Plant Science 165 (2003) 13031313

Low-temperature sensing in olive tree: calcium signalling

and cold acclimation
Simone DAngeli a , Rui Malh b , Maria Maddalena Altamura a,
a Dipartimento di Biologia Vegetale, Universit La Sapienza, P.le A. Moro 5, 00185 Roma, Italy
b Departamento de Biologia Vegetal, Bloco C2, Campo Grande, 1749-016 Lisboa, Portugal

Received 27 March 2003; received in revised form 22 May 2003; accepted 17 July 2003

Abstract

Olive tree is a warm-temperature evergreen tree with low tolerance to frost, although cultivars which differ in terms of cold acclimation
have been empirically selected. In herbaceous species, free cytosolic calcium ([Ca2+ ]c ) is involved in cold acclimation. The objective of this
study was to measure [Ca2+ ]c signalling in the olive tree during cold acclimation and to assess the possibility of using [Ca2+ ]c as an early
genotype-selection marker of cold susceptibility. To this end, non-cold-acclimated and cold-acclimated leaf protoplasts of cultivars differing
in terms of cold susceptibility were analysed.
Cold shocks of various amplitude applied to non-cold-acclimated protoplasts caused consistent and transient increases in [Ca2+ ]c . A decrease
of 0.05 C/s (i.e. T/dt = 2.5 C/50 s) was the threshold cooling rate at which a significant increase in [Ca2+ ]c could still be observed. When the
protoplasts were incubated with either 8-(N,N-di-methylamino)octyl 3,4,5-trimethoxy-benzoate (TMB-8; organelle Ca2+ channel blocker) or
Gd3+ (plasma membrane Ca2+ channel blocker), applying the threshold cooling rate, the increase in [Ca2+ ]c was partially inhibited, suggesting
that both an intracellular release of Ca2+ and an influx through the plasma membrane are involved. When applying repeated cold shocks,
the transient increases in [Ca2+ ]c were reduced only when using a non-severe T/dt. In protoplasts subjected to standard acclimation, the
increases in [Ca2+ ]c were further reduced, or inhibited, depending on the cold susceptibility of the cultivar, suggesting that the Ca2+ response
is involved in a long-term adaptation to cold.
2003 Elsevier Ireland Ltd. All rights reserved.

Keywords: Cold acclimation; Cold hardiness and genotype; Cooling rate; Cytosolic calcium; Leaf protoplasts; Olive tree

1. Introduction branes [2] and can result in anatomical changes in specific

Plants exposed to low temperatures can suffer chilling in-
jury (at temperatures above 0 C) and/or freezing injury (at
subzero temperatures). The extent of resistance to low tem-
peratures (i.e. cold hardiness) varies according to the species,
and it plays a determining role in species distribution [1].
The capacity of a species to survive at subzero temperatures
is to a great extent determined by the ability to undergo
cold acclimation, which entails acquiring freezing tolerance
through exposure to increasingly low, yet non-freezing, tem-
peratures [2]. Cold acclimation is a complex process which
involves the accumulation of cryoprotectans, which leads
to the physical and biochemical restructuring of cell mem-
Corresponding author. Tel.: +39-06-4991-2452;
fax: +39-06-446-3865.
E-mail address: mariamaddalena.altamura@uniroma1.it
(M.M. Altamura).
tissues. The leaf seems to be the site of production of sub-
stances essential for the promotion and/or inhibition of cold
acclimation [3], as well as the site of the perception of the
short-day stimulus that initiates acclimation [4].
Most plants (e.g. trees) from regions with cold winters ini-
tiate the process of cold acclimation as soon as shoot growth
has ceased. For the development of maximum cold toler-
ance, growth cessation is usually associated with the induc-
tion of dormancy ([5], and references therein), yet certain
cold-tolerant plants (e.g. winter cereals) do not undergo dor-
mancy [6] and may thus be more prone to suffer significant
losses in plant productivity.
Olea europaea is a Euro-Mediterranean species whose
cultivated variety (i.e. O. europaea sativa, olive tree) is of
economic value for the nutritional and therapeutic charac-
teristics of the oil obtained from its fruit. O. europaea is a
warm-temperature, long-living, evergreen species, which in
nature grows between the latitudes of approximately 30 and

