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REVIEW
Physiological and molecular changes in plants grownat low temperatures
Andreas Theocharis • Christophe Clement •
Essaıd Ait Barka
Received: 30 November 2011 / Accepted: 13 March 2012 / Published online: 20 April 2012
� Springer-Verlag 2012
Abstract Apart from water availability, low temperature
is the most important environmental factor limiting the
productivity and geographical distribution of plants across
the world. To cope with cold stress, plant species have
evolved several physiological and molecular adaptations to
maximize cold tolerance by adjusting their metabolism.
The regulation of some gene products represents an addi-
tional mechanism of cold tolerance. A consequence of
these mechanisms is that plants are able to survive expo-
sure to low temperature via a process known as cold
acclimation. In this review, we briefly summarize recent
progress in research and hypotheses on how sensitive
plants perceive cold. We also explore how this perception
is translated into changes within plants following exposure
to low temperatures. Particular emphasis is placed on
physiological parameters as well as transcriptional, post-
transcriptional and post-translational regulation of cold-
induced gene products that occur after exposure to low
temperatures, leading to cold acclimation.
Keywords C-repeat binding factor � Low temperatures �Plant acclimation � Signal perception
Abbreviations
ABA Abscisic acid
CBF C-repeat binding factor
COR Cold-responsive genes
CRT C-repeat elements
DRE Dehydration-responsive elements
DREB Dehydration-responsive element binding
ICE Inducer of CBF expression
LT Low temperature
Introduction
Among various environmental stresses, low temperature
(LT) is one of the most important factors limiting the
productivity and distribution of plants. Low temperatures,
defined as low but not freezing temperatures (0–15 �C), are
common in nature and can damage many plant species. In
order to cope with such conditions, several plant species
have the ability to increase their degree of freezing toler-
ance in response to low, non-freezing temperatures, a
phenomenon known as cold acclimation. It is well estab-
lished that some of the molecular and physiological chan-
ges that occur during cold acclimation are important for
plant cold tolerance (Hsieh et al. 2004; Zhu et al. 2007).
Accordingly, it has been concluded that cold tolerance that
develops in initially insensitive plants is not entirely con-
stitutive and at least some of it is developed during expo-
sure to low temperatures.
This review addresses plant adaptive responses to cold
stress, with a special emphasis on understanding (i) the
key elements involved in cold signal perception and
transduction, (ii) the major physiological and biochemi-
cal changes that occur following cold exposure, (iii)
cold-inducible gene products that help the plant to
accomplish a synergistic response to cold, and finally
(iv) products that may play major roles in cold accli-
mation and tolerance.
A. Theocharis � C. Clement � E. A. Barka (&)
Laboratoire de Stress, Defense et Reproduction des Plantes,
URVVC, UPRES EA 2069, Universite de Reims
Champagne-Ardenne, BP 1039, 51687 Reims Cedex 2, France
e-mail: [email protected]
123
Planta (2012) 235:1091–1105
DOI 10.1007/s00425-012-1641-y
How do plants perceive cold?
As a result of exposure to low temperatures, many
physiological and biochemical cell functions have been
correlated with visible symptoms (wilting, chlorosis, or
necrosis) (Ruelland and Zachowski 2010). Often, these
adverse effects are accompanied by changes in cell mem-
brane structure and lipid composition (Uemura and
Steponkus 1999; Matteucci et al. 2011), cellular leakage of
electrolytes and amino acids, a diversion of electron flow to
alternate pathways (Seo et al. 2010), alterations in proto-
plasmic streaming and redistribution of intracellular cal-
cium ions (Knight et al. 1998) (Fig. 1). They also involve
changes in protein content and enzyme activities (Ruelland
and Zachowski 2010) as well as ultrastructural changes in a
wide range of cell components, including plastids, thyla-
koid membranes and the phosphorylation of thylakoid
proteins, and mitochondria (Zhang et al. 2011). Brief
exposures to low temperatures may only cause transitory
changes, and plants generally survive. However, prolonged
exposure to stress causes plant necrosis or death.
To overcome stresses generated by exposure to low
non-freezing temperatures, plants can trigger a cascade of
events that cause changes in gene expression and thus
induce biochemical and physiological modifications that
enhance their tolerance (Smallwood and Bowles 2002; Zhu
et al. 2007). This phenomenon is known as chilling or cold
acclimation.
Mechanisms of acclimation to low non-freezing
temperatures
The primary mechanisms involved in cold acclimation are
related to a number of processes discussed below. These
include molecular and physiological modifications occur-
ring in plant membranes, the accumulation of cytosolic
Ca2?, increased levels of ROS and the activation of ROS
scavenger systems, changes in the expression of cold-
related genes and transcription factors, alterations in
protein and sugar synthesis, proline accumulation, and
biochemical changes that affect photosynthesis (Fig. 2).
Modifications to plant cell membranes
Membranes are a primary site of cold-induced injury
(Fig. 1). Several studies have demonstrated that membrane
rigidification, coupled with cytoskeletal rearrangements,
calcium influxes, and the activation of MAPK cascades,
triggers LT responses (Uemura and Steponkus 1999; Orvar
et al. 2000; Xin and Browse 2000; Sangwan et al. 2002).
The lipid composition of the plasma membrane and chlo-
roplast envelopes in acclimated plants changes such that
the threshold temperature for membrane damage is lowered
relative to that for non-acclimated plants (Uemura and
Steponkus 1999). This is achieved by increasing the cold-
adapted membranes’ unsaturated fatty acid content, which
makes them more fluid (Vogg et al. 1998). The process of
Transition phase
Liquid-Crystalline Solid gel
‘’FLUID’’ ‘’SOLID’’
Solute leakage and disrupted ion balance
Injury and death of cells and tissues
Enhanced permeability
Increased activation of energy-bound enzymes
Cessation of protoplasmic streaming
Imbalance in metabolism
Accumulation of toxic metabolites
Reduced ATP supply
Return to normal metabolism
Brief exposure and return to 20 C
Prolonged exposure
Survival
Chilling stress
Cell membranes
°
Fig. 1 A model to explain
symptoms of chilling injury in
chilling-sensitive plants.
