ALTERATION OF CELL MEMBRANE STRUCTURE AND FUNCTIONALITY: THE PRIMARY EFFECT OF CHILLING INJURY

Loss of cell membrane integrity is the primary deleterious effect provoked by chilling injury (Rui et al. Reference Rui, Cao, Shang, Jin, Wang and Zheng2010). Phase transition from a flexible liquid-crystalline stage to a solid-gel one occurs at chilling temperatures, which risks a loss of cell membrane selective permeability. This phenomenon is based on the ‘membrane theory’ to explain chilling injury biological effects in plants sensitive to cold stress (Lyons Reference Lyons1973). At chilling temperatures, membrane selective permeability is negatively affected by a reduction in membrane fluidity, caused by fatty acid peroxidation, increases in the degree of saturation of membrane lipids, degradation of phospholipids and galactolipids, and the rise of the sterol:phospholipid ratio (Sevillano et al. Reference Sevillano, Sanchez-Ballesta, Romojaro and Flores2009). If the whole plant or plant organ is exposed to damaging low temperatures for too long then cell membranes rupture, causing leakage of intracellular water, ions and metabolites, which can be monitored by determination of electrolyte leakage (Sharom et al. Reference Sharom, Willemot and Thompson1994). Electrolyte leakage is an effective parameter to assess membrane permeability and therefore is used as an indicator of membrane integrity (Marangoni et al. Reference Marangoni, Palma and Stanley1996). Lipid peroxidation, which can be evaluated by analysing MDA content, is responsible for this loss of cell membrane integrity (Wise & Naylor Reference Wise and Naylor1987). Malonyl dialdehyde is the end product of fatty acid peroxidation, and is a marker of oxidative stress and damage on cell membrane integrity (Hodges et al. Reference Hodges, DeLong, Forney and Prange1999). Both physiological determinations (electrolyte leakage and MDA content) are widely used in post-harvest research to indirectly assess cell membrane integrity in refrigerated storage assays and therefore to evaluate chilling injury impact (Fig. 1; Wise & Naylor Reference Wise and Naylor1987; Sharom et al. Reference Sharom, Willemot and Thompson1994; Shewfelt & Purvis Reference Shewfelt and Purvis1995).

Fig. 1.

Transition of cell membrane phase from a flexible liquid-crystalline to a solid-gel structure that occurs at chilling temperature, increases the risk of loss of cell membrane selective permeability and leakage of intracellular water, ions and metabolites, which can be monitored by determination of electrolyte leakage. Phospholipase-D enzyme activity is responsible for degradation of unSFA and reduction of cell membrane integrity, therefore augmenting the impact of chilling injury. Loss of function of proteins linked to cell membrane, especially in mitochondrial and chloroplastic membranes, lead to an increase of ROS and decrease of ATP. In addition to the direct effect of chilling temperatures on membrane structure, loss of membrane integrity is itself boosted by oxidative stress, since cold stress increases the production of ROS that stimulate lipid peroxidation in cell membranes. Lipid peroxidation could be carried out by enzymatic oxidation of unSFA by lipoxygenase or by non-enzymatic oxidation by ROS. Lipid peroxidation could be evaluated by the content of MDA. Electrolyte leakage and MDA content, well-known physiological markers of loss of membrane permeability and membrane lipid peroxidation, are widely used in post-harvest research to indirectly assess cell membrane integrity in cold storage assays and therefore to evaluate chilling injury impact.

A key factor in cell membrane integrity and selective permeability is the maintenance of a high content of unsaturated fatty acids (unSFA). During the post-harvest life of fruits and vegetables, membrane unSFA play a dual role: (1) high content of unSFA such as linoleic and linolenic acids enhance chilling injury tolerance by contributing to maintenance of membrane fluidity (Nishida & Murata Reference Nishida and Murata1996) and (2) high unSFA levels are associated with maintenance of the aroma quality of fruits and vegetables, since these unSFA are precursors of volatile ester biosynthesis (Flores et al. Reference Flores, El Yahyaoui, de Billerbeck, Romojaro, Latché, Bouzayen, Pech and Ambid2002).