0168-9452/$ see front matter 2003 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/S0168-9452(03)00342-X
1304 S. DAngeli et al. / Plant Science 165 (2003) 13031313
45 in both hemispheres. In addition to the Mediterranean
Basin, the cultivated variety is present in Portugal, western
Africa, South Africa, the United States, Australia, Azerbai-
jan, and China [7]. Olive tree lacks a true dormancy and
its cultivation is greatly impeded by the inability to survive
temperatures below 12 C, with severe leaf damage occur-
ring at 7 C [8]. Nonetheless, winter chilling results in the
slowing down of the post-maturation process of the fruit,
which improves the quality of the oil [9], and it is essential
for good flowering to occur in the following spring [7].
Olea europea sativa is a cultigen group of forms which
possibly originated through mutations and hybridisations
[10] of Afro-Asiatic wild species that are tolerant to frost (i.e.
Olea chrysophylla and Olea cuspidata) [11,12] and species
growing in warmer habitats, e.g. Olea excelsa [13].
In general, for cold acclimation to occur, specific gene
expression is required [1416], yet in the olive tree no ge-
netic studies on cold tolerance have been published. How-
ever, in Italy, the recurrent winter frosts that have occurred in
the past 50 years have allowed cultivars (and clones within
cultivars) to be empirically identified as cold tolerant (i.e.
hardy), weakly tolerant (i.e. semi-hardy), or cold sensitive
(i.e. not-hardy) [17,18]. With regard to the cultivars that are
typical to the Tuscany Region of Italy, which has cold win-
ters, differences in cold tolerance have been observed: Lec-
cino is considered to be hardy, Moraiolo as not-hardy, and
Frantoio as semi-hardy [17,19]. Furthermore, unlike herba-
ceous plants, in which cold injury symptoms are visually
discernible within hours or days, the olive tree, like other
long-living evergreen woody plants, e.g. conifers [20], ex-
hibits much more complex symptomatology and may take
weeks to express injury. Thus, the best way to understand
cold tolerance in this plant and to aid in the selection of
hardy genotypes is to identify the biochemical markers that
are specifically involved in the sensing mechanism of cold
shock and in cold-acclimation.
It has been hypothesised that the cold-induced increase
in cytosolic calcium concentration ([Ca2+ ]c ) constitutes the
primary sensing mechanism for low temperatures [21]. Cal-
cium (Ca2+ ) is a second messenger used in many signalling
processes, and changes in [Ca2+ ]c are necessary for adap-
tive responses to stress [22]. Calcium pumps located at the
plasma membrane and at the intracellular membranes main-
tain the [Ca2+ ]c at around 100200 nM by translocating
Ca2+ into the external space and the internal pools. Upon
stimulation, an influx of Ca2+ into the cytosol occurs via
Ca2+ -selective ion channels either in the plasma membrane
or in organelles [23]. Only a small fraction of apoplastic
Ca2+ is free and available for influx into the cytosol [24].
By contrast, the intracellular Ca2+ stores (in particular, the
vacuole) constitute a plentiful reserve and a reliable source
of Ca2+ for the transient elevation in the cytosol [25].
In Arabidopsis, tobacco, tomato and Physcomitrella, tran-
sient elevations in [Ca2+ ]c have been detected in response
to low temperatures [2628]. In Arabidopsis, the magnitude
of the [Ca2+ ]c peak during cold shock has been shown to be
a function of the cooling rate (i.e. the ratio of temperature
decrease to the time necessary for this decrease to occur,
T/dt) [29]. In the herbaceous model plants, [Ca2+ ]c has
been shown to increase in response to not only rapid cold
shocks but also gradual reductions in temperature, indicat-
ing that Ca2+ is also necessary for mediating the cellular
changes associated with cold acclimation ([30], and refer-
ences therein). In alfalfa, it has been proposed that Ca2+ sig-
nalling during cold shock is independent of the genotypes
capacity for cold acclimation, in contrast with Ca2+ sig-
nalling along the pathway of cold acclimation, which is
genotype-dependent and leads to cold-acclimation-specific
(cas) gene expression [31]. In woody plants, there is little
information on changes in [Ca2+ ]c related to cold shock and
acclimation. It is known that the application of a Ca2+ fer-
tiliser increases freezing tolerance, at least in white poplar
and hornbeam [32]. In apple and pear, the exogenous appli-
cation of Ca2+ increases Ca2+ concentrations in leaves and
fruit and improves cold hardiness [33]. In conifers, a pre-
disposition to winter injury because of a low level of Ca2+
in the needles has been suggested [34].
In olive tree, younger leaves are more susceptible to cold
than older leaves, and leaf Ca2+ greatly increases with age
[35]. Furthermore, in olive tree, the requirement for exoge-
