J Agro Crop Sci (2014) ISSN 0931-2250

REVIEW

An Overview of Cold Resistance in Plants

L.-J. Chen1, H.-Z. Xiang1, Y. Miao1, L. Zhang1, Z.-F. Guo1, X.-H. Zhao2, J.-W. Lin1 & T.-L. Li1
1 Key Laboratory of Agricultural Biotechnology of Liaoning Province, Key Laboratory of Protected Horticulture(Ministry of Education), College of
Biosciences and Biotechnology, Shenyang Agricultural University, Shenyang, China
2 The Liaoning Academy of Agricultural Sciences, Shenyang, China

Keywords Abstract
cold-resistant; functional genes; protective
enzymes; regulation genes; unsaturated fatty Cold is a typical environmental stress factor that limits the geographical distribu-
acid tion and growth of various plants. It affects crop quality and productivity. In the
contemporary years, with the plant cold-resistant genetic engineering develop-
Correspondence ment, great progress was obtained in this field. This paper gave a general state-
L.-J. Chen
ment about the development of plant cold resistantance on the level of
Shenyang Agricultural University,
unsaturated fatty acids, protective enzymes, functional genes and regulation
60# College of Bioscience and Technology,
120 Dongling Road, Shenyang, genes, aiming to provide some useful information and ideas to researchers who
Liaoning Province, 110866, China work on plant cold breeding and the mechanism of cold resistance. As research
Tel.: +86-24-88487164 continues, we could develop further multiple resistant plant species.
Fax: +86-24-88492799
Email: chenlijing1997@126.com
T.-L. Li
Shenyang Agricultural University,
40# College of Horticulture,
120 Dongling Road, Shenyang,
Liaoning Province, 110866, China
Tel./Fax: +86-24-88487004
Email: litianlai@126.com

Li-jing Chen and Heng-zuo Xiang considered

as joint first authors.

Accepted May 9, 2014

doi:10.1111/jac.12082

Introduction
Plants have sufficient adaptability and resistance to envi-
ronmental changes. This resistance is controlled by the evo-
lution of genetic type and is restricted by physiological and
ecological factors in the development. As one of the impor-
tant environmental factors, temperature limits growth and
yield of plants (Viswanathan and Zhu 2002). In the evolu-
tion of the long-term progress, it has been found that
adaptability of temperature stress, for example, the freezing
tolerance of many plants is induced in response to low,
non-freezing temperatures, a phenomenon known as ‘cold
acclimation’ (Levitt 1980). But for now, the mechanism of
this adaption is not very clear, so exploring the genomic
mechanism of plant cold resistance has enormous signifi-
cance, not only in basic theory, but also to deal with the
problem of actual production.
Biochemistry, cell biology and molecular biology
research data showed that in the process of cold acclima-
tion, the plants can produce a series of changes including:
the change of lipid composition (Steponkus and Lynch
1989), increased sugar and soluble protein content, changes
in hormone levels (Welin et al. 1995, Alghabari et al. 2014)
and the new alleles of enzyme (Hajela et al. 1990) etc. The
freezing tolerance was associated with acclimation pro-
cesses such as cellular osmotic stabilisation, photosynthesis
modifications, antioxidant production, modifications in
hormone metabolism, cell wall composition and dynamics
(Lucau-Danila et al. 2012). Destruction of organelles mem-
brane structure leads to injuries and deaths in plants. Kaye

