas of the world.
Tropical plants are usually sensitive to both chilling and
freezing temperatures (Levitt, 1980). Plants growing in
temperate regions have evolved several strategies to survive
freezing. They can avoid or tolerate freezing. In the first
case, tissue freezing can be delayed or prevented. In the
second case, ice forms in the extracellular spaces, without
damaging cellular structures (Levitt, 1980; Sakai and
Larcher, 1987; Alberdi and Corcuera, 1991).
Supercooling, which is the capacity of cell fluids to be
cooled down to a temperature lower than the freezing
point without immediate freezing, is a frequent avoidance
mechanism against freezing injury in plants from habitats
subjected to frosts. Supercooling is especially important
when frost occurs during periods of high metabolic or
developmental activity (Sakai and Larcher, 1987; Larcher,
2003). Freezing tolerance is usually observed in tropical
environments at high latitude where subzero temperatures
can occur any night of the year, or in zones with a high
seasonal climatic variation (Bravo et al., 2001; Larcher,
2003).
Many plants increase their freezing resistance when
exposed to low non-freezing temperature (below 10 8C),
* To whom correspondence should be addressed. E-mail: [email protected]
Abbreviations: FT, freezing temperature; LT, lethal temperature; IN, ice nucleation; MDH, malate dehydrogenase; SPS, sucrose phosphate synthase; TSS,
total soluble sugars.
ª The Author [2006]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.
For Permissions, please e-mail: [email protected]
3688 Reyes-Dı´az et al.
a process known as cold acclimation or cold hardening
(Levitt, 1980; Larcher, 2003). Arabidopsis thaliana (L.)
Heynh., the best suited model plant for genetic, molecular,
and physiological studies, has been described as a freezingtolerant species (Gilmour et al., 1988; Thomashow, 1994,
1998; Knight et al., 1999). In general, freezing resistance in
this species increases from ÿ3 8C (lethal temperature 50%,
LT50) to an LT50 of about ÿ6 8C after 24 h of cold acclimation at 4 8C. Lower LT50 values (ÿ8 8C to ÿ10 8C)
were obtained after 8–9 d of cold acclimation (Gilmour
et al., 1988). The freezing resistance of 26 mutants of
Arabidopsis ranged from ÿ6.8 8C to ÿ10.6 8C in the absence
of acclimation. One of these Arabidopsis mutants has been described as constitutively freezing tolerant (eskimo1: esk-1)
(Xin and Browse, 1998; Thomashow, 2001).
Many biochemical changes occur in plants during cold
acclimation and are likely to have roles in freezing
resistance. It is documented that the increased levels of
proline and sucrose during cold acclimation are effective
cryoprotective mechanisms in Arabidopsis (McKown
et al., 1996; Wanner and Junttila, 1999) and other plants
(Guy et al., 1992; Alberdi and Corcuera, 1991; Livingston
III, 1996). Soluble carbohydrates and free proline may be
involved in freezing point depression of cell sap, prevention
of plasmolysis during cell dehydration caused by freezing,
and membranes stabilization (Strauss and Hauser, 1986;
Santarius, 1992). Additional changes associated with an
increased stability of the photosynthetic apparatus preventing low temperature-induced photoinhibition have been
reported during cold acclimation (Sane et al., 2003). Nonacclimated esk-1 accumulated some compatible solutes like
proline (Xin and Browse, 1998). Artus and Thomashow
(1992) screened mutants of Arabidopsis deficient in freezing resistance. Mutants sensitive to freezing (sfr) in this
plant species were also reported by Warren et al. (1996) and
Knight et al. (1999). One of these, sfr-4 showed depressed
levels of sugars in leaves (McKown et al., 1996; Uemura
et al., 2003).