Membranes are the primary site
of cold-induced injury, leading
to a cascade of cellular
processes with adverse effects
on the plant. When exposure to
low temperature is brief, the
effects may be transitory and
plants survive. However, the
plant will exhibit necrosis or die
if exposure is maintained
(Lyons 1973; Raison and Lyons
1986)
1092 Planta (2012) 235:1091–1105
123
cold acclimation promotes the stabilization of membranes,
which prevents damage leading to cell death. The accli-
mation process also activates mechanisms that protect
membrane fluidity by ensuring the optimal activity of
associated enzymes (Matteucci et al. 2011).
Photosynthesis and photosynthesis-related pigments
At the physiological level, photosynthesis is strongly affec-
ted by exposure to cold. The cessation of growth resulting
from cold stress reduces the capacity for energy utilization,
causing feedback inhibition of photosynthesis (Ruelland and
Zachowski 2010). In cold-acclimated winter annuals, pho-
tosynthetic activity is maintained by increases in the abun-
dance and activity of several Calvin cycle enzymes (Goulas
et al. 2006). This recovery is associated with elevated levels
of thylakoid plastoquinone A and a concomitant rise in the
apparent size of the intersystem electron donor pool to PSI
(Baena-Gonzalez et al. 2001). Consequently, non-photo-
chemical quenching increases in cold-stressed leaves in
parallel with increased zeaxanthin levels to compensate for
the reduced electron consumption by photosynthesis. Zea-
xanthin protects the PSII reaction centres from over-excita-
tion (Krol et al. 1999). Nevertheless, Ruelland and
Zachowski (2010) reported that energy dissipation via non-
photochemical quenching (NPQ) and electron transport was
not only enhanced following cold acclimation but also con-
tributed to protection from oxidative damage.
Xanthophylls
Although they are not considered photosynthetic pigments
per se, the xanthophylls (notably, violaxanthin, anthera-
xanthin, and zeaxanthin) help in protecting the photosystems
and their abundance increases at low temperatures (Ivanov
et al. 2006). Xanthophylls have structural roles and act as
natural antioxidants, quenching triplet Chl and singlet oxy-
gen, which are potentially harmful to the chloroplast
(Passarini et al. 2009; Han et al. 2010). It has also been
postulated that unbound zeaxanthin and other carotenoids
may also stabilize thylakoid membranes against putative
peroxidative damage and heat stress (Laugier et al. 2010).
Flavonoids
These accumulate in leaves and stems in response to low
temperatures. They are synthesized via the phenylpropanoid
pathway, which is controlled by key enzymes, including
phenylalanine ammonia-lyase and chalcone synthase
(Sharma et al. 2007). Recently, it has been reported that cold
stress induces transcriptomic modifications that increase
flavonoid biosynthesis, including reactions involved in
anthocyanin biosynthesis and the metabolic pathways that
supply it (Crifo et al. 2011).
Calcium and cold temperatures
Calcium acts as a mediator of stimulus–response coupling
in the regulation of plant growth, development, and
responses to environmental stimuli (Sanders et al. 2002; Du
and Poovaiah 2005). Cold stress-induced rigidification of
plasma membrane microdomains can cause actin cyto-
skeletal rearrangement. This may be followed by the acti-
vation of Ca2? channels and increased cytosolic Ca2?
levels (Fig. 3), which may be involved in the cold accli-
mation process (Sangwan et al. 2001; Catala et al. 2003).
The Ca2? released from internal cellular reserves, mediated
by inositol triphosphate, is upstream of the expression of
Accumulation of [Ca2+cyt]
Accumulation of ROS and activation of scavenge system
Modification in plant membranes
Photosynthetic acclimation
Accumulation of cryoprotectantssugars, proline, ...
Changes in gene expression and protein synthesis
Change in lipid composition
Increase in desaturated fatty acids
Increased fluidity of membranes
Reduction of lower threshold temperature in acclimated plants
ColdAcclimation
Fig. 2 Different cellular
processes induced as a
consequence of plant
acclimation to cold
Planta (2012) 235:1091–1105 1093
123
CBFs (C-repeat binding factors) and COR (cold respon-
sive) genes in the cold-signalling pathway(s) (Chinnusamy
et al. 2007, 2010). Recently, Doherty et al. (2009) provided
more evidence for a link between calcium signalling and
cold induction of the CBF pathway, showing that cal-
modulin binding transcription activator (CAMTA) factors
bind to a regulatory element in the CBF2 gene promoter.
As the CAMTA proteins are calmodulin binding tran-
scription factors, they may act directly in the transduction
of LT-induced cytosolic calcium signals into downstream
regulation of gene expression (Doherty et al. 2009). Sim-
ilarly, CRLK1, a novel calcium/calmodulin-regulated
receptor-like kinase, was reported to be crucial for cold
tolerance in plants (Yang et al. 2010).
Role of reactive oxygen species in acclimation
to low temperatures
The role of ROS in abiotic stress management has become
a subject of considerable research interest, particularly
since ROS have been reported to be involved in processes
leading to plant stress acclimation (Suzuki et al. 2011).
This finding indicates that ROS are not simply toxic
by-products of metabolism, but act as signalling molecules
that modulate the expression of various genes, including
those encoding antioxidant enzymes and modulators of
H2O2 production (Neill et al. 2002; Gechev et al. 2003;
Suzuki et al. 2011) (Fig. 3). In addition, LT stress has been
reported to cause significant increases in the levels of the
soluble non-enzymatic antioxidants ascorbate and gluta-
thione, as well as the activity of the main NADPH-gener-
ating dehydrogenases (Airaki et al. 2011).