© 2014 Blackwell Verlag GmbH, 200 (2014) 237–245 237

Chen et al.
et al. (1998) studied this phenomenon through the freezing
experiment by separation of spinach chloroplasts, which
confirm that the chilling injury first destroys the chloro-
plast membrane structure and then leads to the inactivation
of photophosphorylation enzyme. Since then, a large num-
ber of research revealed and confirmed that rupturing of
cell membrane system is the fundamental mechanism of
plant cold injury (Russell 2008, Seo et al. 2010, Mironov
et al. 2012).
The Role of Unsaturated Fatty Acid in Cold
Resistance
A lot of studies have demonstrated that cell membranes
are a primary site of cold-induced injury (Kratsch and
Wise 2000). Lyons (1973) proposed the famous theory
of ‘membrane lipid phase transformation’, and he
thought that the low temperature affects the liquidity
of membrane lipid. Under physiological conditions,
membrane lipid showed liquid crystal phase, but when
ambient temperature was lower than the phase transi-
tion temperature, membrane lipid could be transformed
into liquid phase. The membrane becomes cracked and
permeability was increased, extravasation of contents
resulting in metabolic disorder. Electrical conductivity is
a physiological symptom that measured whether the
insoluble substance diffuses outside and also measured
whether the cytoplasmic membrane damage. Wang
et al. (2008) found that the relative conductivity of
corn seedling increased under low temperature stress. Li
et al. (2012) reported that relative conductivity of tea
leaf was increased with decrease in temperature. Under
low temperature stress, the conductivity increases, and
it is proved that the membrane lipid phase changed at
this stress and transformed from the liquid phase into
the gel phase thus increasing the permeability of
the membrane. The phase transformation of plant
membrane lipid change is closely related to cold stress
resistance.
For now, researchers found that the unsaturated fatty
acid increased after cold treatment. This unsaturation of
membrane lipids was also thought to determine the cold
stress resistance (Campbell and Close 1997, Uemura and
Steponkus 1997, Routaboul and Fischer 2000). Rice, corn,
tobacco and other plants also confirmed that membrane
lipid saturation was proportional to the cold resistance
(Kodama et al. 1995, 1997, Murata and Wada 1995). The
study on ADS2 (ACYL-LIPID DESATURASE2) in cold
stress response pathway of Arabidopsis thaliana pointed out
that ads2 mutant plants show increased sensitivity to freez-
ing temperature, and the unsaturated fatty acid was
reduced, particularly in double mutation (Chen and Thelen
2013). Zhang et al. (2009) found that high-unsaturation
238
Fig. 1 Increase in unsaturated fatty acid (IUFA) variation under cold
treatments.
lipid level is one of the factors that maintain the membrane
function of chloroplasts for cold tolerance during low tem-
perature. The study of winter wheat showed that linolenic
acid and palmitic acid were closely related to cold
resistance, and IUFAvariation is increasing (Fig. 1) for cold
tolerance (Xie et al. 2013).
Earlier study had found that accumulation of polyunsat-
urated fatty acids during cold acclimation, membrane glyc-
erine linoleic acid of the wild potato species S. commersonii
accumulated in leaves, no other potatoes demonstrated this
property (Vega et al. 2004). After cold acclimation, there is
an increase in unsaturated fatty acids (IUFA) ratio com-
pared with saturated ones which is a sign of cold tolerance
in chickpea (Shahandashti et al. 2013).
The above research conclusion well illustrated that the
levels of unsaturated fatty acid in membrane lipid and its
cold resistance were positively correlated. The phase transi-
tion temperature of the membrane lipid reduced accord-
ingly with increase in unsaturated fatty acid content, thus
increasing the membrane fluidity, and the cold resistance
of plants is improved correspondingly.
Role of Protection Enzymes in Freezing Tolerance
The generation of active oxygen within the plants normal
life activities is inevitable, but in order to maintain the nor-
mal physiological activities, the active oxygen removal sys-
tem exists at the same time. Studies showed that with
extended time of low temperature and reinforce the degree
of temperature decline, reactive oxygen removal system
activity will fall, and there is an accumulation of reactive
oxygen. This excessive reactive oxygen has negative effects
on cell structure and metabolism, and also causes damage
to proteins, DNA and membrane lipids which leads to
death (Suzuki and Mittler 2006, Nordberg and Arner
© 2014 Blackwell Verlag GmbH, 200 (2014) 237–245