Freezing tolerance implies a capacity to tolerate ice
formation in the intracellular spaces. As explained above,
A. thaliana (Columbia) is referred as a freezing-tolerant
species (Gilmour et al., 1988; Thomashow, 1994, 1999;
Warren et al., 1996; Xin and Browse, 1998; Knight et al.,
1999). Their experiments and conclusions are based on
LT50 (temperature at which 50% lethality occurred) determinations by exposure to freezing temperature. This type of
experiment only shows the temperature at which the plant
or tissues die, ignoring at what temperature ice was formed
in the tissues. To conclude that a plant is freezing tolerant, it
must be shown that is able to survive ice formation (Levitt,
1980; Sakai and Larcher, 1987; Larcher, 2003). This is
achieved by a combination of thermal analyses and lethal
temperature (LT) determinations. Thermal analysis permits
the determination of the supercooling capacity and ice
nucleation and freezing temperatures of tissues. To the best
of our knowledge, these experiments have not been done
in Arabidopsis thaliana. Thus, it is not known whether its
freezing resistance is due to a capacity to tolerate freezing
or to avoid ice formation by supercooling. The main
purpose of this paper is to determine whether A. thaliana,
ecotypes Columbia, Landsberg erecta (Ler) and their
mutants, esk-1 (freezing resistant) and frs-1 (freezing sensitive), respectively, are freezing tolerant or avoid freezing by
supercooling under cold-acclimated and non-acclimated
conditions. In addition, changes in freezing behaviour of
leaves of A. thaliana were correlated with the accumulation
of sugars in the apoplast.
Materials and methods
Plant material and growth conditions
Wild types of Arabidopsis thaliana (L.) Heynh. Columbia WT-2 and
Arabidopsis thaliana Landsberg erecta (Ler) seeds were obtained
from Lehle Seeds (Round Rock, TX, USA). Their mutants eskimo-1
(esk-1) (freezing tolerant) and frs-1 (freezing sensitive), respectively,
were kindly provided by Drs Zhanguo Xin and John Browse
(Institute of Biological Chemistry, Washington State University,
Pullmann, USA) and Julio Salinas (INIA, Madrid, Spain), respectively. Both mutants have been described elsewhere (Xin and
Browse, 1998; Llorentes et al., 2000). Surface-sterilized seeds were
sown in plastic pots in a 1:1 mixture of vermiculite: soil. Pots were
maintained at 21–23 8C with 16/8 h light/dark day length and
a photosynthetic photon flux density (PPFD) of about 150 lmol
photons mÿ2 sÿ1. The light source consisted of cool-white fluorescent
tubes F40CW (General Electric, Charlotte, NC, USA). Plants were
watered twice a week and fertilized with Phostrogen (Solaris,
Buckinghamshire, UK) using 0.12 g lÿ1, once every 2 weeks.
Relative humidity was around 60–70%. These plants were referred
as non-acclimated or controls.
Cold acclimation treatment
For cold acclimation, plants at the vegetative stage (basal or rosette
leaves with approximately 10–15 leaves, about 2-week-old plants)
were used. Plants were transferred to a growth chamber set at 4 8C
with the other conditions the same as those mentioned for controls at
different times. Cold acclimation was performed only for 1, 7, 14, and
21 d. At each time of cold acclimation, whole plants were used to
determine freezing resistance (LT50) and leaves from the first or
second basal node of the rosette were removed for determinations of
thermal analyses, carbohydrates, and pigments (chlorophyll and
carotenoids). Plant material was collected 2 h after the beginning of
the photoperiod.
Thermal analysis
Leaves (first or second basal node of the rosette) from five different
plants were removed and attached to a thermocouple (Gauge 30
Copper-constantan thermocouples; Cole Palmer Instruments Co.,
Vernon Hills, Illinois, USA), and immediately enclosed in small,
tightly closed cryotubes to avoid changes in tissue water content.
Temperature was continuously monitored (1 measurement sÿ1) with
a ACjr data acquisition board connected to a multi-channel temperature terminal panel (Strawberry Tree Inc. Sunnyvale, California,
USA). The tubes were placed in a cryostat and the temperature was
lowered from 0 8C to ÿ17 8C at a rate of approximately 2 8C hÿ1
(Bravo et al., 2001). Temperature records were made and the freezing
exotherms were analysed. The temperature at the initiation of the
exotherm corresponds to the ice nucleation temperature, while the
Arabidopsis thaliana avoids freezing
highest point of the exotherm represents the freezing temperature of
water in the apoplast (including symplastic water driven outwards by
the water potential difference caused by apoplastic ice formation)
(Larcher, 2003). Thermal analyses experiments were executed
without adding an ice nucleating agent. When AgI was added, no
second low temperature exotherm was observed in these plants down
to ÿ15 8C.
