Review

Received: 7 August 2008 Revised: 10 October 2008 Accepted: 17 October 2008 Published online in Wiley Interscience: 15 January 2009

(www.interscience.wiley.com) DOI 10.1002/jsfa.3468

Physiological, hormonal and molecular

mechanisms regulating chilling injury in
horticultural species. Postharvest technologies
applied to reduce its impact
Laura Sevillano,a Mara T Sanchez-Ballesta,b Felix Romojaroc
and Francisco B Floresc

Abstract
The storage of fruits and vegetables at low temperature near the freezing point is the foremost technology applied to retard
postharvest ripening and to extend the shelf-life period of agricultural products. However, most tropical and subtropical produce
is sensitive to chilling injury, which constitutes a set of physiological alterations caused by exposure to low temperatures for
variable time periods, to the detriment of quality. This article is a thorough review of the physiological, hormonal and molecular
mechanisms involved in the induction and development of this physiopathy. Also, the different postharvest technologies of a
chemical, physical or biotechnological nature assayed in research or applied in the agro-food industry with the aim of inhibiting
or delaying the emergence of chilling injury in sensitive plant produce of agricultural interest are reviewed.

c 2009 Society of Chemical Industry

Keywords: chilling injury; oxidative stress; phytohormones; transcription regulation; heat shock proteins; postharvest technologies

INTRODUCTION
Low-temperature storage is a postharvest technology used widely
to extend the postharvest life of horticultural produce. Refrigerated
storage of fruits and vegetables allows the preservation of their
quality after harvest, because low temperatures decrease the
speed of cell metabolism and delay plant senescence in general
and fruit ripening in particular.1,2 However, certain tropical and
subtropical fruits and vegetables are not suitable for this type
of storage, since it causes the appearance of physiological
disorders that negatively affect their quality. The whole set of
these alterations is known as chilling injury (CI). This physiological
disorder is manifested at temperatures above 0 C, around 8 C for
subtropical plant species and around 12 C for tropical ones.3
The economic repercussion of this physiopathy on agriculture
has been recognised and studied for more than a century.4 6
Therefore crops which are susceptible to CI often have a short
storage life, since low temperatures cannot be used to delay
deterioration and pathogen growth. The exposure of sensitive
species to low temperatures above the freezing point causes the
alteration of multiple metabolic processes, the manifestation of
visible symptoms and, finally, cell death if the tissue is exposed
to the damaging temperature for too long a period of time.1,3,7
However, these processes are reversible if the tissue is placed at a
higher temperature before damage occurs.6,8
The CI symptoms can be observed in all developmental stages
of the plant, but their intensity depends on the time and
temperature of the cold treatment, the type of plant organ and
the developmental stage. The visual symptoms caused by this
such as fruits and vegetables, vary as a function of the species and
even of the variety of a single species and depend also on the
environment (soil, climate, watering) where the plants have been
grown.3
Although there are many different variables affecting the
significance of CI, the symptoms, at a microscopic level, are similar
in all plant organisms. These symptoms include swelling and
disorganisation of the mitochondria and the chloroplasts, where
thylakoid dilation and unstacking of grana occur, reduction in the
size and number of starch granules, accumulation of lipid droplets
inside the chloroplasts and condensation of nuclear chromatin.9
The macroscopic symptoms of CI in fruits and vegetables are
diverse, and two categories can be distinguished, which often
develop simultaneously. The first, of a qualitative nature, includes
developmental or metabolic disorders such as incomplete or
impaired ripening and deficient flavour and aroma. The second
Correspondence to: Francisco B Flores, Departamento de Biologa del Estres
y Patologa Vegetal, Centro de Edafologa y Biologa Aplicada de Segura
(CEBAS-CSIC), Apartado de Correos 164, E-30100 Espinardo, Murcia, Spain.
E-mail: borjaflores@cebas.csic.es
a Departamento de Bioqumica y Biologa Molecular II, Facultad de Farmacia,
Universidad de Granada, Campus Cartuja s/n, E-18071 Granada, Spain
b Departamento de Ciencia y Tecnologa de Productos Vegetales, Instituto del
Fro (IF-CSIC), C/Jose Antonio Novais 10, E-28040 Madrid, Spain
c Departamento de Biologa del Estres y Patologa Vegetal, Centro de Edafologa
y Biologa Aplicada de Segura (CEBAS-CSIC), Apartado de Correos 164, E-30100
555

physiopathy, developed after harvest on isolated plant organs Espinardo, Murcia, Spain

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includes the real physiological symptoms that become apparent
in different ways, e.g. skin depression, abnormal skin yellowing,
tissue decomposition, internal or surface browning (scald), water
infiltration in intercellular spaces, development of a woolly or
dry texture in the pulp, browning of carpelar membranes and
lower resistance to mechanical injury and fungal and microbial
attack.10,11 However, some symptoms, e.g. internal bleaching and
browning or development of an abnormal texture in the pulp,
are not externally visible. This different symptomatology indicates
that the problem of CI is not simple, because the mechanisms
involved differ as a function of the crop and become apparent
in many different ways. Some studies suggest that the texture
changes of certain CI-sensitive fruits during low-temperature
storage are due mainly to disorders of the cell wall metabolism,
including a decrease in the solubilisation and depolymerisation of
pectins.12 16
Also, it has been observed that the symptoms of this
physiological disorder become more evident when fruits and
vegetables are transferred to room temperature and that they can
even be undetectable until this reconditioning occurs.17,18
Preharvest factors influencing CI are mostly related to tem-
peratures experienced during fruit development or at harvest.
Generally, it has been observed that exposure of fruits on the
tree to high temperatures, particularly close to or at harvest, may
induce tolerance to CI during postharvest storage.19 Hass avoca-
dos exposed to direct sunlight on the tree, which in New Zealand
frequently reached 35 C, had lower levels of CI.20 However, there
are exceptions to this general observation. In Delicious apples, re-
duced susceptibility to superficial scald, defined as a CI symptom,21
has been related to relatively low preharvest temperatures, rainfall
and sunshine, while late season high temperatures resulted in
higher susceptibility to scald.22 Also, in Tomua kiwifruit, acclima-
tisation by low preharvest temperatures reduces its susceptibility
to CI.23
In the particular case of fruits, their CI sensitivity during low-
temperature storage seems to be dependent on the stage of
ripening, but in different ways according to the plant species.
In the case of banana the fruits most sensitive to CI come from
bunches at an advanced ripening stage.24 However, in avocado,
honeydew melon and tomato the CI tolerance is higher in the
postclimacteric stages, once autocatalytic ethylene production
has taken place and the fruits are in the last stages of ripening.25 27
These observations point to the existence of a more or less complex
link between developmental stage, ethylene production and CI.28
ALTERATIONS IN CELL MEMBRANE CONFOR-
MATION AND STRUCTURE: THE FIRST PHYSIO-
LOGICAL RESPONSE TO LOW TEMPERATURES
Alterations in biomembrane conformation and structure are
considered the first events at the molecular level of CI having
an effect on membrane permeability.3,8,29 Different forms of cold
damage to the cell membranes have been shown, with increase
in electrolyte leakage,27,30 32 lipid phase transitions at critical
temperatures, shown in Arrhenius diagrams,33 and changes in
lipid composition being among the most significant. The changes
in the lipid composition of the membrane during storage at
low temperatures are similar to those during senescence.32
Among these modifications, the most significant are lipid
peroxidation, increase in the saturation index of fatty acids,
556
degradation of phospholipids and galactolipids and increase in
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the sterol/phospholipid ratio.34 42 All these changes in the lipid
composition of the membrane cause a decrease in fluidity that
finally turns into a decrease in the functionality of the membrane
and of the proteins associated with it.32
Of all these changes, the degree of saturation of membrane
lipids is one of the most significant parameters with regard to
assessment of the functionality of biological membranes and
therefore the survival of organisms at low temperatures.43 It
has been observed that organisms adapt themselves to low
temperatures by increasing the proportion of unsaturated fatty
acids and therefore membrane fluidity. This adaptation implies
the induction of fatty acid desaturase (FAD, EC 1.14.19.13)
activity,44 which participates in fatty acid unsaturation, and some
specific isoforms of glycerol-3-phosphate acyltransferase (GPAT,
EC 2.3.1.15).45 The GPAT enzyme is associated with changes in the
fatty acid composition of plastid membranes, where it is localised in
the mature protein.45,46 The significance of the degree of saturation
of membrane lipids in the sensitivity to low temperature has been
demonstrated using genetically modified plants that overexpress
or silence one or some of the enzymes involved in lipid metabolism.
Murata et al.45 observed a higher tolerance to CI in transgenic
tobacco plants having a higher level of unsaturated fatty acids due
to the overexpression of a transgene that codifies for an isoform of
GPAT of Arabidopsis thaliana, but the contrary was observed when
the transferred and overexpressed transgene was a GPAT isoform
from squash. Thus, depending on the GAPT isoform, the effects on
CI sensitivity are opposite. Wolter et al.46 observed that transgenic
plants of Arabidopsis that overexpressed a gene for a GPAT from
Escherichia coli showed lower levels of unsaturated fatty acids and
a phenotype of high sensitivity to CI. In this last case the transferred
and overexpressed gene, known as plsB, encodes an isoform for
GAPT specific for saturated fatty acids.46 Depending on the type
of fatty acids that the GPAT isoform uses as substrate, it is able to
stimulate CI sensitivity or tolerance: if the fatty acids are saturated,
then CI sensitivity is induced; if they are unsaturated, the opposite
occurs.