Overexpressing genes encoding for the fatty acid desaturase (FAD) enzyme leads to a rise in the degree of unsaturation of fatty acids: this is a biotechnological approach not only to improve chilling injury tolerance due to the resulting maintenance of membrane fluidity but also to enhance the organoleptic quality of the produce because of the impact of unSFA on the aroma profile of fruits and vegetables mentioned above. Domínguez et al. (Reference Domínguez, Hernández, Pennycooke, Jiménez, Martínez-Rivas, Sanz, Stockinger, Sánchez-Serrano and Sanmartín2010) observed that overexpression of the endogenous FAD3 and FAD7 genes in tomato plants by transformation with genetic constructions containing the respective coding sequences under the control of CaMV35S promoter resulted in a rise of the ratio of 18 : 3 to 18 : 2 fatty acids in leaves and fruits, as expected since the gene products catalyse the conversion of linoleic acid (18 : 2) to linolenic acid (18 : 3). This increased degree of unsaturation led to an augmentation of the hexenal content, which positively affected the fruit aroma profile. But transgenic tomato plants also exhibited a marked tolerance against chilling temperatures (Domínguez et al. Reference Domínguez, Hernández, Pennycooke, Jiménez, Martínez-Rivas, Sanz, Stockinger, Sánchez-Serrano and Sanmartín2010). Therefore, the generation of fruits and vegetables with high levels of unSFA by biotechnological methods not only enhances chilling injury tolerance but also contributes to improved aroma quality.

OXIDATIVE STRESS: THE SECONDARY EFFECT CAUSED BY CHILLING INJURY

Oxidative stress is a common secondary stress occurring in diverse biotic and abiotic stress, such as low temperatures in susceptible plant species. The alterations in membrane integrity are themselves boosted by oxidative processes, since cold stress stimulates overproduction of reactive oxygen species (ROS), which are responsible for lipid peroxidation in cell membranes, contributing to loss of its selective permeability (Sevillano et al. Reference Sevillano, Sanchez-Ballesta, Romojaro and Flores2009).

Plant defence against oxidative stress is accomplished in two ways. The first is by inducing the expression of genes encoding proteins involved in ROS avoidance, such as alternative oxidase (AOX). The AOX pathway is a branch of the respiratory electron transport chain in mitochondria that emerges from the cytochrome. When overreduction occurs in this respiratory electron transport chain, AOX intervenes in order to inhibit the excessive reduction of ubiquinol and thus assisting in avoiding ROS accumulation (Møller Reference Møller2001). Increases in the levels of AOX gene expression in pepper fruit treated with methyl salicylate and methyl jasmonate have been found to enhance chilling injury tolerance (Fung et al. Reference Fung, Wang, Smith, Gross and Tian2004). The induction of AOX gene expression in treated pepper fruits led to an increase in chilling injury tolerance via maintenance of the balance between ROS production and the general antioxidant system activity (Purvis Reference Purvis1997; Fung et al. Reference Fung, Wang, Smith, Gross and Tian2004).

The second way in which plants defend themselves against oxidative stress is by inducing enzymatic activity or gene expression for ROS scavengers such as superoxide dismutase, catalase, APX, glutathione peroxidase, glutathione-S-transferase, monodehydroascorbate reductase, dehydroascorbate reductase and glutathione reductase antioxidant enzymes (Møller Reference Møller2001). Higher activity of the enzymatic antioxidant system leads to reduction of ROS, therefore improving membrane integrity and ultimately stimulating resistance towards abiotic stresses such as chilling injury (Mittler Reference Mittler2002).