Freezing resistance
This was evaluated as survival of plants after frost at various
temperatures. Whole plants in their pots (30 for each frost temperature) were subjected for 2 h at temperatures between 0 8C and ÿ15 8C
in a sealed metallic cell submerged in a cryostat. At each temperature, plants were removed and thawed overnight at 4 8C and then
introduced into a growth chamber set at 23 8C for survival evaluation
by monitoring the fate of the frozen leaves and the progress of new
leaf growth over the next 10 d. Plants that produced at least one new
leaf were considered as survivors. LT was determined essentially
as described by Neuner and Bannister (1995), which consisted
of plotting the percentage of survival against frost treatment
temperature. Temperature that kills 10% (LT10), 50% (LT50), and
90% (LT90) of the exposed plants were obtained from the curve
and used as indexes of incipient, medium, and severe damage,
respectively.
Freezing resistance mechanism
From the comparison of the temperatures that cause injury or
significantly depress plant survival (LTs) and the ice-nucleating
temperatures, the mechanism of freezing resistance can be established
(Levitt, 1980; Sakai and Larcher, 1987; Squeo et al., 1991). If the ice
nucleation temperature is lower than the temperature that causes
incipient damage (LT10), the freezing-resistance mechanism is
freezing avoidance. Conversely, when the ice-nucleating temperature
is significantly higher than the damaging temperature (LT10), the
tissue is able to tolerate ice formation.
Apoplastic fluid extraction
Extractions were undertaken as described by Livingston and Hanson
(1998), 4 g of non-acclimated (control) and cold-acclimated fresh
leaves from first or second node of the rosette of A. thaliana were cut
in pieces of around 0.5 cm and submerged in distilled water to avoid
desiccation, and then dried with filter paper for fluid extraction. For
extraction, the plant material was placed in the bottom 3 cm of a 50 ml
syringe barrel. A 4 ml HPLC insert vial was placed on the syringe tip
to collect apoplastic solution. The syringe barrel containing the leaf
tissue with the attached 4 ml vial was placed in a 50 ml centrifuge
tube and centrifuged at 2000 g for 1 h at 4 8C. To evaluate
contamination of the apoplastic fluid, the activity of MDH (malate
dehydrogenase), as a cytoplasmic marker, was determined by the
NADH oxidation oxalacetate-dependent reaction, according to Duke
et al. (1975). The extinction was spectrophotometrically measured in
a Shimadzu UV-1203 spectrophotometer at 310 nm. Levels of
cytoplasmic contamination in the apoplastic fluid were 2%, which
is within the range reported by Husted and Schjoerring (1995).
Soluble carbohydrates determinations
They were measured in leaf tissue and apoplastic fluid. Soluble
carbohydrate was extracted from fresh leaf tissue (0.1 g) in 86% v/v
ethanol with overnight agitation and then centrifuged at 12 000 g for
10 min. The supernatant was depigmented with chloroform in
a mixture 1:3 v/v (extract:chloroform). The supernatant was freezedried overnight. The dry residue was resuspended in 500 ll of
methanol. Total soluble sugars (TSS) content was determined
spectrophotometrically by the Resorcinol method (Roe, 1934), at
520 nm, using sucrose as standard.
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Sucrose content was determined by high performance thin layer
chromatography (HPTLC) using a silicagel plate (60 F254, Merck)
pretreated with 0.1 M methanolic potassium phosphate solution.
After loading and running the samples, the plates were developed five
times in acetonitrile:water (85:15 v/v). For calibration, a standard
solution containing 0.1 mg mlÿ1 of sucrose, glucose, and fructose
was used. The position of individual carbohydrates was visualized
by spraying with diphenylamine/aniline reagent and then heated
to 105 8C (Lee et al., 1979). The image was scanned at 520 nm
and the amounts were expressed in ng of carbohydrate per area.
These values were then converted to mg sugar gÿ1 dry weight.
Chlorophyll and carotenoid determinations
Chlorophyll and carotenoids were extracted with 80% acetone at
0 8C and spectrophotometrically determined at 663, 646, and
470 nm according to Lichtenthaler and Welburn (1983).
Dry weight determinations
Samples of leaves from the first or second node of the rosette were
removed from plants at the stage of vegetative phase. This tissue was
weighed and then dried at 60 8C for about 2 weeks until constant
weight was obtained. Similar tissue was used at the same time to
determine sugars and pigments content. Water content was determined and values of sugars were expressed on dry weight bases.
Pigment contents were expressed on fresh weight bases.