Cold-mediated transcription regulation
Plant acclimation to LT depends on changes in the
expression of specific genes encoding products that confer
increased cold tolerance (Gilmour et al. 1998; Doherty
et al. 2009) (Fig. 4). The process involves modifying pre-
existing proteins and up- or down-regulating gene expres-
sion and protein synthesis. Several studies have suggested
that activity of cold/chilling-induced genes may facilitate
the metabolic changes that confer LT tolerance (Gilmour
et al. 1998; Doherty et al. 2009). They may also be
involved in the signal transduction of the stress-response
(Ingram and Bartels 1996; Thomashow 2010). HOS15, one
of 237 predicted WD40-repeat proteins in Arabidopsis,
functions as a repressor that modifies chromatin and
thereby controls the expression of genes involved in cold
tolerance (Zhu et al. 2008). In addition, HOS1 is a RING-
type ubiquitin E3 ligase that mediates the ubiquitination
and proteosomal degradation of ICE1 and thus negatively
regulates CBF regulons. The constitutive HOS9 and
HOS10 regulons have a role in the negative regulation of
CBF-target genes (Fig. 4).
The CRT/DREB1 regulatory pathway
A wide variety of COR genes have been isolated from cold-
acclimated plants (Svensson et al. 2006). The cloned
genes’ products can be classified as (i) proteins that protect
cells against environmental cold/chilling stress, and (ii)
proteins that regulate gene expression during the adaptation
response (Fowler and Thomashow 2002). Further classifi-
cation divides the gene products into (i) mediators of
Inactivation of enzymes
Lipid peroxidation
Protein degradation
Damage to DNA
PS I, II
Plasma membranes
Cessation of cytoplasm streaming
Inhibition of ethylene biosynthesis
Activation or repression of phosphatase activity
Changes in cytoplasm viscosity
Inhibition of photosynthesis
Induction of cold related genes (COR )
Elevation of proline andtotal soluble sugars contents
Chilling stress
ROS
[Ca2+cyt]
Fig. 3 Ca2? and reactive
oxygen species (ROS) responses
to chilling in sensitive plants.
ROS are not simply toxic
by-products of metabolism; they
also act as signalling molecules
by modulating the expression of
various genes, including those
encoding antioxidant enzymes
and modulators of H2O2
production. Changes to the
plasma membrane can cause
actin cytoskeletal
rearrangements that may be
followed by the activation of
Ca2? channels and increased
cytosolic Ca2? concentrations,
triggering a series of reactions
such as the expression of
cold-regulated genes
1094 Planta (2012) 235:1091–1105
123
biochemical and physiological changes required for growth
and development at low temperatures and (ii) gene prod-
ucts with a direct role in chilling and freezing tolerance
(Thomashow 2010). A transcriptome analysis of resistant
Arabidopsis treated at 13 �C indicated that 20 % of about
8,000 genes were affected by treatment, particularly those
involved in protein biosynthesis (Provart et al. 2003;
Chinnusamy et al. 2010). The mRNA profiles for the
chilling-lethal mutants were very similar and included
extensive chilling-induced and mutant-specific alterations
in gene expression. The expression pattern of the mutants
upon chilling suggests that the normal function of the
mutated loci is to prevent a wide-ranging damaging effect
of LT on transcriptional regulation (Provart et al. 2003).
Role of CBF transcription factors in cold acclimation
The signal transduction pathways that control COR
expression incorporate a regulatory network (Fig. 4) in
which a few regulatory genes control those involved in the
cold response (Fowler and Thomashow 2002; Yamaguchi-
Shinozaki and Shinozaki 2006; Chinnusamy et al. 2010;
Thomashow 2010). Attempts to isolate the regulatory ele-
ments responsible for the initiation of the COR gene tran-
scription under LT have primarily focused on Arabidopsis.
The ability to express all of the COR genes in concert at
warm temperatures was described following the discovery
of the CBF family of transcriptional activators (Gilmour
et al. 2004; Skinner et al. 2005; Chinnusamy et al. 2010),
which are also known as DREs (drought responsive ele-
ments) or LTREs (low temperature responsive elements)
(Shinwari et al. 1998; Shinozaki and Yamaguchi-Shinozaki
2000; Yamaguchi-Shinozaki and Shinozaki 2006). The
CBF1, CBF2, and CBF3 genes follow one-another in
sequence on Arabidopsis chromosome 4 (Shinwari et al.
1998; Gilmour et al. 2004). DRE/LTREs stimulate gene
expression in response to cold, high salinity and drought,
but not in response to exogenously applied abscisic acid
(Shinozaki and Yamaguchi-Shinozaki 2000). Using the
yeast one-hybrid system, the DRE/CRT elements have
been used as bait to isolate DRE/CRT-binding proteins.
The five different DRE binding proteins isolated to date
have been classified into two groups, DREB1 and DREB2
(Liu et al. 1998), which bind specifically to the DRE/CRT
elements and transcriptionally activate the expression of
COR genes. The promoters of CBF/DREB-regulated COR
genes contain a cold- and dehydration-responsive DNA
regulatory element known as CBF/DRE (Shinozaki and
Yamaguchi-Shinozaki 2000).
Overexpression of CBF1 in Arabidopsis has been shown
to activate the expression of the entire battery of known
CBF/DREB-regulated COR genes and to enhance whole
plant freezing survival without a low temperature stimulus
(Jaglo-Ottosen et al. 1998). In Arabidopsis, overexpression
of CBF1 and CBF3 activates COR gene expression and
enhances freezing tolerance (Liu et al. 1998; Maruyama
et al. 2004). Additionally, when overexpressed in trans-
genic Arabidopsis plants, the homolog of the CBF/DREB1
protein CBF4 activates a C-repeat/dehydration-responsive
element containing downstream genes that are involved in
cold acclimation (Haake et al. 2002).
The three CBF genes are cold-induced. Indeed, CBF
transcript levels start to increase within 15 min of exposing
plants to low temperatures, and transcripts from the tar-
geted CBF/DRE-regulated COR genes start to accumulate
within approximately 2 h (Mantyla et al. 1995). The pre-
cise mechanism whereby the CBF genes are activated by
LT does not involve autoregulation (Gilmour et al. 1998,
2004) but is controlled by a set of redundant interacting
transcription factors (Vogel et al. 2005; Chinnusamy et al.