Duan et al. (Reference Duan, Feng, Wang, Li and Meng2012) observed that overexpression of a thylakoid APX gene in tomatoes (LetAPX) enhanced resistance against chilling injury and suggested that this behaviour could be associated with the increased APX activity and the maintenance of higher ascorbate/dehydroascorbate (AA/DHA) and glutathione/glutathione disulphide (GSH/GSSG) redox ratios. Also, transgenic plants presented lower levels of hydrogen peroxide and MDA, reduced electrolyte leakage, higher net photosynthetic rate (Pn) and finally higher maximal photosystem II (PSII) photochemical efficiency determined by chlorophyll fluorescence (Fv/Fm). Shu et al. (Reference Shu, Wang, Duan, Deng and Meng2011) generated transgenic tomato plants where the expression of a glutathione reductase gene (LeGR) was silenced by means of an antisense construction and the concomitant diminution of glutathione reductase activity enhanced the sensitivity of the transgenic plants to low temperatures. Glutathione reductase played a key role in maintaining the high activities of APX and superoxide dismutase under chilling conditions. Since the AA/GSH cycle is critical in ROS scavenging, Shu et al. (Reference Shu, Wang, Duan, Deng and Meng2011) suggested that the APX/glutathione reductase system is crucial in stimulating chilling injury tolerance via enhancement of the activity of the above cycle.

Wu et al. (Reference Wu, Lin, Liu, Wang, Lim, Oh, Park, Rajashekar, Whitham, Cheng, Hirschi and Park2012) reported that the overexpression of AtGRXS17, an Arabidopsis gene encoding a glutaredoxin belonging to the thioredoxin protein family, enhanced heat-stress tolerance in tomato plants and this effect was accompanied by mitigation of cell membrane damage and chlorophyll photo-oxidation. The overexpression of AtGRXS17 induced catalase activity and diminished hydrogen peroxide accumulation in tomato leaves, a trend suggesting that AtGRXS17 plays a crucial role in activation of ROS scavenging systems.

In AtGRXS17 overexpressing tomato plants, genes coding for HSP and related HSTF were also up-regulated, an event suggesting that activation of the HSTF/HSP pathway contributes to heat tolerance in the transgenic plants (Wu et al. Reference Wu, Lin, Liu, Wang, Lim, Oh, Park, Rajashekar, Whitham, Cheng, Hirschi and Park2012). Heat-shock transcription factors are present in the cytoplasm of unstressed cells as a latent monomer that lacks both DNA binding and transcriptional activities, but are translocated to the nucleus under stressful conditions (Al-Whaibi Reference Al-Whaibi2011). Also, AtGRXS17 protein has the same cytolocalization if there is no stress but it is translocated from cytoplasm to the nucleus during heat stress. Since both types of proteins, AtGRXS17 and HSTF, accumulated in the nucleus under heat stress, Wu et al. (Reference Wu, Lin, Liu, Wang, Lim, Oh, Park, Rajashekar, Whitham, Cheng, Hirschi and Park2012) suggested that the nuclear accumulation of AtGRXS17 under heat stress could participate in the induction of HSTF transcriptional activity and ultimately this fact could be considered as responsible for the enhanced thermotolerance. Wu et al. (Reference Wu, Lin, Liu, Wang, Lim, Oh, Park, Rajashekar, Whitham, Cheng, Hirschi and Park2012) considered that AtGRXS17 enhancement of catalase activity may be achieved directly via physical protection or indirectly by enhancing HSP accumulation, which would stabilize catalase via its chaperone activity.

When these overexpressing AtGRXS17 tomato plants were exposed to chilling conditions, a higher degree of tolerance to the stress compared with the wild-type ones was observed (Wu Reference Wu2012). As mentioned elsewhere, chilling injury is associated with ROS accumulation and in this regard AtGRXS17 seemed to protect plant cells against the secondary oxidative stress by enhancement of catalase activity (Wu Reference Wu2012), in an analogous way to that observed in the transgenic plants subjected to high temperatures stress (Wu Reference Wu2012). Wu (Reference Wu2012) suggested that genetic engineering of glutaredoxins may be a useful strategy to improve tolerance to chilling/heat/oxidative stress. According to these results from Wu et al. (Reference Wu, Lin, Liu, Wang, Lim, Oh, Park, Rajashekar, Whitham, Cheng, Hirschi and Park2012) and Wu (Reference Wu2012), the HSTF/HSP functional pathway as well as catalase activity seems to be under the regulation of glutaredoxin. Therefore, biotechnological use could be made of glutaredoxin to improve heat and cold tolerance, as well as tolerance to oxidative stress in fruits, vegetables and ornamentals, this beneficial effect on stress tolerance being achieved via induction of HSP (Wu et al. Reference Wu, Lin, Liu, Wang, Lim, Oh, Park, Rajashekar, Whitham, Cheng, Hirschi and Park2012; Wu Reference Wu2012).