Fluorescence measurements
Room temperature fluorescence signals were generated by a pulseamplitude modulated fluorometer (FMS 1, Hansatech Instruments
Ltd., U.K.). Fully developed leaves (attached to the plant) from the
first or second internodes of cold-acclimated and non-acclimated
plants were dark adapted for 30 min (to obtain open centres) with
leaf-clips provided by a mobile shutter plate. Then the fibre-optic and
its adapter were fixed to a ring located over the clip at about 10 mm
from the sample and the different light pulses (see below) were
applied following the standard routines programmed within the
machine. Signal recording and calculations were performed on
a personal computer using the data analyses and control software
(Hansatech, Instruments Ltd. UK). Minimal fluorescence Fo (terminology according to Van Kooten and Snel, 1990) was determined
applying a weak modulated light (0.4 lmol mÿ2 sÿ1) and maximal
fluorescence was induced by a short pulse (0.8 s) of more than
9000 lmol mÿ2 sÿ1. The following fluorescence parameters were
measured as indicators of the functionality of the photosynthetic
apparatus: the potential maximal PSII quantum yield (Fv/Fm=variable
fluorescence/maximal fluorescence), where Fv=FmÿFo (maximal
fluorescence–minimal fluorescence).
Statistics
The reported values correspond to the means of three different
experiments, with three replicates each. Differences between the
values (P <0.05) were determined by a two-way ANOVA. A Tukey
test was used to identify those values with significant differences.
Sigma Stat 2.0 software (SPSS Inc., Chicago, IL, USA) was used
for both analyses.
Results
Freezing resistance
Ice nucleation temperature (IN), the temperature at which
ice crystals are initiated, corresponds to the temperature
at the beginning of the freezing exotherm. The highest
3690 Reyes-Dı´az et al.
temperature reached after all apoplastic available water has
frozen is the freezing temperature of the apoplastic fluid
(FT) (Fig. 1). These results showed that all A. thaliana
genotypes studied decrease their IN and FT after 21 d of
cold acclimation (Fig. 1). IN of cold-acclimated leaves of
A. thaliana genotypes fluctuated from ÿ6 8C to ÿ11 8C,
corresponding to the lowest temperature for esk-1 (Fig. 1B).
The non-acclimated wild genotypes (Columbia and Ler)
exhibited similar values of freezing temperature (FT)
(ÿ1.3 8C and ÿ1.6 8C, respectively). The mutants esk-1
and frs-1 presented lower FT values than their wild types
(ÿ2.7 8C and ÿ2.9 8C, respectively). During cold acclimation, the genotype described as freezing tolerant exhibited
the lowest FT value after 21 d of cold acclimation (ÿ6.8 8C).
This variation with respect to the non-acclimated control
represents twice the variation observed in its wild type
Columbia. The genotype described as freezing sensitive
frs-1 showed a small variation in FT value during cold
acclimation (at 21 d it was ÿ7.8 8C). Its wild genotype
Ler exhibited a similar variation with an FT of ÿ7.9 8C at
21 d (Fig. 1C). These results suggest two interesting
phenomena: first that supercooling capacity can increase
about 4 8C in the esk-1 mutant after cold acclimation and
only about 1–1.5 8C in the other A. thaliana genotypes
studied. Second, a higher solute accumulation in esk-1
mutant during cold acclimation could be predicted from
the high freezing point depression caused by cold acclimation in this mutant.
The range of temperature associated with 10–90%
damage was determined in the A. thaliana genotypes
Fig. 1. Thermal analyses performed in non-acclimated and cold-acclimated genotypes of A. thaliana. Leaves of A. thaliana cv. Columbia (A), mutant
esk-1 (B), cv. Ler (C), and its mutant frs-1 (D) were attached to fine thermocouples and cooled down in a programmable cryostat at about 2 8C hÿ1. Leaf
temperatures were recorded twice per second and freezing exotherms were studied to determine ice-nucleation temperatures (IN) and freezing
temperatures (FT). Single representative temperature recordings are shown for each treatment. Temperatures (IN and FT) in the boxes represent the
average of five independent assays.
Arabidopsis thaliana avoids freezing
studied (Fig. 2). LT10 and LT90 oscillated closely and
almost symmetrically around LT50 with a variation ranging
from 1–2 8C in most of the genotypes studied. The
exception was the ecotype Ler which exhibited in the
non-acclimated state and also after 1 d of cold acclimation
a high LT10 (about ÿ1.58C) compared with its LT50, which
oscillated around ÿ7.5 8C and LT90 around ÿ8.5 8C (Fig.