nous Ca2+ peaks in the period from the end of the summer
to late autumn, depending on the cultivar [35]. Nonethe-
less, in olive tree, two key issues regarding the possible role
of Ca2+ during low-temperature signalling and acclimation
need to be resolved. Specifically, it remains to be deter-
mined whether genotypes that differ in terms of cold sus-
ceptibility undergo different Ca2+ signalling in response to
the same cold shock(s), and whether low-temperature Ca2+
signalling changes as a result of cold acclimation, and how,
in the various genotypes. To this end, changes in [Ca2+ ]c
were evaluated in non-cold-acclimated and cold-acclimated
leaf protoplasts of cultivars known to differ in terms of cold
susceptibility which were exposed to cold shocks of vari-
ous amplitude. The results showed that olive tree leaf proto-
plasts respond to rapid temperature decreases with transient
increases in [Ca2+ ]c which involve both an efflux of the ion
from the organelles and an influx through the plasma mem-
brane. In the acclimated protoplasts, Ca2+ transients were
reduced, or inhibited, depending on the cold susceptibility
of the cultivar. The possible role of Ca2+ in mediating the
cellular changes associated with cold acclimation in trees
lacking dormancy and the use of Ca2+ signalling as an early
biochemical marker of genotype selection for cold hardiness
are discussed.
2. Materials and methods
2.1. Protoplast isolation and acclimation
Protoplasts of O. europaea L. cv. Leccino, Frantoio, and
Moraiolo were obtained from leaves randomly collected in
 S. DAngeli et al. / Plant Science 165 (2003) 13031313
the late summer (i.e. before the onset of decreases in tem-
perature in the field) during two successive years. The leaves
were collected from the fourth node of the apex of the
youngest twigs (i.e. those that had not experienced winter
cold), and were excised from different trees grown in the
same field in northern Lazio (Italy) (an area bordering Tus-
cany), cut in thin sections, excluding the midrib region, and
incubated in an enzymatic solution (10%, w/v) for 16 h un-
der darkness (23 C) with orbital shaking at 100 rpm. Pro-
toplasts were used instead of whole cells so as to exclude
the possible interference of the large fraction of apoplastic
Ca2+ bound to the cell wall [24].
The enzymatic solution was composed of Macerozyme
R-10 (Yakult Honsha, Japan) 1% (w/v), Cellulase (C1184,
Sigma) 0.5%, Pectinase (P2401, Sigma) 0.5% (w/v), Hemi-
cellulase (H7649, Sigma) 0.7% (w/v), Pectolyase (P3026,
Sigma) 0.05% (w/v), and mannitol (Sigma) 0.6 M, dissolved
in ultra-pure water (Milli-Q). The cellular suspension was
filtered through a nylon grid (200 m) before use. After
protoplasts were isolated, their viability was determined by
staining with fluorescein diacetate, according to Harrison
and Harrison [36], and it was verified that 90% of the pro-
toplasts were still alive.
Protoplasts were incubated in the same enzymatic solution
of the extraction. Protoplast suspensions were acclimated
using standard procedure, that is, through incubation for 3 h
at 4 C.
To allow for fixing of the protoplasts, the slides for the
microscope analysis were rinsed in HCl (37%) for 10 min,
washed in ultra-pure water, and dried. Polylysin (0.1%, w/v)
was scattered on the slide surface, with a cover-glass, and
the slide was again dried and washed with ultra-pure water.
The slides were used soon after the preparation.
2.2. Histology
Thirty leaves per cultivar, collected as above, were fixed
in 70% ethanol, dehydrated, embedded in paraffin (melt-
ing point: 5658 C, Carlo Erba), and sectioned at 8 m in-
tervals with a HM 350 SV automatic microtome (Microm,
Germany). The sections were washed with 70% ethanol,
stained with toluidine blue for 3 min, and mounted with Eu-
kitt (Kindler, Germany).
The anatomical features of the leaf were evaluated away
from the midrib (i.e. in the same region from which the pro-
toplasts were extracted). The histological images were ac-
quired by means of a Zeiss Axiolab microscope, equipped
with a JVC TK1280E video camera (JVC, Japan), and anal-
ysed with Optilab 2.6 (Graftek, France), as image analysis
software. The values were statistically compared (2 - and
Students t-test), with similar results for the 2 years of the
study period.
2.3. Fluorescence measurements
Considering that micro-injection of the dextran-coupled
or free-acid versions of the fluorescent Ca2+ indicators,
1305
Indo1 and Calcium Crimson, is virtually impossible (due to
the large vacuole and reduced cytosol in olive tree proto-
plasts), we decided to use the ester version (AM) under the
conditions described by Camacho et al. [37]. Thus, the pro-
toplast solution (1 ml) was incubated with either Indo1-AM
(5 M) or Calcium Crimson-AM (5 M), in the presence
of Pluronic (0.3% (w/v) in dimethylsulfoxide, DMSO) for