With regard to the action of FAD, Hugly and Somerville47
detected CI-hypersensitive phenotypes in mutant plants of Ara-
bidopsis which showed lower desaturase activity due to enzymic
lesions in diverse FAD isoforms. The authors found a correlation
between the chlorosis observed in the mutants at low temperature
and the degree of reduction in polyunsaturated lipids.47 Gener-
ation of desaturase-overexpressing and knockout plants showed
that these genes could contribute to the degree of perception of
low temperatures.48 In Arabidopsis the transcript of the FAD8 de-
saturase gene accumulates upon low-temperature treatments.49
Likewise, the down-regulation of a putative aminophospholipid
translocase gene, known as ALA1 (from aminophospholipid AT-
Pase1), in Arabidopsis resulted in cold-affected plants which were
much smaller than wild-type ones.50 The ALA1 gene product is
involved in the generation of membrane lipid asymmetry, and the
authors suggested the existence of a link between regulation of
transmembrane bilayer lipid asymmetry and cold adaptation in
plants. However, in other studies, no differences were observed
regarding the cold sensitivity of mutant plants of Arabidopsis with
lower contents of unsaturated fatty acids when they were kept for
7 days at 2 C, but when this period was prolonged to 2 weeks, a
delay in the growth of the mutant plants was found, and when this
period lasted 4 weeks, the mutant plants died while the wild-type
plants survived.51
Sharom et al.52 only observed a significant increase in electrolyte
leakage in tomatoes stored at 5 C when they were transferred
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to 25 C. This observation supports the assertion mentioned in
the introduction stating that the CI effects, in this case on cell
membranes, are not observed until the fruits are transferred to
room temperature. Similar conclusions have been attained from
experiments on cell membrane integrity and lipid composition in
peppers stored at 2 C and transferred afterwards to 20 C.38
In order to explain this physiological disorder of cell membranes
and its manifestations at the molecular level when CI occurs, Lyons3
proposed the so-called membrane theory, which postulates that
thermotropic changes in membrane lipids constitute the primary
response to the physiological disorders unleashed by cold and
are responsible for the alterations in the molecular and structural
organisation of the lipid matrix of cell membranes. This theory
supports the idea that the immediate effect of low temperatures
is a global increase in the microviscosity of this matrix due
to a diminution of random rotation or folding movements of
the aliphatic chains of fatty acids and to a reduction in the
degree of desaturation of these acids. Below a critical value of
temperature, called the transition temperature, this event leads to
a reorganisation of the membrane lipids in a rigid structure called
solid gel. If the storage period at low temperature is too long,
then the membrane regions affected by these rigid structures
spread to the whole membrane, causing losses of elasticity that
lead to functional disorders of the membrane proteins and finally
to ruptures of the membrane itself, causing leakage of water, ions
and metabolites from inside the cell.
As a consequence of the previous phase change of cell
membranes, which is the primary physiological response of
sensitive species to cold, a cascade of secondary responses arises,
such as loss of turgor, electrolyte leakage, loss of metabolic energy,
disintegration of the photosynthetic systems and, finally, cell lysis.3
To end this section, it is worth mentioning the possible rapport
between changes in the lipid composition of mitochondrial
membranes during CI development and the resulting loss of
metabolic energy of the affected cells. The fact that CI causes
disorders in mitochondrial respiration has been known for a long
time.29,53 Regarding the metabolic energy, Lyons and Raison,29
in a study of the oxidative activity of mitochondria from cells
of cold-sensitive plant tissues, posed the hypothesis that the
loss of integrity of mitochondrial internal membranes, due to a
phase transition from liquid crystalline to solid gel caused by low
temperatures, was responsible for the decoupling of the oxidative
phosphorylation, i.e. of the electron transport chain and the ATP
biosynthesis. This series of events only occurred when the cell
membrane damage due to the persistence of low temperatures
was irreversible. The mitochondria are not affected per se if the cold
period is relatively short. Only a slight decrease in the respiration is
observed in this case, but in general the oxidative phosphorylation
is not affected.29,54,55 The decrease in the mitochondrial respiration
seems to be related to changes in the fatty acid composition of
the mitochondrial membranes, which affects the temperature of
the cell membrane phase transition.55,56
OXIDATIVE STRESS: THE SECONDARY
RESPONSE TO CI
Apart from the direct effect of low temperatures on the molecular
organisation of membrane lipids, the loss of integrity of the
membrane itself is boosted by oxidative processes, since, as
has been described previously, low-temperature stress increases
the levels of reactive oxygen species (ROS). These molecular
species are responsible for some of the alterations caused by
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low temperatures in plants.57 Oxidative stress occurs when
cell homeostasis is perturbed and there is a disproportionate
increase in ROS production.57 ROS are partially reduced forms of
atmospheric oxygen derived from its excitation to produce singlet
oxygen or from the transference of one, two or three electrons to
generate a superoxide radical, hydrogen peroxide or a hydroxyl
radical respectively. All of these can oxidise uncontrollably the
different cellular components and lead to oxidative destruction of
the cell. The abnormal upsurge of the ROS content comes mainly
from shifts in the photorespiration process, the photosynthetic
mechanism and the mitochondrial respiration.58 If the plant cannot
counteract this abnormal proliferation of ROS caused by oxidative
stress, then cell oxidative death occurs owing to induction of
oxidative processes such as cell membrane disintegration via lipid
peroxidation, protein oxidation, enzymatic activity inhibition and,
finally, damage occurring to DNA and RNA.57,58
In plants the chloroplast is a particularly abundant source of
ROS owing to the highly energetic reactions of photosynthesis
and the abundant supply of oxygen. A high light intensity can lead
to an excess of photosystem I reduction in such a way that the
rate of fixation of CO2 cannot be maintained and the deposition
of NADP+ is reduced. Under these conditions, oxygen competes
for the electrons of photosystem I, causing the formation of ROS.
When the CO2 fixation is limited by an environmental stress such as
CI, owing to the loss of functionality of membrane proteins caused
by alterations in their structure and conformation, an excess of
photosystem I reduction and ROS production is observed.59 It has
been observed that chloroplasts are especially CI-sensitive cell
organelles in CI-sensitive species such as tomato and pepper.37,38
As has been mentioned already, the decoupling of the electron
transportation chain from the oxidative phosphorylation also
occurs in the mitochondria, and, since the metabolism of electron
transference is affected by alterations in the functionality of
membrane proteins involved in this process, an induction of
ROS production occurs.58,60 These changes in the constitution
and functionality of mitochondria, together with those of the
chloroplast, cause an abnormal production of ROS and therefore
an oxidative stress in CI-sensitive tissues.58,59
Another source of ROS production is the activation of NADPH
oxidases (EC 1.6.3.1) localised in cell membranes, which brings
about a massive production of the superoxide anion.61,62 This event
leads to peroxidation of membrane lipids.63 The multiplication of
ROS along with the resulting lipid peroxidation in damaged leaf
tissue due to low-temperature stress is a common symptom in
species and cultivars sensitive to this physiopathy.64 66
Different studies have proven that the response of phospholipid
catabolism to low temperatures is associated with a rise in the
activities of phospholipase D (EC 3.1.4.4) and lipoxygenase (LOX,
EC 1.13.11.12). LOX is a key enzyme in the triggering of lipid
peroxidation of the plasma membrane, causing a decrease in
the lipid desaturation level and membrane fluidity.42 The CI
development in different plant species is accompanied by an
increase in LOX activity.67 71 Heat treatment at 37 C for 24 h prior
to low-temperature storage caused a decrease in LOX activity and
CI incidence in cucumber,70,71 thus confirming the significance of
LOX activity in the development of this physiopathy.