In summary, it can be concluded that biotechnological applications for enhancing the unSFA: fatty acid ratio, inducing high FAD gene expression and activity, conferring high levels of cell metabolic energy (adenosine triphosphate (ATP) and adenylate energy charge) and lower levels of phospholipase D and lipoxygenase activities along with boosted antioxidant enzymes activities (AOX or superoxide dismutase, catalase, glutathione peroxidase, glutathione-S-transferase, APX, dehydroascorbate reductase, monodehydroascorbate reductase and glutaredoxin) could contribute to the general goal of inducing chilling injury tolerance. However, inducing HSP accumulation via biotechnology has a positive effect on chilling injury tolerance, given the beneficial effects of these proteins. Heat shock proteins have a multifaceted nature, thanks to their molecular chaperone activity, their contribution to ROS scavenging and to membrane stabilization, and all these events play a part in increasing membrane fluidity and integrity.

LOW-TEMPERATURE SENSING AND SIGNALLING

Understanding how plants perceive low temperatures and trigger the signal transduction pathway in response to this environmental effect will be of great help in designing biotechnological solutions to counteract the negative effects of this stress, aiming at manipulating cold-susceptible plants in order to stimulate their tolerance to chilling injury. So deciphering how plants survive low temperatures is critical for setting up genetic engineering strategies to improve tolerance to this abiotic stress (Solanke & Sharma Reference Solanke and Sharma2008).

Cell membranes are fluid-mosaic in nature and are made up of a bilayer of phospholipids. The membrane is plastic and permeable in normal conditions but when it is subjected to stressful low temperatures it undergoes a phase transition from liquid crystalline to solid gel, which leads to membrane rigidification. Rigidification of membrane leads in turn to loss of fluidity, a key factor in maintaining selective permeability which, if lost, would cause uncontrolled solute leakage. Therefore, since the cell membrane is the primary organ in plants that is affected negatively by cold stress at the cellular level, it is logical to think that it may also be the primary perception site for low temperature (Orvar et al. Reference Orvar, Sangwan, Omann and Dhindsa2000). As mentioned above, chilling injury occurs because of a decrease in membrane fluidity; this phenomenon is called membrane rigidification and it is highly dependent on the degree of saturation of fatty acids, essential components of the lipid bilayer that makes up the membrane (Hayashi & Maeda Reference Hayashi and Maeda2006).

For all the above reasons, the membrane can be hypothesized as the primary site for perception of low temperatures by the plant cell. Cell membrane rigidification is perceived by proteins inserted or attached to it, which function as primary sensors (Mikami & Murata Reference Mikami and Murata2003). Thus membrane proteins really ‘sense’ the phase transition from liquid crystalline to solid gel occurring in this essential cell component (Hayashi & Maeda Reference Hayashi and Maeda2006). As a second step in this perception process, the actin cytoskeleton undergoes rearrangement following phase transition of the cell membrane. It has been reported that depolymerization of microtubules and actin microfilaments occurs under low temperatures (Sangwan et al. Reference Sangwan, Foulds, Singh and Dhindsa2001; Al-Fageeh & Smales Reference Al-Fageeh and Smales2006). This whole set of events leads to an intracellular influx of calcium ions (Ca²⁺) (Orvar et al. Reference Orvar, Sangwan, Omann and Dhindsa2000; Sangwan et al. Reference Sangwan, Örvar, Beyerly, Hirt and Dhindsa2002); Ca²⁺ permeable channels responsible for the rise of cytosolic Ca²⁺ concentration under cold stress are also considered as potential sensors of this stress, and the activation of these channels by cold is thought to be the result of physical alterations in the cell membrane (Monroy & Dhindsa Reference Monroy and Dhindsa1995).