30 min, as suggested by the manufacturer (Molecular Probes,
Eugene, OR, USA). Aliquots of 100 l of this solution were
used for measuring [Ca2+ ]c (3 protoplasts per aliquot were
analysed, out of an average of 10 protoplasts).
The cell permeability of both fluorescent indicators was
evaluated by means of an Olympus IX-50 epifluorescence
inverted microscope with a 40 Plan Apo objective. Imag-
ing with the appropriate filter settings (see below) was
performed at 5 s intervals with a 12-bit V-Scan Cool CCD
camera (Photonic Science) at 400 ms exposure, and anal-
ysed with Image Pro-Plus software (Media Cybernetics).
The variations in [Ca2+ ]c caused by cold shocks of 20 and
10 C decreases (T) were evaluated by means of a Zeiss
Axiolab epifluorescent microscope (40 achrostigmat ob-
jective, 0.65 NA), equipped with a COHU 4720 video cam-
era (COHU Electronic Division, CA, USA). An excitation
filter (BP) of 510560 nm, a beamsplitter (FT) of 580 nm,
and an emission (LP) filter of 590 nm were used for the de-
tection of Calcium Crimson-AM, and a BP of 340380 nm,
an FT of 400 nm, and an LP of 430 nm for Indo1-AM de-
tection. Using Optilab 2.6 software (Graftek, France), the
Ca2+ fluorescence signal was quantified and expressed as
average pixel intensity.
The variations in [Ca2+ ]c caused by cold shocks of 4.5,
2.5 and 1.5 C (T) were evaluated by means of a laser
scanning confocal microscope (MRC-600, Bio-Rad Micro-
science Ltd., UK), equipped with a Kr:Ar laser, and en-
hanced photomultiplier tubes. A 1% laser intensity was used
to excite Calcium Crimson-AM. Images were collected with
a zoom factor of 2.0 and in the F2 mode using an Olym-
pus 40 Plan Apo objective. The Ca2+ fluorescence signal
was quantified in terms of average pixel intensity by using
COMOS/MPL software (Bio-Rad).
Variations in the average pixel intensity were statistically
compared using Students t-test. Increases in the Ca2+ flu-
orescence signal with respect to the initial value were ex-
pressed as percentage increase.
2.4. Cold shocks and temperature measurements
For the experiments on non-acclimated protoplasts, only
cv. Frantoio was used; this cultivar was chosen because
it does not represent either of the extremes in terms of
cold-hardiness (i.e. it is semi-hardy). The non-acclimated
protoplasts (aliquots of 100 l solution) were exposed to
cold shocks of various amplitudes (i.e. T of 20, 10, 4.5,
2.5 and 1.5 C, starting from room temperature). The cold
shocks were expressed as cooling rates (T/dt), in accor-
dance with Plieth et al. [29].
1306 S. DAngeli et al. / Plant Science 165 (2003) 13031313
To obtain a T of 20 C within 150 s (T/dt =
20 C/150 s) an ice spray (Pharmamed) was used, directing
the spurt for 3 s directly on the lower surface of the slide.
To obtain a 10 C (T) within 90 s (T/dt = 10 C/90 s), a
piece of ice of 10 cm3 was placed in contact with the lower
surface of the slide for 60 s. To obtain a T/dt of 4.5 C/60 s,
2.5 C/50 s, and 1.5 C/40 s, ice was placed near the lower
surface of the slide for 50, 20 and 10 s, respectively.
The variations in temperature during the cold application
were measured at 10 s intervals until the desired tempera-
ture was reached. Temperature was measured by immersing
the tip of the thermometer probe (range 200.0 to 760.0 C)
(Hanna Instruments 98804) in the protoplast solution, which
had been placed on the slide without the cover-glass. Tem-
perature measurements were repeated five times for each
cold shock experiment, with similar results.
For the non-acclimated protoplasts, repeated cold shock
was performed by applying the shock three times, each after
the protoplast solution had returned to room temperature (i.e.
Fig. 1. Variations in [Ca2+ ]c over time, expressed as pixel intensity (A.U., arbitrary units), in a cv. Frantoio protoplast incubated with either Indo1-AM
(a) or Calcium Crimson-AM (b) and the ionophore A23187 at room temperature.
after 35 min). The repeated cold shock consisted of T of
10, 4.5 and 2.5 C, within 90, 60 and 50 s, respectively. For
the acclimated protoplasts, cold shock was applied either
twice (T of 10 C) or three times (T of 2.5 C).
2.5. Ionophore and inhibitor applications
To evaluate the cell permeability of the fluorescent indi-
cators, the Ca2+ ionophore A23187 (Sigma) was added at
a concentration of 50 M to the protoplast solution soon
after incubation with the fluorescent indicator. To highlight
the role of Ca2+ efflux from the organelles, the protoplasts
were incubated with the organelle Ca2+ channel blocker
8-(N,N-di-methylamino)octyl 3,4,5-trimethoxy-benzoate
(TMB-8, Sigma) at a final concentration of 5 M. To high-
light the role of Ca2+ influx through the plasma membrane,
the plasma membrane Ca2+ channel blocker, gadolinium
(Gd3+ ) (gadolinium-chloride, Sigma) was added at a final
concentration of 20 M.
 S. DAngeli et al. / Plant Science 165 (2003) 13031313 1307