Plants are protected against the effects of ROS by a complex
antioxidant system including antioxidant metabolites such ascor-
bate, glutathione, -tocopherol and -carotene and antioxidant
enzymes such as superoxide dismutase (SOD, EC 1.15.1.1), catalase
(CAT, EC 1.11.1.6), ascorbate peroxidase (APX, EC 1.11.1.11) and
557
glutathione reductase (GR, EC 1.8.1.7).58,72,73 In the particular case
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of the enzymatic systems, when the balance between the activities
of antioxidant and pro-oxidant enzymes is lost, an accumulation
of ROS occurs. The oxidative stress is stimulated by inhibition
of the activity of the antioxidant enzymes responsible for ROS
removal, as has been observed in citrus fruits74 and corn72 which
were stored at low temperatures and suffered CI. The antioxidant
enzymes whose activity seems to have a major influence on the
degree of sensitivity or tolerance to this physiopathy are CAT,
peroxidases and SOD.69,75 78 CAT seems to be one of the first and
main enzymes of the plant antioxidant system that is activated as a
response to oxidative stress caused by CI. Different findings seem
to confirm this hypothesis. In the tomato cultivar Micro-Tom, this
enzymatic activity exhibited a patent increase in fruits stored for
4 weeks at 4 C and then reconditioned at room temperature. Of
the other antioxidant enzyme activities analysed, SOD, APX and
GR, only the latter showed an increase in activity, but it was not
as high or sustained and, unlike that of CAT, took place during the
period of storage at 4 C.79 In a study of low-temperature storage
of CI-tolerant and sensitive mandarin varieties, Sala74 observed
a similar increase in SOD activity in both groups, but the toler-
ant varieties showed higher activities of CAT, APX and GR. In an
experiment carried out using genetically modified Charentais can-
taloupe melon with inhibited autocatalytic ethylene production,
the emergence of CI resistance was observed in comparison with
untransformed fruit. This resistance was reflected in an increase in
CAT and SOD activities, in the former case during low-temperature
storage and subsequent room temperature reconditioning and in
the latter case after reconditioning.80 A study of peach storage
in modified atmospheres indicated that the diminution of the
activity of these two antioxidant enzymes was associated with the
triggering and development of CI in this fruit.69
Several experiments using a strategy of genetic transformation
have been carried out which seem to confirm that both enzymes,
CAT and SOD, are somehow involved in the defence mechanisms
activated when CI occurs in plants. An assay with genetically
modified tomato plants seems to confirm that the catalase isoform
CAT1 is a key element of the antioxidant systems triggered as a
response to oxidative stress in this species, whether associated
with CI or not. In tomato plants modified genetically by means of
antisense RNA technology, with the aim of blocking CAT1 gene
expression, the sensitivity to oxidative stress per se, imposed
by application of 30 mL L1 H2 O2 , increased significantly in
comparison with untransformed control plants. Likewise, these
genetically modified plants were very sensitive to CI and were
unable survive to an exposure to 4 C, unlike control plants.76 Also,
the genetic modification of tobacco plants by the introduction
into their genome of a clone of a Cu/Zn-SOD isoform from pea
induced an increase in tolerance to oxidative stress caused by
exposure to low temperature (3 C).81
The ascorbateglutathione cycle is also an important mech-
anism in the removal of ROS in plants. Its activation seems to
produce a positive effect by inhibiting the development of CI. Sato
et al.82 showed that the induction of APX activity by means of a
heat treatment is a key element in the protection of rice against a
later exposure to low temperatures. The glutathione (GSH) content
and GR activity are also significant in plants showing tolerance to
low temperatures. Cold-tolerant genotypes of tomato,83 Sorghum
bicolor,84 corn85 and wheat86 accumulate more GSH, and, during
cooling, their GR activity is higher than that of sensitive genotypes.
Moreover, the rise in GSH content and GR activity in cold-sensitive
558
genotypes of corn caused a diminution of CI.87 Finally, as men-
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tioned above, GR activity is induced during reconditioning at room
temperature in Micro-Tom tomatoes kept previously at 2 C.79
Another way of reducing ROS levels in plants is the prevention of
their formation. This can be achieved by the activation of a group
of enzymes called alternative oxidases (AOXs). AOXs are able to
divert the flow of electrons from the electron transport chain and
use them instead for reducing O2 to water. In this way, AOXs
reduce ROS production by means of two mechanisms: preventing
the reduction of O2 to superoxide radical and reducing the amount
of O2 , the substrate for the formation of ROS in cell organelles.58
Some studies have pointed out the significance of this enzyme
in CI development and have shown that AOX mRNA levels and
enzymatic activity increase during low-temperature storage of
pepper and tomato treated with methyl salicylate and methyl
jasmonate, this AOX genetic expression and protein activity being
associated with cold tolerance in these crops.88,89 Moreover, in
zucchini stored in controlled atmospheres with high O2 levels, a
decrease in CI has been observed. This diminution of CI impact is
associated with higher AOX transcripts levels and the activity of
other antioxidant enzymes such as SOD, CAT and APX.90
THE POSSIBLE ROLE OF POLYAMINES AS
PROTECTIVE AGENTS AGAINST CI
The possible implication of the plant growth regulators polyamines
(PAs) in plant defence mechanisms against different types of
stress91 93 has generated much interest with regard to plant
stress research. PAs such as the tetramine spermine (Spm), the
triamine spermidine (Spd) and the diamine putrescine (Put)
exist ubiquitously in plant organisms, and all of them behave
as antisenescent agents.94 It has been considered that they are
able to bind to negatively charged molecules as biologically
essential as phospholipids, proteins and nucleic acids owing
to their polycationic nature at physiological pH.94 Because of
the interaction of PAs with the anionic groups of membrane
phospholipids, the binding of these molecules to this group
of lipids could stabilise cell membranes under stress conditions
and therefore delay their disintegration.95 Likewise, it has been
observed that PAs have antioxidant properties as agents for
removal of ROS.96 In summary, the capacity of PAs to bind
phospholipids, firstly, and their antioxidant properties, secondly,
could explain their ability to protect cell membranes against lipid
peroxidation, one of the main symptoms of most abiotic stresses.97
Put is synthesised directly via the decarboxylation of
L-ornithine in a reaction catalysed by ornithine decarboxylase
(ODC, EC 4.1.1.17). It can also be produced indirectly from the de-
carboxylation of L-arginine, catalysed by arginine decarboxylase
(ADC, EC 4.1.1.19), through the formation of agmatine and some
other intermediate products such as N-carbamoylputrescine. The
addition of an aminopropyl moiety to one or both amino groups
of Put results in the formation of Spd and Spm respectively.
These consecutive reactions are catalysed by the Spd-synthase
(EC 2.5.1.16) and Spm-synthase (EC 2.5.1.22) enzymes respectively.
The donor molecule of the aminopropyl moiety is decarboxylated
S-adenosylmethionine (SAM), decarboxylation being achieved in
a reaction catalysed by SAM decarboxylase (SAMDC, EC 4.1.1.50).98
SAM is also a precursor of the biosynthesis of the plant hormone
ethylene; therefore an increase in PA production due to the
SAMDC activity affects the degree of ethylene production. As the
concentrations of PAs are much higher than those of ethylene
and its immediate precursor 1-aminocyclopropane-1-carboxylic
acid (ACC), changes in PA concentrations are more likely to affect
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ethylene and ACC biosynthesis than the other way round.98 As will
be mentioned in the next section, ethylene seems to be implicated
in the induction and development of CI.
With regard to the problem of low-temperature storage of fruits
and vegetables, it was observed a long time ago that the impact
of CI entailed an increase in Put in pepper, lemon and grapefruit.99
Moreover, it has been found that the rise in PA content as a
physiological response to low-temperature stress is sharper in CI-
tolerant plants than in CI-sensitive ones.100 104 These observations
seem to indicate, although not to prove, an involvement of PAs
in the induction of CI tolerance, but the ways in which these
molecules boost that tolerance are not clear.93
In general, when different postharvest technologies such
as room temperature preconditioning, hot water dipping and
modified atmosphere packaging are applied separately or in
combination in order to prolong the storage life of horticultural
products, an increase in PA content and a delay in the appearance
of CI symptoms occur simultaneously, with variations depending
on the plant species analysed, lemon, zucchini or pepper.105 107
In an experimental study of cucumber plants with a chilling-
sensitive variety and a chilling-tolerant one, Shen et al.108 observed
that the content of PAs, mainly Spd, increased significantly in the
latter variety when plants were kept at temperatures at which the
former suffered CI. Moreover, pretreatment of the sensitive variety
with Spd inhibited the oxidative stress associated with CI as well
as mitigating the general CI damage, while pretreatment of the
resistant variety with an inhibitor of PA biosynthesis caused the
appearance of CI and associated oxidative stress, so that the plants
became CI-sensitive.108
Kim et al.109 found an accumulation of Put when tomato plants
were kept at low temperatures, and they posed the hypothesis
that this PA could play a role as an inductor of CI tolerance. The
authors based their assumption on the observation that plants
treated with this PA experienced a reduction of around 30% in
the increment of electrolyte leakage, the augmentation of which
is a typical chilling-induced phenomenon, as we have already
commented elsewhere. This physiological response depends on
the dose, as Martnez-Tellez et al.110 found in zucchini treated with
the three PAs Put, Spd and Spm. This behaviour is characteristic
of biomolecules that function as plant growth regulators, as is
the case for PAs.94 Moreover, when plants were treated with an
inhibitor of Put biosynthesis, the content of this PA decreased
and the percentage of electrolyte leakage increased, an event also
dependent on the concentration applied.109
This hypothetical protective role of PAs, in particular Spd, against
chilling in plants seems to be confirmed by the observation that
transgenic plants of A. thaliana that overexpress a gene for Spd-
synthase present a manifest tolerance to different types of stress,
especially chilling.111
All these results seem to indicate that the upsurge of PA content
in fruits, other plant organs and whole plants showing tolerance
to CI during cold storage is a cause and not a consequence of
the tolerance to this stress; that is, it is a defence action of plants
against chilling rather than a symptom caused by it.
Owing to the link between PAs and protection of cell membranes
and that between CI and membrane degradation, it is reasonable
to question if there is a relationship between PAs and CI, something
we have tried to explain in this section. However, this is not the only
association that can be established between these plant growth
regulators and low-temperatures stress. As has been mentioned
already, PA levels affect the production of the plant hormone
ethylene, and, as discussed in the next section, this hormone
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seems to be a principal participant in the development of this
physiopathy.