2007, 2010).
Many of these genes are also induced by abscisic acid
(Knight et al. 2004) or by dehydration (Shinozaki and
Yamaguchi-Shinozaki 2000), which is consistent with the
fact that both of these factors can increase freezing toler-
ance in transgenic plants (Jaglo-Ottosen et al. 1998; Liu
et al. 1998). However, the existence of CBF-parallel
pathways involved in cold acclimation has been supported
by transcription profiling of plants overexpressing the three
members of the CBF family (Fowler and Thomashow
2002). On the other hand, the Arabidopsis mutant eskimo1
displays freezing tolerance in the absence of cold treat-
ment, without changes in expression of the components of
the CBF pathway, but with a high level of proline accu-
mulation. This suggests that distinct signalling pathways
activate different aspects of cold acclimation. The activa-
tion of one pathway can result in considerable freezing
tolerance without activation of other pathways (Xin and
Browse 2000).
Subsequent to their discovery in Arabidopsis, many
CBF homologues have been found in both monocotyle-
donous and dicotyledonous species capable of cold accli-
mation, but they are also found in species that are not
(Ruelland et al. 2009). For instance, grapevines have five
CBF/DREB1-like genes (CBFs), CBF1 to CBF4 (Xiao
et al. 2006, 2008) and one undefined CBF-like transcription
factor, CBFL (XM_002270601). CBF expression was
reported to be induced within a few hours of exposure to
low temperatures, particularly CBF4 (Xiao et al. 2008).
More recently, Takuhara et al. (2011) have demonstrated
that LT enhanced the expression of VvCBF2, VvCBF4, and
VvCBFL within 3 h, but not VvCBF1 or VvCBF3 expres-
sion. CBF1, 2 and 3 transcripts also accumulated in
response to drought and treatment with exogenous abscisic
acid (ABA), indicating that grapevines contain unique CBF
genes. The expression of the endogenous Vitis CBF4 genes
Planta (2012) 235:1091–1105 1095
123
was low at ambient temperature but increased on exposure
to LT (4 �C). This expression was maintained for several
days, which is uncommon for CBF genes. Further, in
contrast to the previously described Vitis CBF1-3, vCBF4
was expressed in both young and mature tissue. Altogether,
these results suggest that CBF4 represents a second type of
CBF in grape that might be more important for the over-
wintering of grapevine plants.
This raises the question as to whether the CBF tran-
scription factors are limited to activating the expression
of COR genes encoding cryoprotective polypeptides, or
alternatively have a role in activating multiple components
of the cold acclimation response. When both CBF1 and
CBF3 are shut down, the cold induction of all CBF targets
is lower, suggesting that a basal level of CBF proteins
would be needed for the induction of all CBF targets and
for the activation of cold acclimation in Arabidopsis. This
level would be reached only when the CBF1, CBF2, and
CBF3 genes are properly induced in a co-ordinated fashion
(Novillo et al. 2007). On the other hand, overexpression of
CBF3 in Arabidopsis results in multiple biochemical
changes that ultimately increase the concentration of
LOW TEMPERATURES
UNKNOWN SENSORS
Ca2+ influx
ICE1
MYB15
MYBRS
Kinase/Phosphatase
COLD ACCLIMATION/TOLERANCE
ICE1box
ICE1 like
ABRE
METABOLISM
ROS
PROTECTIVE PROTEINS and METABOLITES
Proteolysis
Unknown cis-elements
MYB/MADS/NAC/others
HOS1
HOS1
STZZAT12
LOS2
RAV1
Target
HOS9HOS10
Rd29aCor15a
ZAT10
EP2
RAP 2.6
SFR6
Rd29aRd29b
Rd22AtADH1
ICE1
CBF2CBF3 CBF1
ICE1
COR/KIN/RD/LT? MYCR/MYBR
CBF
U
P
S
CRT/DRE ?
1096 Planta (2012) 235:1091–1105
123
proline and total soluble sugars, including sucrose, raffi-
nose, glucose and fructose (Gilmour et al. 2000). Never-
theless, transcriptome analysis of transgenic Arabidopsis
overexpressing CBF revealed that only about 12 % of the
cold-responsive genes are components of the CBF regulon
(Fowler and Thomashow 2002), suggesting that other
transcriptional activators/repressors also play a significant
role in cold acclimation.
Inducer of CBF expression (ICE), a regulator
of cold acclimation
Since CBF genes are cold-induced, it may be that an
upstream transcription factor present in the cell at normal
growth temperatures is activated by cold stress and, in turn,
induces the expression of CBFs. A constitutive transcrip-
tion factor, inducer of CBF expression 1 (ICE1), which
acts upstream of the CBFs in the cold-response pathway,
has been identified (Chinnusamy et al. 2003). ICE1 binds
to the CBF3 promoter and may activate CBF3 expression
upon cold treatment (Figs. 3, 4). The dominant ice1
mutation blocks the cold induction of CBF3, but not CBF1
or CBF2, and decreases the expression of many CBF-target
genes (Chinnusamy et al. 2003, 2004). The serine 403
residue of ICE1 is involved in regulating the transactiva-
tion and stability of the ICE1 protein. Interestingly, the
substitution of serine 403 by alanine was reported to
enhance the transactivational activity of ICE1 in Arabid-
opsis protoplasts (Miura et al. 2011). These authors also
reported that overexpression of ICE1(S403A) conferred
more freezing tolerance than ICE1(WT) in Arabidopsis,
and the expression of cold-regulated genes such as CBF3/
DREB1A, COR47 and KIN1 (cold-induced gene) was
enhanced in plants overexpressing ICE1(S403A). Whereas
ICE1 primarily affects CBF3/DREB1A expression
(Chinnusamy et al. 2003), the protein encoded by ICE2 (a
homolog of ICE1; At1g12860) primarily influences the
expression of CBF1/DREB1B but has little effect on CBF3/
DREB1A (Fursova et al. 2009). Therefore, ICE1 and ICE2
play pivotal roles in the transcriptional regulation of the
CBF/DREB1 genes (Miura et al. 2011).