2C). Columbia and its mutant (esk-1) exhibited a similar
LT50 in the non-acclimated state (Fig. 2A, B). The genotype
that presented the lowest LT50 in the non-acclimated state
was Ler (LT50 ÿ7.260.09 8C, Fig. 2C). Its mutant,
described as freezing sensitive (frs-1), showed the highest
LT50 value (LT50 ÿ4.6 8C, Fig. 2D) in the non-acclimated
state. LT50 decreased significantly (P <0.05) in Columbia,
esk-1, and frs-1 under cold acclimation, reaching the
lowest values in esk-1 after 21 d (LT50= ÿ12.2 8C,
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Fig. 2B). This contrasted with Ler, which maintained its
LT50 around ÿ7.5 8C (Fig. 2C). Cold acclimation only
caused a significant reduction in LT10 from ÿ1.5 8C
to ÿ6.0 8C (Fig. 2C).
Freezing-resistance mechanisms can be established,
contrasting the IN temperature with temperatures at which
plants are killed or damaged (Fig. 2). With the exception of
esk-1 after 7 d of cold acclimation, in which plants injured
by the frost treatment were observed at a lower temperature than IN, most of the genotypes at most of the acclimation stages were unable to tolerate ice formation without
a significant number of plants being killed (Fig. 2). These
results indicate that the main mechanism for surviving
frost in A. thaliana is avoidance of freezing by moderate supercooling. Nonetheless, esk-1 is able to tolerate
freezing after 7 d of cold acclimation (Fig. 2B).
Fig. 2. Freezing resistance of A. thaliana cv. Columbia (A) and its mutant esk-1 (B) and cv. Ler (C) and its mutant frs-1 (D). Plants were subjected to
cold acclimation (4 8C), 16/8 h light/dark day length and a photon flux density of 150 lmol mÿ2 sÿ1, for different times. 0=start of experiment with nonacclimated plants (23 8C) and the same conditions as above. Bars represent the range of lethal temperatures (LT) that caused 10–90% plant mortality,
LT10, (upper edge of the white portion of each bar) LT50 (division line between white and grey portion of each bar) and LT90 (lower edge of the grey
portion of each bar). Values correspond to the average of three LT determinations using 10 plants per temperature treatment in each LT determination
(n=30 6SE). Thermal analyses were performed using five plants from each acclimation treatment, Ice-nucleation temperature (IN) values are the average
of n=5 6SE).
3692 Reyes-Dı´az et al.
Accumulation of sugars during cold acclimation
Total soluble sugar accumulation (TSS) was evident within
7 d of cold acclimation (P <0.05) in leaves of three
genotypes (Columbia, esk-1, and frs-1) (Fig. 3). TSS
increased around 3-fold in Columbia and in esk-1 and
around 2-fold in frs-1. This genotype exhibited no further
accumulation of TSS after 7 d of cold acclimation while
Columbia and esk-1 exhibited a significant increase from
7–21 d of cold acclimation. The highest TSS accumulation
was observed in esk-1 at 21 d (63.360.6 mg gÿ1 DW). No
significant differences in sugar content were observed in
Ler during cold acclimation (Fig. 3). Sucrose, glucose, and
fructose increased during cold acclimation. However, the
content of most of them tended to decrease towards the
third week of cold acclimation, except glucose, which was
the most abundant sugar in leaves. esk-1 exhibited
a sustained accumulation at 21 d of cold acclimation
reaching 23.161.7 mg gÿ1 DW (Fig. 4). Sucrose is the
main sugar accumulating in leaf apoplastic fluid during cold
acclimation in esk-1, and to a lesser extent, Columbia, but
not in the other two genotypes. esk-1 presented an increase
of about 4-fold in sucrose concentration at the end of cold
acclimation with respect to zero time. Although esk-1 was
also the genotype that presented the highest values of
glucose and fructose concentration in apoplastic fluid, the
difference between it and Columbia and frs-1 is not
statistically significant (Table 1).