THE ROLE OF ETHYLENE IN THE STIMULATION
OF CI AND THE INFLUENCE OF LOW TEMPERA-
TURES ON ITS BIOSYNTHESIS
Since the characteristic symptoms of ripening, such as alterations
in the structure and composition of cell membranes and the
associated oxidative stress, as observed in melon,40,112 are similar
to the effects caused by low temperatures, the hypothesis of
the possible implication of the plant hormone associated with
senescence and ripening, ethylene, in the development of CI has
been posed.7,28 Its action would be, in principle, contrary to that
of PAs, which, as we have mentioned already, are antagonists
of ethylene and antisenescent agents which act to preserve
membrane integrity.94 In many cold-sensitive plant systems,
low temperatures stimulate ethylene biosynthesis together with
development of CI, and these events have been related to an
increase in the precursors of ethylene biosynthesis or the activities
of enzymes implicated in the biosynthetic pathway.113 115
Andersen and Kent116,117 found that increases in the content
of ACC, the immediate precursor of ethylene, and ethylene
production in cucumbers occurred immediately after their
transference to room temperature. Usually, the development of
CI symptoms occurs when the fruits are reconditioned at room
temperature after cold storage, and this is correlated with a sharp
increase in ethylene production, as has been observed in many
different fruits such as cantaloupe melon,80 mango,114 pear,118
zucchini119 and eggplant120 and in different citrus fruits such as
mandarin, orange121 and grapefruit.122 This increased ethylene
production can be interpreted in two different ways, either (i) as a
simple response to low temperatures or (ii) that this ripening and
senescence hormone could induce CI in sensitive species.
The observation that ethylene treatments of different climac-
teric fruits such as avocado123 and non-climacteric ones such
as citrus fruits124 induce the manifestation of CI seems to sup-
port hypothesis (ii). Studies on avocado125 and orange126 treated
with ethylene and its antagonist 1-methylcyclopropene (1-MCP) as
well as similar assays carried out recently with climacteric and non-
climacteric varieties of plum127 seem to confirm the induction of CI
by ethylene. The role of ethylene, together with low temperatures,
in the induction of CI has been revealed in experiments carried out
with a genetically modified cantaloupe Charentais melon having
inhibited autocatalytic ethylene production.80 An apparent resis-
tance to CI, characterised by a low degree of disintegration of
the cell membranes as determined by a lower percentage of elec-
trolyte leakage, has been observed in these genetically modified
fruits, a property also observed during their ripening.80,128
However, this theory of the promoting effect of ethylene with
regard to CI cannot be generalised for every plant species sensitive
to this physiopathy; for example, in some horticultural products
a protective effect of ethylene against CI has been observed.
Wang et al.129 observed a beneficial effect of a pretreatment
with propylene, an ethylene analogue, on the resistance to CI
appearance during cold storage of banana.
Regarding the influence of low temperatures on ethylene
production, Field130 and Wang and Adams131 observed a sharp
decrease in activity of the enzyme ACC-oxidase (ACO, EC 1.14.17.4)
and a sharp decrease in the production of this plant hormone
559
during the cold storage of bean leaves and cucumber fruits at
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11 and 12 C respectively. However, Wang28 noticed that the
exogenous addition of ACC to discs of cucumber leaves stored at
5 C caused an increase in ethylene production, indicating that the
sharp reduction in ethylene biosynthesis during cold storage is
due more to the diminution of ACC synthesis than to the inhibition
of ACO activity.
As has been mentioned previously, a strong stimulation of
ethylene biosynthesis has been reported for fruits suffering CI
once they are transferred from a low temperature to room
temperature, for example in persimmon and grapefruit.122,132
This phenomenon has also been observed in whole plants, which,
under normal conditions, produce little ethylene.133,134 Field130
noted that the marked increase in ethylene production observed
in bean leaves when they were transferred from low temperatures
to room temperature was inhibited if leaves were treated with
1 mmol L1 aminoethoxyvinyl glycine (AVG), an inhibitor of the
enzyme ACC-synthase (ACS, EC 4.4.1.14). The author explained
this observation on the basis that the absence of ACC, a product
of the reaction catalysed by ACS, prevented ethylene production
by ACO.
Therefore it seems that the synthesis of ACC is the key factor
for the characteristic increase in ethylene production caused by
CI. Many studies have shown that ACC synthesis occurs not
during low-temperature storage but after re-warming at room
temperature.116,117,130,135 Wang and Adams135 found that ACS
activity increased rapidly in cucumber during the first few minutes
after transfer from 2.5 to 25 C and that the increase in ACC content
was 50 times higher during the first 7 h of reconditioning after
a storage period of 4 days at 2.5 C. Lelievre et al.136 obtained
analogous results: the transfer of Passe Crassane pear from 0 to
18 C induced a significant increase in ACS activity.
Wang and Adams135 showed that ACS activity, ACC content
and ethylene production in cucumber fruits which were cooled
and later reconditioned at room temperature were inhibited by
cycloheximide (an inhibitor of translation) but were not affected
by cordycepine or -amanitin (inhibitors of transcription).7 These
experiments show that low temperatures stimulate the synthesis of
mRNA encoding ACS and that the translation of these messengers
occurs at the moment of transfer to room temperature. In Passe
Crassane pear the same behaviour has been observed: the ACS
mRNA accumulated during storage at 0 C; when fruits were
transferred to 18 C, the level of the messenger decreased but the
ACS activity increased.136
However, in other pear cultivars such as Eldorado, ACC
accumulates during storage at low temperatures, which is
considered the process responsible for the subsequent stimulation
of ethylene production that induces fruit ripening.118 Something
similar has been demonstrated for Honey Dew melons. When
these fruits were transferred to 20 C, the ACC accumulated
during storage at 2.5 C diminished.137 These results show the
need for further research on the causes of the ability of certain
plant species, and even certain varieties of the same species, to
synthesise ACC during storage at low temperatures and the fact
that other species only accomplish it when they are transferred
from low temperatures to room temperature.
Nevertheless, experiments carried out on cucumber fruits using
labelled methionine and inhibitors of ethylene biosynthesis such
as AVG, described elsewhere,131 clearly indicate that the synthesis
and activity of the ACS enzyme are stimulated by CI and constitute
the limiting step in the ethylene biosynthesis stimulated by this
physiopathy.28 From the studies carried out with translation and
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transcription inhibitors,135 it seems that ACS transcription takes
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place during storage at low temperatures and translation when the
fruits are transferred to room temperature. This induction of ACS
transcription has also been observed in etiolated hypocotyledons
of bean maintained at 4 C for 3 h.138 The results obtained by
Wong et al.139 for low-temperature storage of citrus fruits support
this hypothesis. These authors observed an increase in ACC and
ethylene levels when fruits were transferred to room temperature
after cold storage and that these levels fell as the number of ACS
transcripts decreased.
In short, the effects of cold on the biosynthesis of the ripening
hormone are, firstly, a reduction in ACO activity, but without this
being a limiting factor for ethylene production, and, secondly,
an increased transcription of ACS that leads to an increase in
its translation upon transfer to room temperature; this, together
with an increase in ACO activity, implies an activation of ACC and
ethylene productions. It can also be asserted, from the experiments
already described using treatments with exogenous ethylene, its
analogue propylene and its antagonist 1-MCP in different plant
species, that the ripening and senescence hormone plays a role as
an inducer of CI, probably in combination with low temperatures,
although there are plant species where the contrary occurs,
ethylene seeming to act as a CI inhibitor.
The response of plant organisms to ethylene at the molecular
level is reflected in the induction or inhibition of the expression
of genes belonging to diverse families with different phylogenetic
relationships. A series of cis and trans elements of regulation of
the gene expression, characteristic of the molecular response to
ethylene, are involved in the underlying molecular mechanism.
The genes regulated by ethylene contain in the promoter
sequence a series of cis elements called EREs (ethylene-responsive
elements).140 These EREs are also known as the GCC box owing
to the sequence AGCCGCC that is essential for recognition by
the corresponding trans ERFs (ERE-binding factors).140,141 These
transcription factors, previously known as EREBPs (ERE-binding
proteins), were isolated as GCC box-binding proteins in tobacco,140
and their expression pattern has been studied in detail.142
The analysis of the genetic sequences of the A. thaliana
genome and the databanks of expressed genetic sequences ESTs
(expressed sequence tags) in different plant species has allowed
the isolation and identification of multiple genes of different plant
species that encode ERFs. These ERF genes represent a large
multigene family with many members, both monocotyledonous
and dicotyledonous. They include the different ERFs of tobacco,
Pti4, Pti5 and Pti6 in tomato and TINY, AtEREBP, abscisic acid-
insensitive 4 (AB14) and ETHYLENE RESPONSIVE FACTOR 1 (ERF1) in
A. thaliana.140,143 147 All of them include a DNA-binding region of
58 or 59 amino acids known as the ERF domain.140,148
In Arabidopsis the five ERFs isolated have the mentioned ERF
domain and display binding activity to the specific GCC box, but
their amino acid structures differ, and, while some are inductors
of the gene expression dependent on the GCC box, others repress
this expression.141 For this review of CI in plants and, in particular,
for our search for an explanation at a molecular level of how this
physiopathy could stimulate ethylene action and production, it is
interesting to highlight the fact that the gene expression for these
ERFs is regulated by ethylene but also by other environmental
factors such as low temperatures.141
These considerations on the relationship between ethylene and
CI at the molecular level lead us to a more general discussion of
the regulation of gene expression by low temperatures.