All of the experiments described in Section 2, for both
acclimated and non-acclimated protoplasts, were repeated
at least 15 times (i.e. using 45 protoplasts per experiment
and cultivar). The changes in the Ca2+ fluorescence signal
were very similar in the 2 years of the study. The data pre-
sented in the figures refer to the protoplasts whose response
was representative of the mean [Ca2+ ]c percentage variation
(S.E.) (data from the second year).
3. Results
not show significant variations during the observation time,
which lasted about 7 min (Fig. 1a and b), indicating that
there were no significant losses in the fluorescence signal
due to sequestration and/or dye leakage. To control the
responsiveness of the loaded dyes, the protoplasts were in-
cubated with the Ca2+ ionophore A23187 (Fig. 1a and b),
which caused the [Ca2+ ]c to increase on average by 18%
for Indo1 and 19% for Calcium Crimson (Fig. 1a and
b), and the amplitude of this increase did not significantly
change with the duration of incubation (from 30 s to 2 min)
(Fig. 1a).

3.1. Stability of fluorescence signal 3.2. Response of non-acclimated protoplasts to

The leaf protoplasts were first checked for autofluores-
cence using the fluorescence settings for imaging Calcium
Crimson and Indo1. No autofluorescence was visible, which
indicates that there was no cell-wall residue (data not
shown). The stability of the fluorescence signal was then
evaluated at room temperature on cv. Frantoio protoplasts.
The fluorescent signal was evident in the cytosol and did
cold shocks
The results of the experiments on non-acclimated proto-
plasts, which were carried out on cv. Frantoio only, showed
that the most severe cold shock (i.e. T/dt = 20 C/150 s;
decrease of 0.133 C/s) caused the Ca2+ signal to peak after
30 s, showing a sharp increase with respect to the initial
fluorescence (i.e. a mean percentage increase of 53% (1.4)

Fig. 2. Variations in [Ca2+ ]c over time, expressed as pixel intensity (A.U., arbitrary units), in a cv. Frantoio protoplast incubated with either Calcium
Crimson-AM (a) or Indo1-AM (b), after a cold shock of T/dt = 20 C/150 s; the peak [Ca2+ ]c represents a 54% (a) and 60% (b) increase with respect
to the initial value.
1308 S. DAngeli et al. / Plant Science 165 (2003) 13031313