Perception of low temperatures leads to activation of phospholipases such as phospholipase-D and -C, which catalyse the hydrolysis of phospholipids that yield phosphatidic acid, a known second messenger (Ruelland et al. Reference Ruelland, Cantrel, Gawer, Kader and Zachowski2002). Phospholipase-D anchors cytosolic microtubules to the plasma membrane, a process that delivers conformational changes in the cytoskeleton and actin filaments rearrangement, then opening Ca²⁺ permeable channels (Gardiner et al. Reference Gardiner, Harper, Weerakoon, Collings, Ritchie, Gilroy, Cyr and Marc2001; Dhonukshe et al. Reference Dhonukshe, Laxalt, Goedhart, Gadella and Munnik2003). The rise of cytosolic Ca²⁺ level under low-temperature stress operates as a second messenger in the low-temperature signal transduction pathway. The activities of phospholipase-C and -D direct the production of inositol-(1,4,5)-triphosphate (InsP₃), which is also recognized as a second messenger molecule in cell signal transduction and is in turn involved in the augmentation of cytosolic Ca²⁺ level. This function is accomplished by releasing the ion from the intracellular compartments where it is stored in normal conditions (Knight et al. Reference Knight, Trewavas and Knight1996; DeWald et al. Reference DeWald, Torabinejad, Jones, Shope, Cangelosi, Thompson, Prestwich and Hama2001; Ruelland et al. Reference Ruelland, Cantrel, Gawer, Kader and Zachowski2002). Finally, this accumulation of cytosolic Ca²⁺ triggers the signal transduction process through proteins acting as Ca²⁺ sensors and functioning as kinases, so the signal is transmitted downstream by a chain of phosphorylations (Monroy et al. Reference Monroy, Castonguay, Laberge, Sarhan, Vezina and Dhindsa1993). Calmodulins interacting with calmodulin domain-containing protein kinases (CDPK), and calcineurin B-like proteins interacting with calcineurin B-like interacting protein kinases (CIPK) are the main intracellular transduction pathways activated by this rise of cytosolic Ca²⁺. As sensor relays, calmodulin and calcineurin B-like proteins bind Ca²⁺ and conformational changes take place within the structure of these proteins, which allows for their interaction with the sensor responders CDPK and CIPK. These responders transmit the message to their downstream targets by interplay of consecutive phosphorylations (Reddy & Reddy Reference Reddy and Reddy2004; Klimecka & Muszyńska Reference Klimecka and Muszyńska2007).

All this series of intracellular events are involved in the phenomenon of low-temperature acclimation in plants, a process of adaptation whereby certain plants increase their tolerance to cold stress (Sangwan et al. Reference Sangwan, Foulds, Singh and Dhindsa2001; Catalá et al. Reference Catalá, Santos, Alonso, Ecker, Martínez-Zapater and Salinas2003). The final target of this signal transduction pathway, triggered by cold, is induction of the expression of specific cold-responsive genes, whose transcription is activated by a particular family of transcription factors known as C-repeat (CRT)-binding factors that belong to the AP2/EREBP superfamily of transcription factors (Gilmour et al. Reference Gilmour, Zarka, Stockinger, Salazar, Houghton and Thomashow1998). All members of this superfamily have a common feature: the DNA-binding domain, discovered in Arabidopsis thaliana APETALA2 protein and in tobacco EREBP proteins, involved in floral morphogenesis and ethylene response, respectively (Jofuku et al. Reference Jofuku, den Boer, Van Montagu and Okamuro1994; Ohme-Takagi & Shinshi Reference Ohme-Takagi and Shinshi1995). These C-repeat binding factors exert their regulatory function by binding to the specific cis-element known as C-repeat because of the nucleotide sequence that characterize it (TACCGACAT) (Baker et al. Reference Baker, Wilhelm and Thomashow1994), which is located in the promoter regions of the cold responsive genes (Chinnusamy et al. Reference Chinnusamy, Zhu and Sunkar2010).