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INDUCTION OF TRANSCRIPTION AS A RE-
SPONSE TO STRESS CAUSED BY LOW TEM-
PERATURES IN PLANTS
In recent years, genes and metabolic pathways involved in the
perception and signal transduction of plant responses to extreme
temperatures have been identified.149 However, most of the
knowledge in this field has arisen from studies conducted on
the chilling-tolerant A. thaliana during cold-induced freezing
acclimation,150 but little is known about the molecular basis
of cold responses in agronomically important, chilling-sensitive
plants. The analysis of the molecular phenotypes of chilling-
sensitive mutants exposed to 13 C before any visible alteration
was brought about in the plants has led to the identification
of 634 chilling-responsive genes with aberrant expression in
all the chilling-lethal mutants, including genes related to lipid
metabolism, chloroplast function, carbohydrate metabolism and
free radical detoxification.151
Different studies focused on the analysis of the perception of
low temperature and the signal transduction to the nucleus for
induction of gene expression have shown a correlation between
calcium availability, cold acclimatisation-specific gene expression
and the development of freezing tolerance, implicating calcium
as an important second messenger in low-temperature signal
transduction.152 It has been observed that cells display differential
regulation of the members of a calcium-dependent protein kinase
gene family during the early stages of cold acclimatisation.153
The plant genes with an expression regulated by low temper-
atures form a superfamily called COR (cold-regulated), and it is
believed that they are involved in the intensification of the resis-
tance of plants against freezing or CI temperatures.154 Many genes
of this superfamily have been isolated and characterised.155,156
Among them, two genes that encode for ACS are found in
orange.139 The molecular analysis of the genes of this super-
family shows the existence of a regulator cis element that controls
the coordinated gene expression of this COR gene group in re-
sponse to cold and dehydration, known as CRT (C-repeat)157 or DRE
(dehydration-responsive element).158 The consensus sequence of
this element is made up of five highly preserved nucleotides (CC-
GAC) and can be found in the promoter of many genes responding
to cold and dehydration, including COR.159
In recent investigations into freezing tolerance in A. thaliana, a
family of transcription factors or trans elements that specifically
bind to these CRT/DRE promoter sequences, denoted CBF
(CRT/DRE-binding factor) or DREB (DRE binding), has been
isolated.150 In Arabidopsis, this family includes three members,
CBF1, CBF2 and CBF3160,161 or DREB1b, DREB1c and DREB1a162,163
respectively. These transcription factors are members of the
superfamily of DNA-binding proteins known as AP2/EREBP.164
It seems that these transcription factors are also involved in the
molecular response to CI stress in plants, as has been observed
in molecular biology experiments with a CI-sensitive species,
tomato.165 The overexpression of the Arabidopsis gene CBF1
in tomato is apparently associated with the expression of the
gene CAT1 that encodes an isoform of catalase, an antioxidant
enzyme implicated in the response to oxidative stress. Also, the
transgenic tomato displayed resistance to CI, as the evolution
of different parameters characteristic of the activation of this
physiopathy, such as electrolyte leakage, showed.166 When these
plants were kept at low temperatures, the reduction in both the
expression of CAT1 and the associated CAT activity was lower
than in unmodified control plants, where the gene expression and
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enzymatic activity even disappear.166 This observation constitutes
proof at the molecular level of a link between the triggering of a
stress due to low temperatures and the activation of antioxidant
systems by plants as a defence, and of the stimulation of an
oxidative stress as an effect of CI.
These results, where heterologous expression of the CRT/DRE-
binding factor 1 from Arabidopsis enhanced tolerance to chilling
and oxidative stress in chilling-sensitive tomatoes,166 indicate
that certain conserved mechanisms may still operate in both
chilling-sensitive and tolerant plants and even in both freezing
and chilling tolerance. Regarding this aspect, a gene homologous
to CBF1 of Arabidopsis has been isolated in tomato.167 This gene,
which encodes a CBF transcription factor of tomato, exhibits a
transitory increase in its expression induced by low temperatures,
but whether it activates the expression of COR genes or not could
not be studied, because the existence of members of this genetic
superfamily in tomato was not known at the time.167
With regard to other COR genes, several studies have led to
the identification of scores of diverse members of this gene
superfamily, many of which encode apoplastic antifreezing
proteins (AFPs), late embryogenesis abundant (LEA) proteins
or other novel polypeptides.159 Diverse attempts at improving
tolerance to low temperature by the overexpression of some of
these genes have been fruitless, which probably suggests that the
cooperation of the different products of the genes inducible by
cold is necessary for the expression of a tolerant phenotype.43
The superfamily of DNA-binding proteins previously mentioned,
AP2/EREBP, to which CBF belongs, received its name owing to
a particular region called AP2 that recognises and binds the
DNA.168,169 This superfamily has also, confusingly, received the
name of the transcription factors EREBP that bind the cis elements
called ERE, characteristic of the genes regulated by ethylene.140
However, at present, it is believed that the region of interaction
with DNA, AP2, and that of the ERF proteins are different, the
mode of recognising DNA and the recognising sequences being
different in each region.141
IMPLICATION OF ABSCISIC ACID IN THE RE-
SPONSE TO CI STRESS
Cellular abscisic acid (ABA) accumulates in response to a number of
environmental stresses, including low temperatures, and increases
in its concentration can lead to a set of physiological adaptations,
including stomatal closure and growth inhibition, as well as
induction of the expression of specific genes. In the particular
case of CI, its intervention in the triggering of this physiopathy has
been considered for a number of years.170 Promoter analysis of
cold-induced genes and expression analyses have shown that ABA
is involved in the response of plants to low-temperature stress.
From gene expression analysis it has been found that ABA is
involved in the induction of the expression of different COR genes
in A. thaliana.171 174 Recent studies on Arabidopsis indicated the
ability of ABA to induce COR gene expression via cis CRT/DRE
elements and CBF13 transcription factors, trans elements for
which activation of their genetic expression was due only to
low temperatures.175 Knight et al.175 observed that ABA was able
to induce the expression of the luciferase LUC (EC 1.13.12.7)
marker gene in genetically modified plants of A. thaliana where
a construction containing the LUC gene bound to four copies
of the CRT/DRE cis element had been introduced. The authors
also showed that ABA could bring about the expression of
561
endogenous COR genes such as LT178, also known as COR78
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or RD29A, considered as paradigm gene, the expression of which
is regulated by osmotic stress and low temperatures.158,162,176
Regarding the induction of the genetic expression of CBFs
themselves by ABA, Knight et al.175 showed that, in another type
of transgenic Arabidopsis transformed with constructs containing
the promoter of CBF13 genes bound to the -glucuronidase GUS
(EC 3.2.1.31) marker gene, this plant hormone is not only involved
in the stabilisation of the transcripts of these trans elements but
also in the activation of the CBF gene promoter, thus participating
in the induction of its transcription.
Low-temperature stress is characterised by a water deficit;
thus, in principle, ABA should have a beneficial effect on CI
tolerance, since it plays an important role in the control of
stomatal closure and therefore in the transpiration rate and
maintenance of cell water relations. In pear an increase in its
concentration was observed when fruits were stored at low
temperatures after harvesting.177 This observation was also made
during the induction of pear ripening on the tree by cold.178,179 In
zucchini an accumulation of this phytohormone during storage at
CI temperatures has also been observed.180
The role of ABA in the stimulation of CI tolerance through
the maintenance of cell water levels has been confirmed by
a comparative study of storage at 4 C for maize seedlings of
different genotypes. The CI-tolerant genotypes showed a higher
leaf accumulation of ABA, which was related to a lower water loss.
The pretreatment of these genotypes with an inhibitor of ABA
biosynthesis caused a slight reduction in their CI tolerance. This
behaviour could be counteracted if the seedlings were treated
with exogenous ABA. Therefore ABA seems to participate in the
induction of CI resistance by regulating stomatal closure, thus
decreasing dehydration, a typical symptom of low-temperature
stress.181
Kawada et al.182 observed that seasonal levels of ABA in the
flavedo of Marsh grapefruit were correlated with resistance to
CI. However, Lafuente et al.183 did not find any relation between
changes in ABA and CI impact during fruit maturation in the
highly CI-susceptible Fortune mandarin. In addition, it has been
demonstrated in this citrus cultivar that treatments that increase
ABA levels favour development of CI.183,184
In leaves of tomato plants, application of exogenous ABA caused
a significant decrease in electrolyte leakage, one of the first
detectable symptoms of CI.109 Likewise, these authors observed
an accumulation of Put caused by low temperatures. Treatments
with this polyamine and an inhibitor of its biosynthesis have
shown that this plant growth regulator is also involved in the
tolerance to low temperatures, as already mentioned elsewhere,
but it is important to note that the role of ABA in the induction
of resistance to CI seems to be much more significant than that
of Put, since the increase in electrolyte leakage in tomato leaves
treated with an inhibitor of Put synthesis was counteracted when
they were treated with ABA.109 A correlation between ABA and
Put levels and CI tolerance in different varieties of rice has been
observed by Lee et al.,185 measuring electrolyte leakage; these
authors suggested that the ABA and Put concentrations could be
physiological markers for the resistance of rice to CI.