when detected with Calcium Crimson and of 60% (1.2)
when detected with Indo1); within 23 min, the signal de-
creased to its initial value, as exemplified in Fig. 2a and b.
The Ca2+ signal after a cold shock of T/dt = 10 C/90 s
(decrease of 0.111 C/s) also peaked after 30 s showing
mean increases that were very similar to those following the
more severe cold shock (Fig. 3a and b).
Given that the use of the two fluorescence indicators pro-
vided similar results in the above experiments, only Calcium
Crimson was used when performing the milder cold shocks;
this fluorochrome was chosen because its spectrum is com-
patible with the laser scanning confocal microscope used in
this study (see Section 2). When inducing a cold shock of
T/dt = 4.5 C/60 s (i.e. decrease of 0.075 C/s), the Ca2+
signal peaked after 10 s, with a mean increase of 43%
(1.8) compared to the initial fluorescence; this increase
was not significantly different from the increases observed
with the more severe cold shocks (Fig. 4a). When inducing a
cold shock of T/dt = 2.5 C/50 s (i.e. 0.05 C/s), the Ca2+
signal peaked after 40 s, with a mean increase of 10%
(0.4) (Fig. 4b), which was much lower than the increase
observed after the more severe cold shocks (Figs. 2a and b,
3a and b, and 4a). A cold shock of T/dt = 1.5 C/40 s (i.e.
0.037 C/s) caused a non-significant increase (5% or less)
after 10 s (data not shown).
For the protoplasts that had been incubated with TMB-8,
the peak signal after a mild cold shock (i.e. T/dt =
2.5 C/50 s) was significantly lower than that observed for
the protoplasts that had not been incubated (Fig. 5a, com-
pared to Fig. 4b). Similarly, the peak signal of the protoplasts
that had been incubated with Gd3+ was significantly lower
than that observed in the absence of this blocker (Fig. 5b).
With regard to repeated cold shocks, when inducing three
successive shocks of T/dt = 10 C/90 s at 180 s intervals,
a conspicuous and very similar increase in [Ca2+ ]c occurred
after each shock, with the [Ca2+ ]c returning to the initial
value after each shock (Fig 6a). When inducing repeated cold
shocks of T/dt = 4.5 C/60 s, [Ca2+ ]c increased by 39%
(1.2%) by average after the second shock, yet it did not
return to the initial value, even after prolonging the interval
between this shock and the third shock to 300 s. After the
third shock, [Ca2+ ]c increased by 18% (0.9%), by average,
with respect to the lowest value reached after the second
shock (Fig. 6b).
3.3. Response of acclimated protoplasts to repeated
cold shocks
The finding that the amplitude of the Ca2+ signal in the
non-acclimated protoplasts gradually decreased with the

Fig. 3. (a) Variations in [Ca2+ ]c over time, expressed as pixel intensity (A.U., arbitrary units), in a cv. Frantoio protoplast incubated with Calcium
Crimson-AM, after a cold shock of T/dt = 10 C/90 s; the peak [Ca2+ ]c represents a 53% increase with respect to the initial value. (b) Epifluorescence
image showing variations in the Ca2+ signal over time in a cv. Frantoio protoplast incubated with Indo1-AM after a cold shock of T/dt = 10 C/90 s;
the most intense fluorescence corresponds to a 59% increase with respect to the initial value (bar = 10 m).
 S. DAngeli et al. / Plant Science 165 (2003) 13031313 1309

Fig. 4. Variations in [Ca2+ ]c over time, expressed as pixel intensity (A.U., arbitrary units), in a cv. Frantoio protoplast incubated with Calcium Crimson-AM,
after a cold shock of T/dt = 4.5 C/60 s (a) or T/dt = 2.5 C/50 s (b); the peak [Ca2+ ]c represents a 43% (a) and 10% (b) increase with respect to
the initial value.

repetition of cold shocks of T/dt = 4.5 C/60 s may indi-
cate that an acclimation process had begun. This led us to
perform repeated cold shocks on protoplasts of cultivars that
differed in terms of cold hardiness (i.e. Leccino, Moraiolo,
and Frantoio) after undergoing a standard acclimation pro-
cedure, as described in the Section 2. Before the cold shock
was induced, leaves that were less than 1-year-old were his-
tologically examined for anatomical differences among the
three cultivars, in the attempt to identify cultivar-specific
features that could be related to cold hardiness. The only
difference found was that the leaves of cv. Frantoio were
thinner than those of the other cultivars (data not shown).
When T/dt = 10 C/90 s was repeated on cold-accli-
mated protoplasts of cv. Frantoio (semi-hardy), the Ca2+
signal increased after each shock by 23% by average (i.e.
23 1.7% after the first shock, and 24 0.9%, after the
second shock, Fig. 7a), yet this increase was only about half
that of the non-acclimated protoplasts (Fig. 7a, in compar-
ison with Fig. 6a). When the same T/dt was applied to
acclimated protoplasts of the other cultivars, differences in
Ca2+ signalling were recorded. Specifically, the acclimated
protoplasts of the hardy cv. Leccino showed only a 10%
(0.6%) increase after the first shock and no increase after
the second shock, whereas the protoplasts of the not-hardy
cv. Moraiolo showed a consistent increase of similar magni-
tude after each shock (291.1 and 290.9%, Fig. 7a). When
a T/dt = 2.5 C/50 s was induced three times, the proto-
plasts of cv. Leccino showed no increase after each shock;
those of cv. Frantoio showed no increase after the first two
shocks and an increase of 11% (0.8%) after the third shock
(Fig. 7b), whereas the protoplasts of cv. Moraiolo showed
an increase after each shock, ranging from 10 to 15% (i.e.
10 0.7, 12 0.9, and 14 0.7%, respectively, Fig 7b).
4. Discussion
The results of this study shows that the non-acclimated
leaf protoplasts of olive tree respond to rapid temperature
decreases with transient increases in [Ca2+ ]c , which involve
1310 S. DAngeli et al. / Plant Science 165 (2003) 13031313