Fluorescence measurements and pigments
determinations
As an indicator of the physiological status of the plants
during cold acclimation, the state of the photosynthetic
apparatus was evaluated by determining a fluorescence
parameter and pigments. Maximum photochemical efficiency (Fv/Fm, variable fluorescence over maximal fluorescence) remained around normal values during cold
acclimation. In the mutants genotypes, Fv/Fm values
decreased slightly to 0.79 after 7 d of cold acclimation,
recovering progressively to the initial values after 21 d. The
two wild-type genotypes showed a similar decrease (around
0.79), but without recovering by the end of the cold
acclimation period. These normal values show that the
photosynthetic apparatus was functional in all genotypes
during the cold treatment (Fig. 5A). This is consistent with
a slight increase in total chlorophyll content observed in
both mutants after 2 weeks of cold acclimation (Fig. 5B).
Carotenoid contents were also slightly higher in both
mutants (esk-1, 0.25–0.29 mg gÿ1 FW; frs-1, 0.19–0.42
mg gÿ1 FW) compared with their respective genetic
Fig. 3. Total soluble sugars content of non-acclimated and coldacclimated leaves of plants of A. thaliana [(Columbia and Landsberg
erecta (Ler)] and their mutants esk-1 and frs-1, respectively. Acclimation
conditions were: 4 8C, 16/8 h light/dark day length and photon flux
density of 150 lmol mÿ2 sÿ1 for different times. Non-acclimated plants
(time zero) grew at 23 8C and the same other conditions as coldacclimated plants. Values are means of three independent samples 6SE
(vertical bars). Vertical bars are not shown when they not exceed the size
of symbols. Different lower case letters on top of each bar indicate
statistically significant differences (P <0.05) between each acclimation
time for a same genotypes, determined by Tukey test, and asterisks (*)
indicate statistically significant differences (P <0.05) between genotypes
for a same time of acclimation.
Fig. 4. Total sucrose (A), glucose (B), and fructose (C) contents in leaves of plants of A. thaliana [(Columbia and Landsberg erecta (Ler)] and their
mutants esk-1 and frs-1, respectively. Acclimation and non-acclimation conditions were the same as in Fig. 3. Each value is the mean of three
independent samples 6SE (vertical bars). Vertical bars are not shown when they not exceed the size of symbols. Different lower case letters indicate
statistically significant differences (P <0.05) between each acclimation time for a same genotypes and same sugar, determined by Tukey test, and
asterisks (*) indicate statistically significant differences (P <0.05) between genotypes for a same time of acclimation and same sugar.
Table 1. Apoplastic sugar concentration of four genotypes of Arabidopsis thaliana
Plants of A. thaliana cv. Columbia and Landsberg erecta (Ler) and their respective mutants esk-1 and frs-1 were cold-acclimated at 4 8C, 16/8 h light/dark day length and a photon flux density of
150 lmolmÿ2 sÿ1, for 21 d. 0=start of experiment with non-acclimated plants (23 8C). Values are expressed in lg mlÿ1 of apoplastic extract. Values correspond to means 6SE of three replicates
each. SE lower than 0.01 are indicated by an asterisk. Suc, sucrose; Gluc, glucose; Fruc, fructose. Different lower case letters indicate statistically significant differences (P <0.05) between each
acclimation time for the same genotypes and the same sugar (columns), determined by Tukey test: (+) indicates statistically significant differences (P <0.05) between genotypes for the same time of
acclimation and sugar.
Time of
acclimation
0
7
14
21
Columbia (lg mlÿ1)
Suc
Gluc
1.660.1 (a)
4.36* (b)
6.56* (c)
5.06* (c)
9.06*
1.36*
060
4.06*
esk-1 (lg mlÿ1)
(a+)
(b+)
(c)
(d)
Ler (lg mlÿ1)
frs-1 (lg mlÿ1)
Fruc
Suc
Gluc
Fruc
Suc
Gluc
Fruc
0.860.4 (a)
1.06* (a)
060 (b)
3.36* (c)
6.163.8 (ab+)
7.06* (a+)
10.06* (b+)
22.560.8 (c+)
060 (a)
060 (a)
0.86* (b)
4.260.9 (c)
10.660.4 (a+)
060 (b)
6.16* (c+)
3.760.7 (d+)
1.060.1 (a)
0.16* (b)
0.26* (b)
0.26* (b)
0.16* (a)
0.86* (b)
1.060.2 (b)
1.060.1 (b)
0.016*
0.26*
0.16*
0.36*
Suc
(a)
(b)
(c)
(b)
2.56*
0.26*
0.76*
0.36*
Gluc
(a)
(b)
(c)
(b)
8.06*
1.06*
4.26*
4.76*
Fruc
(a)
(b)
(c+)
(c+)
6.36*
0.46*
3.06*
3.56*
(a)
(b+)
(c)
(c)
Arabidopsis thaliana avoids freezing
3693
Fig. 5. Changes in Fv/Fm (A) and total chlorophyll (B) of nonacclimated (23 8C, time zero) and cold-acclimated (4 8C) plants of A.
thaliana. Chlorophyll fluorescence and content were analysed in plants
of cv. Columbia, Landsberg erecta (Ler) and their respective mutants
esk-1 and frs-1, at different times of cold acclimation.
backgrounds (Columbia, 0.19–0.22 mg gÿ1 FW; Ler,
0.16–0.17 mg gÿ1 FW).