The physiological mechanism by which ABA reduces CI impact
in plant tissues is not known in detail. Exogenous application of
ABA has been used to induce stomatal closure prior to refrigerated
storage in order to prevent water loss and therefore CI.186,187
Interestingly, in tobacco callus, which has no stomata, ABA also
reduced CI.188 Owing to the lipophilic nature of ABA, it has been
562
speculated that the hormone could interact with or insert itself
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among the hydrophobic tails of the cell membrane structure,
increasing the transition temperature and therefore fluidity.189
THE SYNTHESIS AND ACTION OF SPECIFIC
PROTEINS IN RESPONSE TO CI STRESS
In response to abiotic stress, all living organisms synthesise new
proteins.190,191 All organisms respond to high temperatures and
other stress conditions with the induction of the synthesis of
a group of proteins known as heat shock proteins (HSPs).192 194
Often the accumulation of HSPs not only confers protection against
the stress that causes their biosynthesis but also against any other
subsequent stress situation.195,196
Under normal growth conditions, most HSPs are usually
undetectable in vegetative tissues, but they accumulate rapidly
in response to heat shock. Synthesis and accumulation of HSPs
are proportional to the temperature and the duration of the
stress period.194,197 Although HSPs were first characterised in
response to high temperatures,198,199 this observation being
the origin of their name, heat shock is not the only stimulus
triggering HSP gene expression and protein biosynthesis. Among
the other physiological conditions in plants inducing accumulation
of HSPs are fruit ripening,200 203 low temperature204 206 and
oxidative stress.207 210 Precisely, these conditions are involved in
CI development in sensitive organisms.
Sabehat et al.211 and Lurie et al.212 observed that a short
exposure of tomato fruits to a moderately high temperature
(38 C) protected them from CI during their later storage at low
temperatures. This high-temperature stress over a short period of
time (38 C for 4872 h) induced the synthesis of HSPs that, in a
classical response of cross-tolerance, induced resistance against
CI. When heated fruits were transferred to room temperature for
4 days prior to storage at low temperature, the level of HSPs
decreased markedly.211,212 If, after 4 days at 20 C, fruits were then
transferred to 2 C for 21 days, the level of HSPs was not recovered
and fruits lost their ability to tolerate low-temperature storage.205
In a previous experiment with tomatoes exposed to heat shock
(3 days at 38 C), Lurie et al.213 detected the accumulation of
mRNA of both high-molecular-weight (hsp70) and low-molecular-
weight (hsp17) HSP transcripts during storage at 2 C for 21 days.
These tomatoes ripened normally after transferring them from 2
to 20 C and did not develop CI symptoms. Meanwhile, control
tomatoes not subjected to heat treatment suffered CI and ripening
impairment after cold storage.213 Following on from the results
of Sabehat and Lurie described above, other workers have
published studies associating the induction of HSP biosynthesis
with acquisition of tolerance to low temperatures.214 220
In addition to high temperatures, treatments with methyl
derivatives of jasmonic acid, a plant growth regulator, and/or
salicylic acid, a molecule participating in cell signalling, prior
to low-temperature storage induce HSP biosynthesis and, at
the same time, CI tolerance in tomato201 and peach.219 These
chemical treatments that induce HSP biosynthesis and inhibit CI
development are widely described at the end of the next section.
Although the mechanism of cell protection against stress
activated by HSPs is not clear, there is much evidence show-
ing that they could play their protective role as molecular
chaperones.194,221 223 Molecular chaperones are proteins that
assist other proteins in maintaining or recovering their native
conformation by the stabilisation of partially denatured protein
conformations. During stress situations there is a denaturation and
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dysfunction of many proteins. HSPs play their role in the preven-
tion of these processes by exerting a control on the proper folding
and conformation of both structural and enzymatic proteins.224
HSPs are also involved in the renaturation of partially denatured
proteins and the elimination of fully denatured ones. This chaper-
one activity of HSPs has been shown both in vivo and in vitro225 230
and is reviewed in Ref. 223.
The protective effect of low-molecular-weight HSPs (small
HSPs, sHSPs) against stress is due not only to their chaperone
activity but also to their function as membrane-stabilising
factors.44,231,232 Tsvetkova et al.232 described how the interaction
of sHSPs with membrane lipids modifies the properties of
these biomolecules, inducing the augmentation of solid gel
membrane structure fluidity, with an associated protective effect
against low temperatures, and rigidifying the liquid crystalline
membrane state, with an associated protective effect against high
temperatures. Therefore, owing to their roles as both molecular
chaperones and molecules stabilising cell membranes, HSPs are
able to exert their protective effect against CI.
POSTHARVEST TECHNOLOGIES FOR INHIBIT-
ING OR DELAYING CI
The disorders caused by low temperatures during ripening and
senescence of chilling-sensitive horticultural products negatively
affect their quality and therefore their marketing.233 236 Today
there are different postharvest technologies for delaying or
inhibiting the development of CI during the storage of sensitive
plant species. Some of these technologies have a physical nature
and consist mainly of changes in temperature, relative humidity
or gaseous composition of the atmosphere during the storage
of fruits and vegetables. They are employed most commonly
at the industrial level; for example, conditioning at moderate
temperatures,75 pretreatment at high temperatures,237 treatments
with CO2 before or during storage,238,239 intermittent warming240
and storage in controlled atmospheres (CA)241,242 or modified
atmospheres (MA).243,244
MA and CA postharvest technologies base their effectiveness
on obtaining atmospheres around the fruits or vegetables with
low levels of O2 and high levels of CO2 during storage at low
temperature.245 By controlling the concentrations of these two
gases, we can optimise storage and minimise the impact of CI, as
has been observed in different fruits such as avocado, nectarine,
guava and bitter-melon.246 249 The MA technology is widespread
and gives excellent results in the prolongation of the cold storage
of fruits and vegetables. Its use during postharvest handling is
greater than storage in CA and other technologies, mainly owing
to its low cost, easy implementation and high benefits resulting
from an increased storage period without quality losses.245,250
MA storage, by generating an atmosphere rich in CO2 and poor
in O2 , reduces the respiratory rate and the production of and
sensitivity to ethylene, thus delaying ripening and senescence, and
contributes to the maintenance of a high relative humidity (RH)
around the fruit, which prevents or delays water loss.233,245,250 252
Although this is also the basis of CA storage, with MA there
is a lower control of the gaseous concentrations around the
horticultural product.233,245,250 In this case the use of plastic films
of a polymeric nature with selective permeabilities towards CO2
and O2 has revolutionised the technology of MA storage, where
the final equilibrium atmosphere is achieved naturally inside the
sealed package and is the result of both the balance between the
consumption of O2 and the production of CO2 by the produce
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and the gaseous diffusion through the polymeric film of the
package.233,245,250,253 255 The beneficial effects of this kind of
storage have been tested using different plastic films, e.g. the
classical polyethylene and the newest Xtend , with positive results
regarding the inhibition of the development of CI symptoms in
different horticultural products of high commercial interest such as
American cantaloupe melon,256 tomato,257 pepper,258 banana,259
avocado260 and mango.244
However, the packaging of pepper in perforated films also
results in a delay in the appearance of CI when compared with
non-packaged control fruit.261,262 Similar results were obtained
for cucumber and avocado.243,260 An MA is not generated by
using perforated films, so the explanation of the inhibition of
CI development is mainly the creation of storage conditions
with high RH, which prevents an excessive loss of water by the
fruit.261 Precisely, a high water loss by the fruit, apart from the
appearance of skin depressions and discolourations, is one of
the main symptoms of CI in peppers.262 These results indicate
that, in some situations and for some fruits, there is also a
possibility of simplifying even more this storage technology by
using perforated films, although there are some exceptions. In the
case of Paraguayo peach the CI symptoms are not reduced by
using packaging with macroperforated polypropylene films.263 In
orange and grapefruit the reduction in CI symptoms was greater
when microperforated Xtend films that generate MA were used
in comparison with macroperforated Xtend films that do not
generate MA.264
There are two other technologies related to CA storage
which have been applied for reducing CI impact in horticultural
products. Both are based on short pretreatments of the produce
with atmospheres of particular gaseous compositions prior
to low-temperature storage. The first consists of anaerobiosis
pretreatment with atmospheres of very low O2 . The pretreatment
of avocado with atmospheres of 3% O2 for 24 h at room
temperature reduced CI symptoms after storage at 2 C for
3 weeks, and treated fruits showed lower respiration, ethylene
production and electrolyte leakage than untreated ones.265
The second consists of pre-storage CO2 treatments of the
CI-sensitive horticultural product. In experiments with lemon
fruits, pretreatments with 40% CO2 for 3, 6 or 9 days reduced
membranosis and rind pitting, typical CI symptoms in citrus fruit,
after 30, 60 and 90 days at 0 C.238 The risk in both technologies
is the induction of fermentation and therefore the appearance of
off-flavours.266,267
Postharvest treatments with high temperatures have been
employed for controlling insects, avoiding fungal rot, mod-
ifying the response of fruits to other kinds of stress and
maintaining the quality of fruits during storage.234 236,268,269
Thus hot water treatments and curing are currently prac-
tised and recognised as methods of controlling postharvest
diseases by direct inhibition of the pathogen and by stimu-
lating certain host defence responses.270 Treatments at high