The MYB transcription factor and the ABA-independent
cold acclimation pathway
ABA-dependent gene expression is regulated by tran-
scription factors that belong to the bZIP (ABRE binding
factors—or AREBs), MYC and MYB families. In A. thali-
ana, the MYB family transcription factor PAP2 regulates
the flavonoid biosynthesis pathway, which is reported to be
involved in cold tolerance (see above). OsMYB4, a member
of the MYB family of transcription factors, has also been
shown to be inducible by cold, but not by ABA in rice
(Park et al. 2010). Surprisingly, Su et al. (2010) reported
that MYBS3 repressed the DREB1/CBF-dependent cold-
signalling pathway in rice at the transcriptional level.
Further, DREB1 responded quickly and transiently,
whereas MYBS3 responded slowly to cold stress, suggest-
ing distinct pathways acting sequentially to acclimate rice
to cold stress. In A. thaliana the abundance of MYBC1
transcripts was not affected by overexpression of CBF1,
CBF2, and CBF3, suggesting that MYBC1 is not down-
regulated by these CBF family members (Zhai et al. 2010).
Abscisic acid (ABA)-dependent cold signal pathway
and other phytohormones
(a) Abscisic acid: ABA serves as a secondary signal that
plays at least some role in the transduction of cold signals
via second messengers, such as H2O2 and Ca2?. This is
demonstrated by the los5 (low expression of osmotically
responsive genes) mutant, which exhibits significantly
decreased cold- and salt/drought-induced expression of
COR. ABA enhanced antioxidant defence and slowed
down the accumulation of ROS caused by low tempera-
tures (Liu et al. 2011). ABA can also induce the expression
Fig. 4 Schematic diagram of the regulatory network involved in low
temperature responses. Plants sense low temperatures and activate a
calcium signal mediated via protein kinases, and other species that
activate multiple transcriptional cascades, one of which involves
ICE1 and CBFs. The CBF genes play important roles in cold
acclimation and are regulated by multiple pathways. They activate the
transcription of CBFs and repress MYB15. ICE1, a constitutively
expressed gene, is activated by cold stress via sumoylation and
phosphorylation. HOS1 is a RING-type ubiquitin E3 ligase that
negatively regulates cold-induced DREB1/CBF expression. The
constitutive HOS9 and HOS10 regulons have a role in the negative
regulation of CBF-target genes. CBFs regulate the expression of CORgenes that confer freezing tolerance. The expression of CBFs is
negatively regulated by MYB15 and ZAT12. HOS1 mediates the
ubiquitination and proteosomal degradation of ICE1 and, thus,
negatively regulates CBF regulons. Transcription of CBFs might be
cross-regulated. Transcription factor binding sites are represented at
the bottom of the diagram, with the representative promoters listed
below. Yellow arrows indicate post-translational regulation; solidarrows indicate activation, whereas broken lines show negative
regulation; small circles indicate post-transcriptional modification,
such as phosphorylation; question marks indicates unknown cis-
elements. ABRE ABA responsive element, CBF C-repeat binding
factor (an AP2-type transcription factor); COR cold-responsive genes,
CRT C-repeat elements, DRE dehydration-responsive elements,
HOS1 high expression of osmotically responsive genes1, HOS9 and
HOS10, ICE1 inducer of CBF expression 1, KIN cold-induced genes,
LOS2 low expression of osmotically responsive genes 2 (a bifunc-
tional enolase with transcriptional repression activity), LTI low
temperature-induced genes, MYB myeloblastosis, MYBRS MYB
recognition sequence, MYCRS MYC recognition sequence, RDresponsive to dehydration) genes, ROS reactive oxygen species,
SIZ1 SAP and MiZ1 (a SUMO E3 ligase), P phosphorylation,
S SUMO (small ubiquitin-related modifier), U ubiquitin
b
Planta (2012) 235:1091–1105 1097
123
of the CBF1, CBF2, and CBF3 genes, but to a significantly
lower level than that caused by cold (Knight et al. 2004).
(b) Polyamines: There are relatively few reports
regarding the involvement of polyamines in LT stress
(Alcazar et al. 2010; Gill and Tuteja 2010). Putrescine
accumulation under cold stress is essential for proper cold
acclimation and survival at freezing temperatures (Cuevas
et al. 2008). Indeed, Arabidopsis mutants defective in
putrescine biosynthesis (adc1, adc2) exhibit reduced
freezing tolerance compared to wild-type plants, suggest-
ing that the detrimental consequences of putrescine
depletion during cold stress are at least partially due to
changes in the concentration of ABA. On the other hand,
the accumulation of putrescine in the early stages of mango
fruit ripening promoted by LT stress did not prevent
chilling injury (Nair and Singh 2004). During treatment,
the spermidine content of leaves increased substantially in
cold-tolerant cucumber cultivars but not in sensitive ones,
while the concentrations of putrescine and spermine did not
change (Shen et al. 2000). The depletion of endogenous
spermidine and spermine in response to chilling and the
reduced severity of chilling injuries in mango fruits that
had been treated with these polyamines prior to storage
both suggest that polyamine biosynthesis influences cold
sensitivity. Polyamines have also been reported to have a
role in alleviating oxidative stress: inhibiting polyamine
synthesis causes increased oxidative damage in cold-trea-
ted plants (Groppa and Benavides 2008).