2001, Ott et al. 2007). Antioxidant system which is com-
posed of many enzymes and small molecules can protect
plants from damage. The activity of antioxidant enzymes
and the increased content of antioxidants play an impor-
tant role in inhibiting membrane proteins and membrane
lipid peroxidation which can improve the cold resistance of
the plant (Prasad 1996). Study on antioxidant activity of
enzymes in annual dormant branches of Acer davidii, Pista-
cia chinensis and Liriodendron chinense found that with
decline of temperature, the rate of water loss and relative
electric conductivity of these plants gradually increased,
and the activities of SOD, POD and MDA were also chan-
ged in a trough and crest manner (Jiao et al. 2013).
Catalase (CAT), peroxidase (POD), superoxide dismu-
tase (SOD) and esterase isozyme (EST) have a close
relationship with cold resistance (Liu et al. 2014). Luo
et al. (2001) explored the relationship between freezing
resistance of tea plants and protective enzymes under the
normal cultivation conditions. The results showed that
varieties exhibiting strong cold resistance have strong activ-
ities of SOD and CAT, while the weakers have weak activi-
ties. But the activities of POD and contents of MDA have
no correlation with the ability of cold resistance in tea
plant. Protective enzyme activities of strong cold-resistant
plants are higher and can maintain for a long time at low
temperature (Patterson et al. 1984, Das et al. 1991, Scebba
et al. 1999). Research on the cold resistance of wheat, rice,
cucumber, tomato and banana found that with the exten-
sion of time in low temperature, SOD and POD activities
were changed in different ranges (Rabinowitch and Sklan
1980, Liu et al. 1985, Lee and Lee 2000, Kondo et al. 2005).
Zhang et al. (2010) observed higher CAT activity in wheat
plasma membrane under cold stress. Under the stress of
low temperature, the turf grass, which has strong cold
resistance had higher SOD activity. This proved that SOD
activity was associated with cold resistance. At the same
time, large number of experiments also proved that the
antioxidant capacity of the plant was likely to be an impor-
tant part of plant cold resistance mechanism (Liu et al.
1998, Yu et al. 2006, Zhu et al. 2011).
Under chilling damages, the hydrolytic enzymes activity
was greatly improved, and protein and various macromo-
lecular substances were decomposed rapidly. The activity of
reactive oxygen removal system is declined, and the active
oxygen thus accumulate causing damage to plant mem-
brane lipid peroxidation (Xu et al. 2010). Sugar as osmotic
regulators and penetration protectant is also accumulating.
This not only can reduce the water potential in the cell and
can effectively protect and maintain the membrane stability
(Purvis et al. 1979, Ewing et al. 1981, Kang and Saltveit
2002).
The hydrolysis of starch is increased under low temper-
ature producing large amounts of soluble sugars, includ-
© 2014 Blackwell Verlag GmbH, 200 (2014) 237–245
Cold Resistance in Plants
ing sucrose, glucose and fructose (Kawakami et al. 2008).
The chilling tolerance might be related to an increase in
protein related to soluble sugar synthesis (Grimaud et al.
2013). Jonak et al. (1996) pointed out that sucrose can
protect the plasma membrane from damage during huge
loss of moisture after low temperature. During cold accli-
mation, the amount of soluble carbohydrate was increased
in the susceptible festulolium (Pociecha et al. 2010).
Research on Camellia sinensis leaves also found that the
content of soluble sugar was rising under low temperature
stress (Zhu et al. 2011). Another study revealed that the
soluble sugar content was increased in different develop-
mental stages of Melilotoides under chilling stress (Du
2007).
Changes in Molecular Level under Freezing
Tolerance
It was reported that cold resistance in plants is a scalar
property manipulated by various genes, which are a kind of
induced genes. These genes only can express under certain
conditions. The cold-resistant ability of plants is improved
by co-expression between these genes. Depending on the
function of genes and their products, these genes can sim-
ply be grouped into two categories: functional genes and
regulation genes (Thomashow 1999). Functional genes are
directly related to improve the plant cold resistance, such
as cold-induced genes, fatty acid desaturases genes and
antioxidant enzyme genes, and they all can protect cell
membrane (Seki et al. 2002). The regulation genes can
improve the cold resistance of plants by regulating the
expression of genes and procession of cold signalling. The
chilling tolerance was related to responses of the CBF, COR
and LEA genes belonging to the CBF regulation (Lucau-
Danila et al. 2012, Al-Issawi et al. 2013). Numerous cold
resistance-related genes has been successively isolated from
plants such as rye, tomatoes,wheat, and their regulation has
been studied preliminarily (Griffith et al. 1992, Ding et al.
2002, Koike et al. 2002, Fung et al. 2006). Plant cold