temperatures prior to cold storage have also been beneficial
in reducing CI symptoms in citrus fruits,78,218,271 275 grapes,217
pomegranates,276 tomatoes,277 279 mangoes,280 avocados,281,282
rice,82,283 cucumber71 and peppers.107 However, in some cases
the response to high temperatures can be specific to each cul-
tivar. For example, Whitaker284 did not find benefits in Rutgers
tomato conditioned at high temperatures prior to storage at low
temperatures. The time and temperature of exposure with re-
spect to improving or maintaining fruit quality will depend on
563
the cultivar, ripening state and growth conditions.285 Other fac-
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tors such as fruit shape and size, which affect the uniformity of
the treatment, must be taken into account, since the warming
is produced as a temperature gradient from the surface to the
centre of the fruit.269 Different methods have been employed for
the application of high temperatures: hot water dipping (HWD),
hot water rinsing and brushing (HWRB), water vapour and hot
air.268,273,285 Different studies recommend the use of short heat
treatments such as HWD and HWRB because they do not modify
the quality parameters of the fruit in comparison with longer
treatments.273,285 Other advantages found in the application of
these short heat treatments are the protection of the fruit against
rots that can appear during postharvest,272,273,278,285 their easy
application and low cost271,273,285 and, since they are useful in
cleaning the fruit, the improvement of the general appearance
of the fruit.273,286 This protection against CI caused by high tem-
peratures before cold storage can be explained by the induction
of HSPs, which, as we have mentioned already, are synthesised
rapidly as a response to high temperatures. However, this protec-
tive effect is not only due to the presence of HSPs, as it has also been
observed that in heat-treated fruit the desaturation index of mem-
brane lipids is higher, and therefore membranes are more fluid,
and the electrolyte leakage during subsequent low-temperature
storage is lower.268 High temperatures also cause an increase
in antioxidant activity.78,82,217,275 Some reports relate the protec-
tive effect of high temperatures to an increase in the synthesis
of polyamines.106,107,276 Likewise, long-term heat-induced chilling
tolerance in citrus fruits seems to be an active process requiring
new transcription factors, activation of secondary metabolism and
stress-related proteins.287
Conditioning at intermediate temperatures prior to storage
at lower temperatures (LTC) has been applied successfully for
reducing CI in grapefruit,218,273,288 loquat289 avocado290,291 and
papaya.292 The key factors for the application of this technology are
the difference in temperature between the conditioning and the
cold storage and the duration of the conditioning treatment.289,290
Although Hofman et al.291 did not observe benefits of the
combined application of the two treatments (treatment with
hot water and LTC), Wang106 and Sapitnitskaya et al.218 observed
that the consecutive application of the two treatments was much
more effective for reducing CI than their separate application. This
suggests that each treatment activates different defence pathways
in response to low temperatures. The heat treatment induces
mainly the expression of different stress genes that encode HSPs
and universal stress proteins, while the conditioning increases
the expression of genes that encode enzymes which modify the
composition of membrane lipids, such as FADs and lipid transfer
proteins.218
Intermittent warming (IW) consists in the periodic interruption
of storage at low temperatures by the exposure of fruits to
short periods of warming. This technology has been effective
for reducing CI in citrus fruits,293 297 pomegranate,298 apple,299
tomato,240,300 peach301 305 and nectarine.306 The main difficulty
for the application of IW is to find the optimal conditions of
temperature, duration and frequency, which depend on the
cultivar, fruit-ripening stage and growth conditions.307 Moreover,
the interruption of cold storage must take place before the
development of CI symptoms, when the physiopathy damage
is still reversible. Although IW has been effective for reducing
CI in different fruits, the mechanism by which it reduces CI
impact is not quite clear. Fernandez-Trujillo and Artes302 and
Fernandez-Trujillo et al.304 observed that IW maintained the quality
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of fruits, allowing their ripening as a result of the production of an
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adequate quantity of ethylene. Likewise, Zhou et al.305 observed
that ethylene production during storage at low temperatures
was higher in IW-treated fruits than in control, non-treated fruits.
These authors also observed that IW treatment increased the
levels of mRNA encoding ACO and ACS, the enzymes of the
ethylene biosynthetic pathway. The authors suggested that IW
prevents inhibition of ethylene production in treated fruit and
helps to maintain the fruits capacity to ripen normally during the
subsequent shelf-life period at 20 C.
As we have mentioned, ethylene is involved in the stimulation
of CI. Some postharvest technologies can reduce CI development
by inhibiting the production and/or action of this hormone. Some
of the technologies described use directly or indirectly the levels
of CO2 and O2 to prevent ethylene biosynthesis and reduce the
stimulation of CI, as with MA and CA storage. The O2 is necessary
for ACO activity and therefore ethylene biosynthesis, and, in this
way, low levels of O2 reduce ACO activity.308 The role of CO2 in the
regulation of ethylene biosynthesis and action is quite complex.
On the one hand it is necessary as a cofactor for in vitro ACO
activity,309 but on the other hand it seems to have a role as an
inhibitor of ethylene biosynthesis, probably at the step in which
its immediate precursor ACC is formed.310
Regarding the manipulation of ethylene biosynthesis and/or
action, two novel strategies can be considered for reducing
CI during cold storage of horticultural products. One is the
application of biotechnology. Throughout this review, different
examples of genetically modified plants with CI tolerance during
low-temperature storage have been mentioned. In this section we
will mention only the particular case of transgenic plants showing
inhibition of autocatalytic ethylene production, and the effect of
that modification on tolerance/sensitivity to CI. The generation
of a transgenic Charentais cantaloupe melon expressing an
antisense ACC oxidase gene inhibited the autocatalytic production
of ethylene by 99%.311 The genetically modified melon, unlike
the control, untransformed melon, shows tolerance to CI both
during and after storage at 2 C.80 Likewise, when MA technology
is also applied, there is an additive effect on the resistance to
CI during cold storage and the subsequent shelf-life period.312
With regard to these results in antisense ACO melon, it can be
assumed that modified tomatoes constructed by overexpression
of a heterologous gene for SAM hydrolase (EC 3.3.1.2)313 or ACC
desaminase (EC 3.5.99.7),314 or by expressing an antisense ACO315
or ACS316 mRNA, should show higher CI tolerance during storage
at low temperatures than control, untransformed fruits.
Another strategy is the use of antagonists of ethylene action
and/or biosynthesis. 1-Methylcyclopropene (1-MCP) is a cyclic
olefin that binds competitively, specifically and irreversibly to
ethylene receptors. It acts as an inhibitor of ethylene perception
or, in other words, it is an antagonist of the action of this
plant hormone.317 319 1-MCP blocks the action of the receptors,
preventing the transduction of the signal and the activation of
ethylene-dependent responses in the cell. However, 1-MCP has
a limited efficiency, because plants can synthesise new receptors
and recover their sensitivity to the hormone. As has been discussed,
ethylene seems to induce or intensify the appearance and
development of CI in different horticultural species, although there
are some exceptions. Therefore the hypothesis that treatment
with 1-MCP stimulates the resistance to the appearance of this
physiological disorder in fruits and vegetables can be considered,
but different results have been obtained. Thus a reduction in
CI symptoms due to treatment with this compound has been
observed in persimmon,320 pineapple321 and cantaloupe melon80
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Postharvest technologies to reduce impact of chilling injury
when it was applied at 0.3, 0.1 and 1 L L1 for 24, 18 and 24 h
respectively prior to refrigerated storage. In climacteric cultivars
of plum that, unlike non-climacteric ones, are sensitive to CI, a
significant reduction in CI incidence has been observed in fruits
treated with 0.4 L L1 1-MCP at 0 C for 24 h.127 In avocado with higher doses (500 mol L1 ).330 Apart from the induction
a reduction in the internal symptoms of CI such as mesocarp
bleaching,125 but not in the external ones such as skin browning,322
has been observed when application of 1-MCP was performed
at 0.1 L L1 for 48 h and 0.5 L L1 for 12 or 24 h, both at the diminution of the decay incidence during low-temperature
relatively low temperatures (315 C). Pretreatments of different
apple cultivars with 0.51 L L1 1-MCP at room temperature
for 712 h, alone or in combination with CA storage, slightly
decreased the incidence of CI symptoms, which in this fruit is
manifested as surface scald.323,324 In the case of pear, pretreatment
with 1 L L1 1-MCP at 0 C for 12 h also inhibited the appearance subsequent cold storage protects against CI, has practical
of this physiopathy, but at the cost of inhibiting fruit ripening.325
By contrast, in orange fruit, treatment with 0.1 L L1 1-MCP for
6 h increased CI symptoms.126 Similar results have been found in
apricot, where 1-MCP applied at concentrations up to 1 L L1
for 20 h at 20 C prior to cold storage does not prevent internal
flesh browning, a characteristic CI manifestation in this fruit.326
Intriguingly, the opposite occurred when 1-MCP treatment was
performed after cold storage.326
All these results make one think that this antagonist of ethylene
can induce resistance to CI in sensitive fruits during and after
storage at low temperatures. From a commercial point of view, in
the agro-food industry, 1-MCP has great advantages in one or more
of the following aspects when compared with other, analogous
agents, namely CO2 , silver thiosulfate and norbornadiene: it is very
effective and stable, it is not needed in ethylene biosynthesis and
it is not toxic. As a conclusion about the use of 1-MCP with the aim
of inhibiting CI, it is important to note that the dose and period of
application of 1-MCP required depend greatly on the considered
horticultural species, and even on the cultivar, ripening stage and
temperature of treatment.