(c) Other regulators affecting cold acclimation: A
number of genes involved in the biosynthesis or signalling
of plant hormones, such as gibberellic acid and auxin, may
be also regulated by cold stress. The regulation of these
genes might be important in co-ordinating cold tolerance
with growth and development. A role for cytokinin in
mediating plant growth rates at LT was recently reported
(Xia et al. 2009). Mutants with elevated cytokinin levels
(amp1) displayed enhanced cell division at 4 �C. More-
over, no changes in CBF expression were recorded in amp1
or NahG plants at LT, suggesting that the effects of cyto-
kinin and salicylic acid (SA) on temperature-regulated
growth are independent of the CBF regulon. In addition to
its role in plant pathogen defences, SA is also involved in
suppressing plant growth during chilling and accumulates
at LT (Scott et al. 2004). However, SA is not required for
thermotolerance (Clarke et al. 2004).
Post-transcriptional regulation
Transcriptomic analyses of gene expression at the mRNA
level have contributed greatly to our understanding of cold
responses in Arabidopsis (Kreps et al. 2002; Zhou et al.
2008, 2011). However, the abundance of individual
mRNAs does not always correlate well with that of the
corresponding proteins, which are the crucial agents in the
cell (MacKay et al. 2004; Tian et al. 2004). Consequently,
it is not sufficient to simply predict protein expression
levels from quantitative mRNA data, mainly due to the
effects of post-transcriptional regulation mechanisms
(Pradet-Balade et al. 2001).
The involvement of post-transcriptional cold-induced
regulation on the abundance of specific mRNAs has been
reported in several species, including Arabidopsis and
alfalfa. Maize plants exposed to cooler temperatures
respond by phosphorylating a minor chlorophyll a/b pro-
tein rather than synthesizing a new protein from a cold-
regulated gene (Bergantino et al. 1995). Although plant cell
membranes exhibit significantly higher levels of unsatura-
tion at lower temperatures, there is no apparent increase in
rate of transcription or stability of fatty acid desaturase
mRNA at lower temperature. The exception is the Ara-
bidopsis fatty acid desaturase gene FAD8 (Matsuda et al.
2005), which suggests that plant fatty acid desaturases are
regulated at the post-transcriptional level.
Expression of homology to pathogenesis-related (PR)
genes and synthesis of antifreeze proteins (AFPS)
Studies in different plant species have shown that several
cold-induced genes encode cryoprotective proteins (Hincha
2002). In the last few decades, research has focused on
specific proteins with antifreeze activity that accumulate in
the apoplast during cold acclimation, thereby offering plant
resistance against freezing (Griffith and Yaish 2004;
Griffith et al. 2005; Yaish et al. 2006) (Fig. 5). These
proteins have been found in many overwintering vascular
plants (Griffith and Yaish 2004; Venketesh and Dayananda
2008), but antifreeze activity is present only after their
exposure to LT and only in plants that tolerate the presence
of ice in their tissues (Griffith and Yaish 2004; Yaish et al.
2006). These proteins were identified as b-1,3-glucanase-
like proteins, chitinase-like proteins, thaumatin-like pro-
teins and as polygalacturonase inhibitor proteins (Wang
et al. 2006; Yaish et al. 2006). Although they were present
in non-acclimated plants, they were found in different
locations and did not exhibit antifreeze activity, which
suggests that different isoforms of pathogenesis-related
proteins are produced under LT conditions (Antikainen
et al. 1996; Wang et al. 2006).
Until now, no plant has been reported to have consti-
tutive antifreeze activity. Rather, all studies have shown
that transcripts and translation products of AFP genes
accumulate during cold acclimation (Yeh et al. 2000;
Huang and Duman 2002; Wang et al. 2006). Recent studies
have shown that many PR genes are induced and disease
resistance is enhanced after exposure to LT, linking cold
signals with pathogenesis in plants (Seo et al. 2010).
1098 Planta (2012) 235:1091–1105
123
By accumulating PR proteins during cold acclimation,
overwintering grasses and cereals acquire a systemic, non-
specific, pre-emptive defence against pathogens and thus
exhibit greater disease resistance.
Cold shock proteins and RNA binding proteins
Cold shock domain proteins (CSDPs) play important roles
in development and stress adaptation in a variety of
organisms, ranging from bacteria to mammals (Chaikam
and Karlson 2010). In higher plants, cold shock domain
proteins are involved in the cold response (Nakaminami
et al. 2006; Sasaki et al. 2007). Recently, it was demon-
strated that CSP Arabidopsis 3 (AtCSP3), which shares a
cold shock domain with the CSDPs, is involved in the
acquisition of freezing tolerance in plants (Kim et al.
2009).
COR/LEA and dehydrins
The accumulation of hydrophilic proteins predicted to form
an amphipathic a-helix is one of the best documented
responses of plants to cold treatment (Eriksson et al. 2011).
Most of these proteins have therefore been named COR
(cold responsive), LTI (low temperature induced), RAB
(responsive to abscisic acid), KIN (cold induced) or ERD
(early responsive to dehydration). These proteins include the
dehydrins, which belong to group II of the late embryogen-
esis abundant (LEA) proteins (Bies-Etheve et al. 2008).
The accumulation of one particular dehydrin (WCO
R410) is correlated with the capacity to develop freezing
tolerance in wheat. However, the overexpression of single
dehydrins does not necessarily lead to enhanced freezing
tolerance. For instance, the overexpression of RAB18, a
cold-induced dehydrin, has no effect on freezing tolerance in
Arabidopsis (Lang and Palva 1992). This suggests that to
fully play their role, dehydrins may need to be activated by a
cold-induced mechanism such as protein phosphorylation.
One of the roles that have been also attributed to dehydrins is
the prevention of membrane destabilization during dehy-
dration. In addition to this postulated function, it has been
proposed that dehydrins possess cryoprotective (Bravo et al.
2003) or antifreeze (Puhakainen et al. 2004) activities.
Cold-induced osmolites/osmoprotectants
In response to cold and other osmotic stresses, plants
accumulate a range of compatible solutes including cer-
erosides, free sterols, sterol glucosides and acylatedsterols,
glucosides, raffinose, arbinoxylans, and other soluble sug-
ars. In addition, plants accumulate other solutes such as
glutamic acid, amino acids (alanine, glycine, proline, and
serine), polyamines and betaines (Hekneby et al. 2006;
Patton et al. 2007; Ruelland and Zachowski 2010). These
different molecules, which are often degraded once the
stress has passed, are referred to as osmolytes, osmopro-
tectants or compatible solutes.