research is further down into genetic engineering. The nor-
mal method is cloning cold resistance-related gene and
then connecting strong promoter or cold inducible pro-
moter. The transgenic plants were obtained by introducing
genes into plants (Ouellet et al. 2001) that is recognised as
transgenic technology. Research and analysis of the func-
tion of these genes can accelerate plant cold-resistant
genetic engineering. It will be an enormous significance in
molecular breeding.
Research of Functional Genes
Cold-induced genes were divided into two species: one is
the gene of coding operated protein, regulating cold signal
239
Chen et al.
Fig. 2 Signaling pathways of functional genes under cold stress.
transduction, expression of cold resistance gene and the
activity of cold resistance protein (Gatz 1997, Kasuga et al.
1999); Another is gene of functional protein that directly
related to cold resistance, such as antifreeze protein gene
(AFP) (Hon et al. 1994). Weiser (1970) suggested that the
expression of gene can be changed in low temperature, and
the adaptability of low temperature might need two condi-
tions: the activation of specific genes and the induction of
new protein synthesis during the process of cold resistance
(Fig. 2). For now, COR genes have been isolated and iden-
tified from Arabidopsis thaliana, rape, alfalfa, barley and
wheat. But expression product of these genes is different,
the fad8 from Arabidopsis thaliana encoding fatty acid
desaturase (Gibson et al. 1994); the blt4 from barley encod-
ing a fat migration protein (Dunn et al. 1998); hsp70 from
Spinach (Anderson et al. 1994); and hsp90 from rape
(Krishna et al. 1995) encoding molecular chaperone to sta-
bilise protein function and to resist from frozen-induced
degeneration phenomenon. Thus, COR improved the cold
resistance of plant by encoding different functional protein.
In the process of chilling stress, gene of various enzymes
also regulates physiological and biochemical reactions of
plants. As the most important of enzymes, superoxide
dismutase (SOD) can scavenge reactive oxygen, and the
increase of content and activity of antioxidant enzyme can
effectively protect the plant from chilling stress. It is one of
240
effective ways that manipulating gene of antioxidant
enzyme into plants can improve the cold stress resistance.
McKersie et al. (1993) introduced the gene of Mn-SOD
into mitochondria and chloroplasts of alfalfa, and the win-
tering rate of transgenic plants was considerably increased.
After introducing the gene of Cu-Zn-SOD into tobacco,
the low temperature tolerance was also improved. Peroxi-
dase (POD) is also a very crucial enzyme to defence plant
resistance (Bowler et al. 1989). Liorente et al. (2002)
cloned RCI from rice, which is related to POD activity and
can make plants resistant to low temperature and other
kinds of stress (Bowler et al. 1991, Gupta et al. 1993, Samis
et al. 2002).
Research of Regulation Genes
Transcription factor gene (Crepeat binding factors, CBFs)
has the major role in plant cold acclimation in recent years,
which were specifically associated with cis-elements in gene
promoter regions of eukaryotes (Gilmour et al. 1998). The
interactions between them and other related protein which
activate or inhibit transcription both ensuring the purpose
of gene expression in a particular intensity with time and
space (Fig. 3). CBF plays an important role in the process
of cold resistance (Fowler and Thomashow 2002). These
genes can specifically interact with the DRE/CRT-binding
transcription factor. Without low temperature, stimulation
can induce excessive expression of COR genes which
enhance cold resistance in plants (Yamaguchi-Shinozaki
and Shinozaki 1994). Its regulation was shown in Fig. 4.
CBF have already been subsequently found in rice, arabid-
opsis and tomatoes (Hsieh et al. 2002, Zhang et al. 2004,
Ito et al. 2006). Transcription factors of CBF contribute to
increase in plant-freezing tolerance (Kim et al. 2013).
Jaglo-Ottosen et al. (1998) imported CBF1 protein in
arabidopsis that can cause induced gene expression, which
improved the cold-resistant ability of this plant. Jofuku
et al. (1994) has isolated APETALA2 (AP2) from Arabidop-
sis thaliana. The protein expressed contains two AP2/ERF
structural domain. The gene was also isolated from many
crops such as corn, wheat, tomato, soybean, rice and others
(Moose and Sisco 1996, Gu et al. 2002, Egawa et al. 2006,
Chen et al. 2009, Matsukura et al. 2010). Chen et al.
(2003) cloned an AP2/EREBP transcription factor that was
induced by minimal temperature and that does not rely on
ABA. It has been proved that the Vitis vinifera C-Repeat
Binding Factor 4 which function in cell wall structure, lipid
metabolism, epicuticular wax formation and stress
responses can enhance freezing tolerance significantly
(Cushman 2013).
A large number of studies have shown that the expres-
sion of AP2/EREBP transcription factors can improve the
cold resistance in plants. Gene expression spectrum analysis
© 2014 Blackwell Verlag GmbH, 200 (2014) 237–245