Up till now, interaction between these Ca²⁺-binding proteins and C-repeat binding factor transcription factor has only been observed experimentally (Doherty et al. Reference Doherty, Van Buskirk, Myers and Thomashow2009). This is the binding of calmodulin proteins to a member of the calmodulin-binding transcription activator (CAMTA) transcription factor family, CAMTA3, which in their turn recognize regulatory elements in the CBF2 promoter and its binding induces the gene expression (Doherty et al. Reference Doherty, Van Buskirk, Myers and Thomashow2009). The results from Doherty et al. (Reference Doherty, Van Buskirk, Myers and Thomashow2009) establish for the first time a role for CAMTA proteins in cold acclimation and provide a possible point for integration of low-temperature Ca²⁺ and calmodulin signalling with the transcriptional regulatory system of cold-responsive genes.

LOOKING FOR A BIOTECHNOLOGICAL ALTERNATIVE TO HEAT TREATMENT

It has been reported that HSP not only play a role under high-temperature stress, but also have a crucial function in response to low-temperature stress (Swindell et al. Reference Swindell, Huebner and Weber2007). Low- and high-temperature stresses enhance the expression of HSP genes. In a systematic analysis of the effects of low-temperature storage on gene expression in peach fruit, Pavez et al. (Reference Pavez, Hödar, Olivares, González and Cambiazo2013) reported that expressions from genes encoding antioxidant enzymes (glutathione reductase, catalase and superoxide dismutase), sHSP and lipid transfer protein increased during cold storage (21 days at 4 °C) and remained high after fruits were subjected to room temperature reconditioning (21 days at 4 °C followed by 5 days at 21 °C). These results hinted at a cryoprotective role of sHSP by maintaining membrane fluidity, and also of higher molecular weight HSP, such as HSP70 and HSP100, by assisting in protein refolding (Sun et al. Reference Sun, Van Montagu and Verbruggen2002; Timperio et al. Reference Timperio, Egidi and Zolla2008). Pavez et al. (Reference Pavez, Hödar, Olivares, González and Cambiazo2013) observed that peach fruits under cold storage are able to uphold the capacity for ROS scavenging via induction of expression of genes involved in the antioxidant system such as glutathione reductase, catalase and superoxide dismutase, as well as sHSP.

Zhang et al. (Reference Zhang, Huang, Pan and Liu2005) observed that hot air treatment (38 °C for 10 h) mitigated chilling injury in grape berries: this emergence in cold tolerance was associated with a reduction of electrolyte leakage and MDA content and an increase of superoxide dismutase and catalase activities. Hot air treatment induced HSP70 expression in the grapes and this occurred prior to the rise in the activities of antioxidant enzymes. Zhang et al. (Reference Zhang, Huang, Pan and Liu2005) suggested that the stimulation of HSP70 expression and corresponding protein accumulation was associated with a rise in the activities of antioxidant enzymes, and that synergistic action between HSP70 and the enzymatic antioxidant system resulted in the maintenance of membrane integrity and the emergence of chilling injury resistance.

Sabehat et al. (Reference Sabehat, Weiss and Lurie1996) observed that treatment of tomato fruit with hot air (38 °C for 48 h) mitigated chilling injury and induced HSP accumulation, which persisted during subsequent low-temperature storage (2 °C), while Lurie et al. (Reference Lurie, Handros, Fallik and Shapira1996) found that treatment of tomato fruit with hot air at 38 °C for 3 days led to stimulation of chilling injury resistance through the rise of HSP17 accumulation and persistence of high levels during subsequent cold storage at 2 °C. Treating tomatoes with hot air (38 °C for 3 days) induced the expression of genes encoding for sHSP, such as tom66 and tom111, and the persistence of their levels during subsequent cold storage (2 °C) was associated with chilling injury resistance (Sabehat et al. Reference Sabehat, Lurie and Weiss1998a , Reference Sabehat, Weiss and Lurie b ).