Another strategy to reduce or inhibit CI appearance is treatment
with regulatory compounds such as PAs, methyl jasmonate (MeJA)
or methyl salicylate (MeSA). Wang and Kramer327 observed in
zucchini and McIntosh apple that exogenous treatment with PAs
induced resistance to CI. In zucchini, pretreatment by infiltration
with increasing doses of different PAs reduced CI impact and
electrolyte leakage during storage at 2 C in comparison with
control, untreated fruits. This decrease in the degree of CI only
occurs in a certain range of doses of PAs, since they are ineffective
when the dose used is too high, behaviour typical of substances
that function as plant growth regulators.110 The application of
10 and 100 mol L1 MeJA for 8 h at 20 C clearly inhibited of lipids, essential components of cell membranes. The difference
the appearance of CI in guava, assessed visually and by the
determination of electrolyte leakage, after storage at 5 C and
during shelf-life at 25 C.328 Likewise, postharvest application
of MeJA (10 mol L1 ) effectively inhibited green mould decay
and reduced CI symptoms after 6 weeks of storage at 2 C
plus 4 days at 20 C in grapefruit, indirectly by enhancing the
natural resistance of the fruit to Penicillium digitatum at low
temperatures.329 In tomatoes treated with 10 mol L1 MeJA or
MeSA for 16 h at 23 C, or with a 48 h heat pretreatment at 38 C,
the appearance of CI resistance in fruits after storage at 5 C and
shelf-life at 20 C was observed. These treatments induced the
genetic expression of different sHSPs (from families class I and
II) and high-molecular-weight HSPs (hsp70), the levels of which
were maintained during subsequent storage at low temperatures
and could be related to the induction of CI tolerance.201 The
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efficacy of the treatment with regard to the induction of CI
resistance depends on the range of concentrations at which
these compounds are applied, as occurs with PAs. Thus resistance
was observed with 10 or 100 mol L1 MeJA or MeSA but not
of HSPs, these treatments also stimulated the expression of
genes that encode proteins related to pathogenesis (PRs), which
apparently are involved in the appearance of CI tolerance and in
storage of tomato fruits.330
The finding that HSP biosynthesis can be induced not only
by high temperatures211,212,217 but also by chemical treatments
with compounds such as MeJA, MeSA and salicylic acid (SA),201,219
and that the persistence of the levels of these proteins during
implications for the postharvest period. A treatment inducing the
accumulation of these proteins in chilling-sensitive horticultural
products would allow their storage at low temperatures without CI
development. The induction and maintenance of HSPs during the
application of postharvest treatments, prior to the appearance of
visual symptoms, could be a useful molecular marker to test their
effectiveness. Polenta et al.220 suggested the quantification of HSPs
as a tool for predicting the performance of a postharvest treatment.
These authors proposed the monitoring of the accumulation of
HSPs in different types of produce that undergo different treatment
intensities, and, in this way, optimal ranges of treatment could be
established in order to avoid CI in each crop. Unfortunately, despite
the practical significance of HSPs, no methods for a rapid, simple
and precise quantification of these proteins in plants have been
described.
CONCLUSIONS
Many tropical and subtropical plant species are sensitive to low
temperatures and suffer different physiological disorders that are
known as CI. The incidence of this physiopathy limits the appli-
cation of cold storage, the strategy employed most widely for
marketing horticultural products from these species. Therefore
the impact of CI on the agro-food industry has serious economic
consequences. Knowledge of the physiological, hormonal and
molecular mechanisms responsible for the activation and devel-
opment of this physiopathy would allow the design of strategies
to avoid or, at least, delay CI appearance.
The mechanisms via which low-temperature stress exerts its
effects are not thoroughly known, but it is accepted that the
primary response is an upsurge in disorders of the physical state
between cold-sensitive and tolerant species is found in the capacity
of cell membranes to resist or adapt to the phase transition, from
a liquid crystalline flexible structure to a solid gel rigid one, that
occurs during exposure to low temperatures. When, in sensitive
species, this transition spreads throughout the cell membranes of
whole tissues, organs or organisms, it provokes the disintegration
of cell membranes and the consequent electrolyte leakage and
loss of turgor and metabolic energy that lead to cell death.
The particular temperature at which this lipid phase transition
occurs (transition temperature) depends mainly on the degree of
desaturation of fatty acids in the membrane lipids.
However, the effect on the structure and composition of the
cell membranes is not the only factor affecting the appearance
of CI. Low temperatures, like most abiotic stresses, cause an
565
increase in ROS that are responsible for oxidative stress, which is
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the secondary response to cold stress. The sources of these ROS
are mainly photorespiration, photosynthesis and mitochondrial
respiration, processes that are altered by the primary response
to low temperatures: changes in the structure and composition
of cell membranes disturb the normal functioning of membrane
proteins in the cell organelles responsible for these processes.
Plants respond to low temperatures with an activation of their
antioxidant defence systems to counteract the production of ROS.
Those with the most efficient antioxidant defence systems will be
able to counteract the harmful effects of low temperatures and
will be most tolerant to cold. This has been employed postharvest
in order to induce CI tolerance in sensitive horticultural products,
since a treatment that increases the antioxidant defences of a
plant or plant organ will produce a protective effect against CI.
From the biotechnological point of view it has been observed also
that the genetic modification of plant species, with the aim of
obtaining plants with a higher degree of fatty acid desaturation in
cell membranes or a higher level of activity of antioxidant enzymes,
results in the appearance of CI resistance.
Plant hormones and growth regulators are also involved in the
development of CI, and their effects can be beneficial or harmful
depending on the plant organ, the growth and development
conditions and the temperature. Among these compounds, those
that seem to play a more significant role in the development
of CI are polyamines, ethylene and abscisic acid. Among the
plant growth regulators that have a major beneficial effect on
the reduction of CI are PAs. PAs have antisenescent properties
and exert a protective effect against low temperatures. Higher
levels of PAs have been found in species and varieties resistant
to low temperatures. Moreover, exogenous treatment with PAs
stimulates the appearance of tolerance to this physiopathy in
sensitive species. The mechanisms by which PAs exert their
protective effect have not been elucidated completely, but it
seems that their capacity to bind to membrane phospholipids,
due to their polycationic nature, and their antioxidant properties
could contribute greatly to the protection against CI. The level
and production of PAs influence the biosynthesis of the ripening
and senescence hormone ethylene, because they share steps in
their respective biosynthetic pathways. In many plant species
sensitive to cold, it has been reported that low temperatures
cause, simultaneously, CI development and an increase in
ethylene production. This ethylene production associated with
the triggering of CI seems to be not just a physiological response
of the cold-affected plant tissue or organ but also to have
an effect on the induction of CI. Moreover, treatment with
ethylene or its analogue propylene stimulates CI appearance.
This stimulation of CI by ethylene can be explained within the
framework of the main function of this hormone, the control of
ripening and senescence. Both of these developmental processes
activate physiological processes that are common to CI, such as
cell membrane disintegration and induction of oxidative stress.
Low temperatures also regulate ethylene production, and the key
point in this regulation is the biosynthesis of the enzyme ACS,
the expression of which is stimulated by low temperatures and
for which the translation of the corresponding mRNA transcripts
is induced in subsequent room temperature reconditioning, as is
ACS activity. Regarding another plant hormone, ABA, its action
seems to be associated with the appearance of CI resistance,
because its application to sensitive plants or organs clearly reduces
CI symptoms, and in tolerant species an accumulation of this
hormone during low-temperature storage has been observed.
566
Although the mechanism of action of ABA against CI is not
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L Sevillano et al.
clear, the regulation of stomatal closure, thereby avoiding the
dehydration which is a main symptom of CI, seems to be a key
point.
The regulation of the transcription of the different genes and
genetic families involved in the response to low-temperatures
stress, especially of those encoding proteins participating in
defence systems against the oxidative stress associated with CI,
has started very recently to be investigated in depth. The control
of the expression of these genes occurs via different cis and trans
elements belonging to molecular regulatory networks controlled
by ethylene, ABA or low temperatures themselves. In the last case
we find the genetic superfamily of COR genes participating in the
molecular response to freezing, dehydration and chilling. Not only
chilling could control the regulatory network of the expression of
COR genes, since it has been found that ABA is able to participate in
this network. Finally, it has been observed that low temperatures
could also control the biosynthesis of transcription factors typically
under the control of ethylene.
Low temperatures induce the accumulation of HSPs, which
protect against CI owing to their stabilising action on cell
membranes and their function as molecular chaperones. This
chaperone activity is crucial in the appearance of resistance to
low temperatures, since the solubility and folding properties of
many proteins are altered by low temperatures. In general, the
production of these proteins takes place at high temperatures, but
once they have been synthesised, they not only protect against
the stress that induced their synthesis, the heat shock, but also
against subsequent stresses such as cold.
Today, different postharvest technologies are employed in order
to inhibit or reduce the impact of CI in sensitive horticultural
products of commercial interest. These technologies can be of
a physical, chemical or biotechnological nature. The reluctance
of consumers regarding the chemical treatment of agricultural
products has promoted the use of physical treatments, which
consist mainly of thermal treatments before or during the
cold storage period and regulation and control of the gaseous
composition around the product during storage. The protection
against CI of these treatments can be explained by the existence
in plants of cross-resistance to different stress conditions. The
exposure of a plant or an organ to a certain condition of moderate
stress not only induces resistance to this kind of stress but also
protects against other kinds of stress. All these treatments applied
before or during storage at low temperatures probably trigger
a response to stress conditions that increases the resistance to
low temperatures. The chemical technologies are based mainly
on the application of an antagonist of ethylene action, 1-MCP,
compounds with antisenescent properties, e.g. plant growth
regulators such as PAs and MeJA, and signalling molecules such
as SA and MeSA. Biotechnology has also offered different ways
to reduce the impact of CI in sensitive horticultural products,
by the generation of genetically modified plants with inhibited
autocatalytic ethylene production or by the overexpression of
genes that encode antioxidant enzymes or whose genetic products
promote an increase in the level of unsaturated fatty acids within
the cell membrane.
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