Fig. 3 Signaling pathways by DREB under cold
stress.
Fig. 4 Regulation of COR genes in plants.
of AP2/ERF family has shown that this kind of transcrip-
tion factors can regulate the development of plant and also
participated in the process of mechanical damage, low tem-
perature, drought, salt and alkali stress responses (Klucher
et al. 1996, Thomashow et al. 2001, Mizoi et al. 2012).
Researchers thought that undue expression of CBF could
indirectly stabilise the cell membrane through the
improved content of the protein, proline and soluble sugar
(Leubner-Metzger et al. 1998). Further researches on cold
resistance have found many low temperature-related genes.
Cold resistance depends on the coordinated expression of
multiple gene, but the mechanism is not clear, and some
resistance plants like C. bungeana can survive at low tem-
perature without CBFs or others (Zhao et al. 2012). We
need to continue work hard to provide more reference data
for this research.
Future Prospects
In recent decades, the development of molecular biology is
rapid, which plays an important role in revealing the mech-
anism of plant cold resistance. Overall, the research of
using molecular biology techniques to study plant cold
resistance is relatively weaker, and there are many problems
to be solved. Some researches have succeeded on the struc-
ture and function of the genes associated with cold resis-
tance and made definite progress by applying genetic
engineering into cold resistance molecular breeding. But
© 2014 Blackwell Verlag GmbH, 200 (2014) 237–245
Cold Resistance in Plants
the current study is focused on cloning of cold resistance
related gene, structure identification and the analysis of
transcriptional regulation function. Although the research
has laid a solid foundation for the application of cold
related genes, the molecular mechanism of cold resistance
research is still in its infancy, especially the expression of
the mechanism of signal transduction, gene regulation and
lack of understanding on the relationship between expres-
sion products and cold resistance in plants. Cold resistance
is the character controlled by a series of directly or indi-
rectly related genes. The majority of current research con-
trapose the single gene. Studying the cold resistance of
plants, including a single gene, and further interaction
mechanism between all kinds of cold related genes is key to
future work that clear a signal transduction and regulation
mechanism. In the process of cold resistance molecular
breeding, in addition to looking for new genes of cold resis-
tance, try to transform multiple gene into plants or do
some conventional hybridisation to obtain new species that
have multiple cold resistance genes. Hoping to develop a
strong cold resistance of new plant varieties by searching
for cold resistance related genes more and more deeply.
Acknowledgements
This work was supported by the Specialized Joint Research
Fund for the Doctoral Program of Higher Education of China
(Grant No. 20112103120005) and by China Postdoctoral
241
Chen et al.
Science Foundation (Grant No. 20100471471), and Sheny-
ang Agricultural University Graduate Student Innovation
Cultivation Project (Grant No. 2013-8).
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