Discussion
The combination of thermal analysis and LT has been
widely used to describe frost resistance strategies in plants
(Neuner and Bannister, 1995; Pearce, 2001). Determinations of IN temperature are essential to establish if plant
tissues can withstand ice crystals without significant injury
(freezing tolerance). The description of A. thaliana ecotype
Columbia as a freezing-tolerant species (Thomashow,
1990, 1999; Lång et al., 1994; Warren et al., 1996;
Gilmour et al., 1998, 2000) is not supported by this study’s
results. The ecotype Columbia is not a suitable genotype
to study freezing-tolerance mechanisms because, when the
ice is formed in the extracellular spaces (at about ÿ6.5 to
ÿ7.5 8C depending on the state of acclimation), nearly 50%
of the plants have died in the non-acclimated state and
a significant number of plants (more that 10%) have been
seriously injured at higher temperatures even in cold
3694 Reyes-Dı´az et al.
acclimated ones (Fig. 2A). The constitutively freezingtolerant mutant (esk-1) (Xin and Browse, 1998) was used
for comparison in this work. Nevertheless, thermal analyses
demonstrated that this mutant can tolerate freezing only
after 7 d of cold acclimation. In the non-acclimated state
it avoids freezing. That means that this genotype is not
constitutively freezing tolerant. The Ler mutant frs-1,
described as freezing sensitive (Llorentes et al., 2000),
has an IN temperature equal to its LT50 even after 21 d of
cold acclimation. Furthermore, 10% of plants were killed at
about 2 8C higher than the beginning of IN, indicating that
this genotype is seriously injured and at least 10–49% of the
plants have died prior to ice formation in its extracellular
spaces. Since this mutant, cannot withstand apoplastic
freezing, it can be properly classified as freezing sensitive.
However, the same criteria should be applied to its genetic
background Ler. Our results contradict published data
which report that Ler is able to cold acclimate from LT50
ÿ3.2 8C to ÿ5.0 8C in a non-acclimated state down to
a LT50 within the range of ÿ8 8C to ÿ10 8C (Gilmour et al.,
1988; Llorentes et al., 2000). According to our results Ler
did not significantly change its LT50 (from ÿ7.2 8C to ÿ7.8
8C) during low-temperature exposure and exhibited even in
non-acclimated state a LT50 near the range of that reported
for cold-acclimated plants. Although this issue was not
extensively addresed in these experiments, it can be
speculated that it could be due to several differences with
this study’s experimental designs. For instance, in these
experiments, the age of plants was lower, growth light
intensity higher (150 compare to 70 lmol photons mÿ2 sÿ1)
and duration of the frost assay shorter than used by
Llorentes et al. (2000) whose assay is the most comparable
to this one because regrowth after freezing was also used as
a survival indicator. It is well established that younger
leaves, grown at higher irradiance and exposed to a lower
duration of frost may significantly increase freezing resistance in Arabidopsis (Wanner and Junttila, 1999). In the
preliminary experiments it was found that plants with more
insulation around the roots are more freezing resistant
(exhibited higher survival) than plants with only minor
insulation (data not shown). Thus, the degree of insulation
used in different laboratories may be a major source of
variation among experiments.
Ler actually experienced a certain degree of cold
acclimation because non-acclimated plants experienced
10% mortality at high temperature (ÿ1.5 8C) and after
7 d of cold acclimation the LT10 was significantly reduced
(Fig. 2). Steponkus (1984) suggested that the accumulation
of compatible solutes in the cytoplasm contributes to
survival by reducing the rate and extent of cellular
dehydration. Sugar accumulation at cold temperature has
been widely documented in many plants, including Arabidopsis (Ristic and Ashworth, 1993). The mutant esk-1
showed higher levels of proline and total sugars in the nonacclimated state with respect to the non-acclimated wild
type (Xin and Browse, 1998), while the freezing sensitive
mutant sfr-4 fails to elevate its levels of soluble sugars in
response to cold (McKown et al., 1996). It is presumed that
the increase in sugars, proline and potentially the RAB18
protein contribute to the enhancement of freezing tolerance
in the esk-1 mutant (Xin and Browse, 1998). Strand et al.