Fig. 5. Variations in [Ca2+ ]c over time, expressed as pixel intensity (A.U., arbitrary units), in a cv. Frantoio protoplast incubated with Calcium Crimson-AM
and TMB-8 (a) or Gd3+ (b), subjected to a cold shock of T/dt = 2.5 C/50 s; the peak [Ca2+ ]c represents a 6% (a) and 5% (b) increase with respect
to the initial value.

both an efflux of the ion from the organelles and an influx
through the plasma membrane. A decrease of 0.075 C/s
was sufficient to cause an increase in [Ca2+ ]c that was
comparable to those caused by more drastic cooling rates,
whereas the response induced with a cooling rate that was
only slightly milder (i.e. 0.05 C/s) was greatly reduced.
This latter cooling rate seems to be the threshold cooling
rate, given that no significant increase in the Ca2+ signal
was observed after subjecting the protoplasts to a milder
cooling rate. Moreover, the cold-induced changes in [Ca2+ ]c
were altered with acclimation, and in a genotype-dependent
manner.
This is the first study to document the involvement of
Ca2+ signalling in the cold response/resistance of a woody
species, in particular, an evergreen species, in which the
lack of a true dormancy makes the plant vulnerable to win-
ter cold, resulting in serious damage [7]. Furthermore, most
of the studies that have demonstrated a transient increase in
[Ca2+ ]c in herbaceous species have adopted relatively severe
cold shocks (i.e. several degree Celsius within less than 1 s)
(see references in [29]). We chose to simulate the less
severe cooling rates to which plants are generally exposed
in nature (i.e. decreases of fractions of a degree Celsius per
second), and the results were similar to those of a study con-
ducted on the herbaceous model plant Arabidopsis thaliana
[29], although the system used was very different from ours
(i.e. intact roots of plants expressing the Ca2+ -indicator
aequorin). Specifically, both studies show that there exists a
threshold cooling rate below which no significant increase
in [Ca2+ ]c occurs and that repeated mild cold shocks atten-
uate the [Ca2+ ]c response to subsequent shocks. In addition,
in the present paper, it was shown that the interval of time
between successive cold shocks is also important. In fact, a
repeated shock of T/dt = 10 C/90 s in non-acclimated cv.
Frantoio protoplasts resulted in a similar increase in [Ca2+ ]c
after each shock (applied at 180 s intervals), indicating that
the protoplast sensed each severe shock as a separate stress,
whereas when a less severe shock (i.e. T/dt = 4.5 C/60 s)
was applied, the amplitude of the Ca2+ peak progressively
decreased after each shock, even when prolonging the in-
terval to 300 s. Furthermore, the results of the experiments
conducted on protoplasts subjected to standard acclimation
 S. DAngeli et al. / Plant Science 165 (2003) 13031313 1311

Fig. 6. (a) Variations in [Ca2+ ]c over time, expressed as pixel intensity (A.U., arbitrary units), in a cv. Frantoio protoplast incubated with Calcium
Crimson-AM and subjected to repeated cold shocks of T/dt = 10 C/90 s; the peak [Ca2+ ]c represents an increase of 45% (first shock), 48% (second
shock), and 46% (third shock) with respect to the initial value of each shock. (b) Variations in [Ca2+ ]c over time, expressed as pixel intensity (A.U.,
arbitrary units), in a cv. Frantoio protoplast incubated with Calcium Crimson-AM and subjected to repeated cold shocks of T/dt = 4.5 C/60 s;
the peak [Ca2+ ]c represents an increase of 48% (first shock), 39% (second shock), and 18% (third shock) with respect to the initial value of
each shock.