Carbohydrate changes
Carbohydrate metabolism has been reported to have greater
instantaneous low temperature sensitivity than other com-
ponents of photosynthesis (Fernandez et al. 2012). Although
the precise function of soluble sugars remains to be deter-
mined, their accumulation in cold-acclimated plants sug-
gests roles as osmoregulators, cryoprotectants or signalling
molecules (Welling and Palva 2006). Sugars play multiple
roles in low temperature tolerance. As typical compatible
osmolytes, they contribute to the preservation of water
within plant cells, thereby reducing water availability for ice
nucleation in the apoplast (Uemura and Steponkus 1999;
Ruelland et al. 2009). Sugars might protect plant cell mem-
branes during cold-induced dehydration, replacing water
molecules in establishing hydrogen bonds with lipid mole-
cules (Uemura et al. 2003; Ruelland et al. 2009). Moreover,
carbohydrates may also act as scavengers of reactive oxygen
Resistance to pathogens
Cold acclimation
Accumulation of antifreeze proteins (AFPs)
Cold acclimation
Chilling stress
Accumulation of transcripts homology to PR genes
Pathogen attack
Accumulation of PR proteins
Expression of genesEncoding for PR proteins
Salicylic acidAbscisic acid
EthyleneJasmonic acid
Fig. 5 Accumulation of antifreeze proteins (AFPs) in cold-accli-
mated plants with antifungal activity (adapted from Yeh et al. 2000;
Huang and Duman 2002; Griffith and Yaish 2004; Yaish et al. 2006).
By accumulating PR proteins during cold acclimation, overwintering
plants may acquire a systemic, non-specific, pre-emptive defence
against pathogens and exhibit greater disease resistance
Planta (2012) 235:1091–1105 1099
123
species and contribute to increased membrane stabilization
(Bohnert and Sheveleva 1998). Sugar signalling is also clo-
sely associated with hormone signalling, the control of
growth and development, and stress responses in plants
(Zeng et al. 2011).
Depending on the plant species, various forms of soluble
sugars are involved in physiological reactions to cold
stress. For example, treatment of rice seedlings with fruc-
tose or glucose prior to LT treatment increases their
resistance to cold. Cotton cotyledon discs floating on a
sucrose solution in the dark were less injured by cold than
those on non-sugar solutions (Couee et al. 2006). On the
other hand, King et al. (1988) reported that cold tolerance
in tomato seedlings decreased following pronounced
reductions in their starch and sugar levels during a dark
period.
The soluble carbohydrate content of grasses can undergo
a tenfold increase within 8 h of transfer from a warm to a
cold environment (Pollock and Lloyd 1987). The oligo-
saccharides raffinose and stachyose are especially associ-
ated with cold hardiness, low temperature and dormancy
(Aıt Barka and Audran 1996; Couee et al. 2006). More-
over, the concentration of sucrose, the most easily detect-
able sugar in cold-tolerant species, increases several fold
during exposure to LT (Bohnert and Sheveleva 1998;
Tabaei-Aghdaei et al. 2003). The accumulation of sucrose
in cane sugar exposed to salt stress or to LT stress supports
the role of this sugar as an osmoprotectant that stabilizes
cellular membranes and maintains turgor (Jouve et al.
2004). In addition, high sucrose levels correlate with the
priming of defence responses in rice that overexpresses the
PRms gene from maize, which encodes a PR-1 type protein
(Casacuberta et al. 1991).
Trehalose is a non-reducing disaccharide of glucose that
is found in a variety of organisms including bacteria, yeast,
fungi, insects and invertebrates, where it serves as a stress
protectant and/or a reserve carbohydrate (Penna 2003;
Fernandez et al. 2010). Although increased levels of tre-
halose are associated with abiotic stress tolerance in
transgenic plants expressing heterologous microbial genes,
the function of endogenous trehalose in higher plants
remains unclear. This sugar possesses the unique capacity
for reversible water absorption and appears to be superior
to other sugars in protecting biological molecules from
desiccation-induced damage (Fernandez et al. 2010). Fur-
ther, transgenic A. thaliana plants that accumulated treha-
lose displayed significantly enhanced freezing tolerance
(Miranda et al. 2007). Increases in trehalose concentration
may also be involved in starch accumulation (Fernandez
et al. 2010).
During exposure to LT, starch content typically declines
following hydrolysis, and there is a corresponding increase
in the concentration of free saccharides (Pollock and Lloyd
1987; Bohnert and Sheveleva 1998). However, in several
cases, increases in the levels of both soluble sugars and
starch have been reported during cold acclimation. For
instance, in cabbage seedlings grown at 5 �C, the concen-
trations of starch and all soluble sugars (myo-inositol aside)
in the leaves increased gradually during cold acclimation
(Sasaki et al. 1996). However, the induced freezing toler-
ance was lost after only 1 day of acclimation at control
temperatures and this change was associated with a large
reduction in sugar content.
Carbohydrate accumulation at LT may be explained
through the activation of specific enzymes (Bohnert and
Sheveleva 1998; Couee et al. 2006). This suggests that
although LT inhibits sucrose synthesis and photosynthesis,
various biochemical and physiological adaptations to LT
counteract these effects. These adaptations include the
post-translational activation and enhanced expression of
enzymes involved in the sucrose synthesis pathways and
those of Calvin cycle—in particular, the cytosolic enzymes
fructose-1,6-bisphosphatase, sucrose phosphate synthase
and sucrose synthase (Stitt and Hurry 2002).