Sevillano et al. (Reference Sevillano, Sola and Vargas2010) reported that hot air treatment (55 °C for 5 h) mitigated chilling injury in cherimoya fruit and was associated with the induction of sHSP gene expression. He et al. (Reference He, Chen, Kuang and Lu2012) also observed that hot air treatment (38 °C for 3 days) alleviated chilling injury in banana fruit, in this case by an interplay of expressions of three genes encoding sHSP; banana sHSP1 expression in peel and pulp and sHSP3 expression in peel increased during heat treatment, and expression of both genes in pulp and sHSP2 in peel increased during subsequent low-temperature storage (8 °C). As with Sevillano et al. (Reference Sevillano, Sola and Vargas2010), He et al. (Reference He, Chen, Kuang and Lu2012) suggested that heat treatment increased resistance to chilling injury in banana by stimulating sHSP gene expression during low-temperature storage. Similar behaviour was observed by Rozenzvieg et al. (Reference Rozenzvieg, Elmaci, Samach, Lurie and Porat2004) in grapefruit. Subjecting the fruit to heat treatment (62 °C for 20 s) led to cold resistance: expression of genes for HSP increased and was maintained during subsequent storage at low temperature (2 °C) (Rozenzvieg et al. Reference Rozenzvieg, Elmaci, Samach, Lurie and Porat2004).

It has been observed that the pace at which HSTF expression is induced with cold stress is lower than when induced by high-temperature conditions (Swindell et al. Reference Swindell, Huebner and Weber2007). Under heat stress, this faster induction of HSTF expression correlates well with the action of HSTF as transcriptional regulators of HSP (Borges & Ramos Reference Borges and Ramos2005). Li et al. (Reference Li, Chang, Lu, Liu, Chan and Charng2003) suggested that the Arabidopsis HSTF AtHSFA1b has a crucial role in the emergence of chilling tolerance when triggered by high-temperature treatment: transgenic tomato overexpressing AtHSFA1b not only exhibited higher tolerance to high temperatures, but also to low temperatures without the need for a previous heat treatment to induce it. Li et al. (Reference Li, Chang, Lu, Liu, Chan and Charng2003) observed that transgenic tomato plants overexpressing the Arabidopsis HSTF AtHSFA1b activate sHSP biosynthesis as well as the expression of genes coding for APX. The results of this set of processes are responsible for the improved cold tolerance of transgenic tomato (Li et al. Reference Li, Chang, Lu, Liu, Chan and Charng2003).

Saltveit & Hepler (Reference Saltveit and Hepler2004) and Pressman et al. (Reference Pressman, Shaked and Firon2006) suggested that fruits and vegetables treated with low temperature should increase their tolerance to heat and vice versa; heat treatment enhances chilling tolerance of sensitive fruits and vegetables, and HSTF play a critical role in this scenario of enhanced cold tolerance. Waters et al. (Reference Waters, Lee and Vierling1996) suggested that the key response of fruits and vegetables to heat treatment at the cellular level was induction of gene expression and protein accumulation of HSPs. Heat treatment also enhanced APX activity conducting increasing chilling injury tolerance in rice seedlings (Sato et al. Reference Sato, Murakami, Funatsuki, Matsuba, Saruyama and Tanida2001). The enhanced expression of pea, Arabidopsis and rice APX gene under heat treatment is explained molecularly by the presence in their promoters of heat-shock cis-acting elements (Mittler & Zilinskas Reference Mittler and Zilinskas1992, Reference Mittler and Zilinskas1994; Storozhenko et al. Reference Storozhenko, De Pauw, Van Montagu, Inzé and Kushnir1998; Sato et al. Reference Sato, Murakami, Funatsuki, Matsuba, Saruyama and Tanida2001). According to these results, it was hypothesized that overexpressing HSFT in chilling-sensitive horticultural species might be a powerful biotechnological tool to use as an alternative to post-harvest heat treatments envisaged to enhance chilling injury tolerance (Fig. 2).