showed that the specific genotype plays a determining role
in the acquisition of a persistent cold memory. The not-hardy
cultivar is unable to acquire true acclimation, i.e. a persis-
tent cold memory, because unable to block the increase in
[Ca2+ ]c . Instead, the acclimation process is effective in the
other cultivars and correlates with a partial (semi-hardy cv.)
or total (hardy cv.) block of [Ca2+ ]c changes.
Concerning the source of the Ca2+ signal, the finding
that the use of Gd3+ and TMB-8 resulted in significant
reductions in the peak of the signal, even after exposure
to mild cold shock, suggests that both an influx of Ca2+
through the plasma membrane and an efflux from the or-
ganelles into the cytosol are involved. These findings are
consistent with suggestions that the influx of extracellular
Ca2+ into the cytosol is responsible for a substantial part of
the cold-induced increase in [Ca2+ ]c ([23], and references
therein). Previous studies have also suggested that the efflux
from intracellular stores is involved. In fact, in Brassica
napus, the activation of the cold-inducible BN115 gene re-
quires both the influx of Ca2+ from the apoplast and the
efflux from the intracellular stores [38]. In Arabidopsis, tar-
geting the Ca2+ -dependent aequorin to the cytosolic face of
the vacuolar membrane, a cold-induced efflux of Ca2+ from
the vacuole has been demonstrated [27]. We were not able
to determine whether or not, in olive tree protoplasts, the
Ca2+ efflux originates in the vacuole. TMB-8 specifically
acts on the InsP3 receptor/Ca2+ -channel complexes, which,
however, are located on both vacuolar and non-vacuolar
membranes [25,39].
Monroy and Dhindsa [31] hypothesised that the
cold-induced changes in membrane fluidity (rigidification),
the activation of Ca2+ channels, and the increase of Ca2+
in the cytosol are independent of the genotypes capacity
to acclimate, whereas further events, induced by an in-
crease in [Ca2+ ]c , are genotype-dependent and essential
for developing a cold tolerance. Our finding that, in the
1312 S. DAngeli et al. / Plant Science 165 (2003) 13031313

Fig. 7. (a) Variations in [Ca2+ ]c over time, expressed as pixel intensity (A.U., arbitrary units), in cv. Moraiolo, cv. Frantoio, and cv. Leccino protoplasts
subjected to standard acclimation, after incubation with Calcium Crimson-AM and exposure to repeated cold shocks of T/dt = 10 C/90 s. The peak
[Ca2+ ]c represents an increase of 29% (first shock) and 29% (second shock) for cv. Moraiolo; of 24 and 25%, respectively, for cv. Frantoio; and of
10% (first shock) for cv. Leccino. (b) Variations in [Ca2+ ]c over time, expressed as pixel intensity (A.U., arbitrary units), in cv. Moraiolo, cv. Frantoio,
and cv. Leccino protoplasts subjected to standard acclimation, after incubation with Calcium Crimson-AM and exposure to repeated cold shocks of
T/dt = 2.5 C/50 s. The peak [Ca2+ ]c represents an increase of 10% (first shock), 11% (second shock), and 13% (third shock) for cv. Moraiolo; and
of 12% (third shock only) for cv. Frantoio; no increase observed for cv. Leccino.

protoplasts exposed to standard acclimation procedure, the Acknowledgements

amplitude of the [Ca2+ ]c signal was inversely related to
the cold-hardiness of the cultivar validates the hypothesis,
demonstrating a strict relationship between Ca2+ signalling
and genotype characteristics for acclimation.
In conclusion, these results suggest that Ca2+ signalling
could be an early biochemical marker of genotype selec-
tion for cold resistance in olive tree, for which no spe-
cific markers have been found to date [7]. It is possible
that these results could be applied to other woody plants
lacking dormancy which, like olive tree, are wild/cultigen
groups of forms/genotypes with differences in cold
susceptibility.
The authors wish to thank Dr. E. Marchetti (Diparti-
mento di Genetica e Biologia Molecolare, Universit La
Sapienza, Rome, Italy) for technical assistance with laser
confocal microscopy. This study was supported by Univer-
sit La Sapienza (Rome, Italy) Progetti Ateneo 2001 (to
M.M.A.) and by PRAL 99/19 project (to M.M.A.).
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