Compatible osmotica other than sugars
(a) Proline: The positive correlation between the accu-
mulation of endogenous proline (Pro) and improved cold
tolerance has been found mostly in LT-insensitive plants
such as barley, rye, winter wheat, grapevine, potato,
chickpea and A. thaliana (Verbruggen and Hermans 2008;
Szabados and Savoure 2010; Kaur et al. 2011). Proline
plays multiple roles in plant stress tolerance, as a mediator
of osmotic adjustment, a stabilizer of proteins and mem-
branes, an inducer of osmotic stress-related genes, and as a
scavenger of ROS (Verbruggen and Hermans 2008;
Szabados and Savoure 2010; Theocharis et al. 2011). The
most probable roles of proline are to (a) regulate cytosol
acidity, (b) stabilize the NAD?/NADH ratio, (c) increase
the photochemical activity of the photosystem II in thyla-
koid membranes and (d) decrease lipid peroxidation
(Kishor et al. 2005). Most chilling-sensitive plants that
accumulate Pro under LT conditions do not acquire cold
tolerance (Kushad and Yelenosky 1987), unless a high
concentration of Pro was applied prior to stress (Xin and Li
1993). It appears therefore that proline possesses the
potential to alleviate LT injury in chilling-sensitive plants,
but for some reason this system fails under natural
conditions.
(b) Glycine betaine: The accumulation of glycine beta-
ine (GB) usually correlates with the plant’s level of stress
tolerance. Both the genetically engineered biosynthesis of
GB in plants that do not naturally accumulate GB and the
exogenous application of GB enhance the tolerance of such
plants to various abiotic stresses (Chen and Murata 2008).
1100 Planta (2012) 235:1091–1105
123
Possible roles for GB include stabilization of the tran-
scriptional and translational machinery. GB stabilizes
protein complexes and membranes in vitro and may indi-
rectly induce H2O2-mediated signalling pathways.
Effect of microorganisms on cold tolerance
Deleterious effects
Both the aerial parts of the plant and the rhizospheric zone
harbour hundreds of species of bacteria, yeast and fungi.
Several bacterial and fungal species have the ability to
nucleate ice at high sub-freezing temperatures. Bacterial
species with ice nucleation activity (Ice? bacteria) such as
Pseudomonas syringae contribute to frost injury in many
frost-sensitive plant species by reducing their ability to
supercool and avoid the formation of membrane damaging
ice crystals (Lindow and Leveau 2002). Other ice nucle-
ating bacterial species include P. fluorescens, Erwinia
herbicola, and some strains of Xanthomonas campestris, as
well as related strains. Some species of Fusarium and
related genera of fungi are also active in ice nucleation.
Beneficial effects
One way to reduce the incidence of LT damage is to use
beneficial microorganisms that enhance plant growth and
improve their resistance to stress. Alternatively, beneficial
bacteria may also be used to eliminate the Ice? bacteria
from plant surfaces. Since the ice nucleation temperature
increases with the population size of Ice? bacteria, pre-
emptive competitive exclusion of Ice? bacteria by natu-
rally occurring non-ice nucleating active bacteria could be
an effective and practical method for managing frost in
cold-sensitive plants (Lindow and Leveau 2002). The
bacteria could also be genetically altered to not carry the
genetic instructions needed to produce the ice nucleating
protein. If such new bacteria (INA-) were sprayed onto
plants at very high concentrations, naturally occurring
bacteria (INA?) would not be able to compete. Recombi-
nant Ice- bacteria, the first engineered microorganisms
released into the open environment in field experiments,
have been used to illustrate the specificity with which
competitive exclusion of Ice? bacteria occurs (Skirvin
et al. 2000).
Several endophytic bacteria have been reported to
induce resistance against biotic stress and tolerance to
abiotic stress in several plants. For instance, a plant
growth-promoting rhizobacterium (PGPR), Burkholderia
phytofirmans strain PsJN, is able to reduce chilling-induced
damage (Ait Barka et al. 2006). Similar conclusions were
reported during the interaction between Chorispora bun-
geana and the endophyte Clavibacter sp. strain Enf12
(Ding et al. 2011). In attempt to explain how some bene-
ficial bacteria may influence cold tolerance, Theocharis
et al. (2011) reported that several stress-related gene tran-
scripts (e.g., StSy, PAL, Chit4c, Chit1b, Gluc and LOX) and
changes in levels of several stress-related metabolites (e.g.,
proline, malondialdehyde and other aldehydes known to be
lipid peroxidation markers, and hydrogen peroxide)
increased earlier, faster, and were more pronounced in
chilled-PsJN-bacterized plantlets. This is consistent with
the ‘priming’ concept (Theocharis et al. 2011), supporting
the establishment of a mutualistic relationship between the
bacterium and the grapevine. The endophyte participates in
the cold acclimation process via a scavenging system (Ding
et al. 2011; Theocharis et al. 2011). The reported increase
in the expression of Chit1b, Chit4c and Gluc was not
surprising because chitinases and glucanases can be clas-
sified as either antifreeze (AFPs, Griffith and Yaish 2004)
or PR (Van Loon and Van Strien 1999) proteins; this
confirms the link between cold signals and pathogenesis in
plants, as shown in Fig. 5. Further, we demonstrated
recently that photosynthesis is modulated by the presence
of beneficial bacteria in grapevine plantlets, suggesting that
the modification of carbohydrate metabolism is one of the
major modes by which PGPR reduces chilling-induced
damage (Fernandez et al. 2012).
Concluding remarks and future perspectives
Plant physiologists and plant molecular biologists have
always been interested in mechanisms involved in plant
tolerance to cold and how plants may react to withstand
damage following stress. The biological and physiological
changes that occur following cold exposure have been
particularly well-studied. Studies conducted in recent years
have analysed the cold signal, the genes that act down-
stream of it to induce cold acclimation, and the overall
cascade of molecular events that occur following cold
perception. This has generated a large amount of data that
requires collation and interpretation. Thus, there is a real
need for a comprehensive model that encapsulates this
multi-step process.
Acknowledgments The first author (A.T.) was supported by a Grant
from the Greek State Scholarship Foundation (I.K.Y.).
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