Fig. 2.

Enhancing tolerance of fruits, ornamentals and vegetables to post-harvest chilling injury by heat treatment theoretically is accessible; but their performance applying different technologies such as hot water, hot air and hot vapour treatments are hindered by capacity limits and energy concerns of the required facilities. The HSTFs genetic engineering could be envisaged to overcome these hurdles and could be considered as the link between basic and applied research. HSTFs genetic engineering can be used in a biotechnological strategy to improve post-harvest chilling injury tolerance of crop plants, via enhancing HSPs accumulation, antioxidant system activity and the accumulation of proline and raffinose, all these events leading to mitigation of secondary oxidative stress and ultimately maintenance of membrane fluidity and integrity, contributing the whole set of processes to enhancement of chilling injury resistance.

HEAT-SHOCK PROTEINS AND MITIGATION OF POST-HARVEST CHILLING INJURY: MECHANISMS OF ACTION

There are environmental and developmental conditions in plants that provoke accumulation of HSP, such as fruit ripening (Lawrence et al. Reference Lawrence, Cline and Moore1997; Medina-Escobar et al. Reference Medina-Escobar, Cárdenas, Muñoz-Blanco and Caballero1998; Ding et al. Reference Ding, Wang, Gross and Smith2001; Neta-Sharir et al. Reference Neta-Sharir, Isaacson, Lurie and Weiss2005), low temperature (Ukaji et al. Reference Ukaji, Kuwabara, Takezawa, Arakawa, Yoshida and Fujikawa1999; Lubaretz & zur Nieden Reference Lubaretz and zur Nieden2002; Lopez-Matas et al. Reference Lopez-Matas, Nuñez, Soto, Allona, Casado, Collada, Guevara, Aragoncillo and Gomez2004) and oxidative stress (Eckey-Kaltenbach et al. Reference Eckey-Kaltenbach, Kiefer, Grosskopf, Ernst and Sandermann1997; Banzet et al. Reference Banzet, Richaud, Deveaux, Kazmaier, Gagnon and Triantaphylidès1998; Lee et al. Reference Lee, Won, Lee, Miyao, Chung, Kim and Jo2000; Jofré et al. Reference Jofré, Molinas and Pla2003). Often the accumulation of HSP not only confers protection against the stress that causes it but also against any subsequent stressful situation, in a phenomenon called cross-tolerance (Lin et al. Reference Lin, Roberts and Key1984; Chen et al. Reference Chen, Chou, Ren, Chen and Lin1988). Knowledge on the structure and function of HSP at molecular level, especially regarding sHSP, has expanded significantly in recent years and potential applications have emerged. By means of its interaction with the cell proteome, sHSP are useful in protecting cells from oxidative stress (Eckey-Kaltenbach et al. Reference Eckey-Kaltenbach, Kiefer, Grosskopf, Ernst and Sandermann1997; Banzet et al. Reference Banzet, Richaud, Deveaux, Kazmaier, Gagnon and Triantaphylidès1998; Lee et al. Reference Lee, Won, Lee, Miyao, Chung, Kim and Jo2000; Jofré et al. Reference Jofré, Molinas and Pla2003; Neta-Sharir et al. Reference Neta-Sharir, Isaacson, Lurie and Weiss2005), heat (Wu et al. Reference Wu, Lin, Liu, Wang, Lim, Oh, Park, Rajashekar, Whitham, Cheng, Hirschi and Park2012) and cold stress (Ukaji et al. Reference Ukaji, Kuwabara, Takezawa, Arakawa, Yoshida and Fujikawa1999; Lubaretz & zur Nieden Reference Lubaretz and zur Nieden2002; Lopez-Matas et al. Reference Lopez-Matas, Nuñez, Soto, Allona, Casado, Collada, Guevara, Aragoncillo and Gomez2004). In this section, examples of manipulation of the expression of sHSP genes for protein production as biotechnological applications for improving tolerance to post-harvest chilling injury in susceptible fruits and vegetables are reviewed.