(2003) reported that the up-regulation of the sucrose
biosynthetic pathway during cold acclimation is an essential element for the development of freezing tolerance in
Arabidopsis thaliana. Plants over-expressing sucrose phosphate synthase showed improved photosynthesis and increased flux of fixed carbon into sucrose. The improvement
in these plants was associated with an increase in freezing
tolerance, showing a significant change in LT50 from ÿ7.2 8C
in the wild type to ÿ9.1 8C in the over-expressing
transgenic plants. The difference in LT50 observed between
non-acclimated and cold-acclimated plants of esk-1 was
higher than the difference observed between transgenic
plants that over-express SPS and the wild-type Columbia.
These results showed that both the freezing resistant mutant
(esk-1) and Columbia accumulated more total carbohydrates than Ler and its mutant under cold acclimation. Total
soluble sugars accumulation was correlated with increased
supercooling (lower ice-nucleating temperatures), especially in Columbia and its mutant esk-1 (r= ÿ0.99 and
ÿ0.95, respectively; P <0.05). Interestingly, the apoplastic
concentration of sugars (especially sucrose) also increased
in the same genotypes concomitantly with a decrease in icenucleation and freezing temperatures. Correlations between
these parameters were statistically significant only for
Columbia and esk-1 (r= ÿ0.8; P <0.05). Although Ler
and frs-1 exhibited a significant increase in total sugars
in their leaves, they showed a decrease in sugar content
(especially sucrose) in the apoplast. This suggests that
accumulation of sugars in the apoplast is essential for increasing freezing resistance. esk-1, the only genotype
studied that after 7 d of cold acclimation was able to
withstand extracellular ice suffering less than 10% plant
death, showed the highest sucrose concentration in the
apoplastic fluid (Table 1). This is consistent with the
highest freezing point depression observed in this genotype.
Similar results were found in cold-acclimated leaves of
barley (Livingston III, 1996; Livingston and Hanson, 1998).
It is also possible that sucrose participates in apoplastic
cryoprotection non-colligatively in cold-acclimated esk-1
(Strauss and Hauser, 1986).
When chilling temperatures reduce the rate of CO2
fixation, thus lowering the overall rate of photosynthesis,
light may exceed the amount that can be utilized in
photosynthetic electron transport (Demmig-Adams and
Adams, 1992). If this excess of energy is not dissipated,
the photosynthetic apparatus can be damaged, resulting in
a decrease of photosystem II activity (Hüner et al., 1993).
The behaviour of this photosystem within chilling conditions was studied in all genotypes. Fluorescence
Arabidopsis thaliana avoids freezing
measurements in these genotypes showed that the maximal
photochemical quantum yield (Fv/Fm) remained under the
normal values of healthy plants (Krause and Weiss, 1991),
suggesting that no significant perturbation of the photosynthetic apparatus during cold acclimation at the photon
fluxes densities used in this work occurred. The maximal
photochemical quantum yield (Fv/Fm) is used as a good
indicator of the photosynthetic apparatus and plant status
(Demmig-Adams and Adams, 1992). It has been suggested
that values of Fv/Fm close to 0.80 are typical for non-stressed
plants (Krause and Weis, 1991; Larcher, 2003). Similarly to
the maintained Fv/Fm values during cold acclimation,
chlorophyll and carotenoid contents hardly changed at all
during cold exposure, indicating that the photosynthetic
apparatus remained without significant alteration. Carotenoid increases are frequently associated with mechanisms
that protect against high irradiance and low temperatures,
avoiding photoinhibition (Demmig-Adams, 1990).
In summary, these results indicate for the first time that
A. thaliana avoids freezing by supercooling. The mutant
esk-1 showed freezing tolerance only after cold acclimation, which appears to be related to sugar accumulation in
the leaves and apoplastic fluid.
Acknowledgements
The authors thank the financial support of Fondecyt 1000610,
DIUFRO 120418, and DID-UACH 200288 for this research and
to Valeria Neira for technical assistance.
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