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Plant Stress Physiology: Opportunities and Challenges for the Food Industry

December 7, 2007

By Galindo, Federico Gomez Sjoholm, Ingegerd; Rasmusson, Allan G; Widell, Susanne; Kaack, Karl

We review and analyze the possible advantages and disadvantages of plant-stress-related metabolic and structural changes on applications in the fruit and vegetable processing industry. Knowledge of the cellular and tissue transformations that result from environmental conditions or industrial manipulation is a powerful means for food engineers to gain a better understanding of biological systems in order to avoid potential side effects. Our aim is to provide an overview of the understanding and implementation of physiological and biochemical principles in the industrial processing of fruits and vegetables. Keywords stress tolerance, freezing, heat, drought, drying, postharvest, minimal processing

INTRODUCTION

Because plants are confined to the place in which they grow, they have a limited capacity to avoid unfavorable conditions in their environment, such as extremes of temperature, water shortage, insufficient or excessive light or mineral nutrients, wounding by herbivores, or attack by pathogenic bacteria, fungi, viruses, and viroids. Plants have developed sophisticated molecular chemical strategies to defend themselves against such abiotic and biotic stress, often combined with changes in growth and development patterns (Boyer, 1982; Gaspar et al., 2002). Stress is usually defined as an external factor that exerts a disadvantageous influence on the plant. This concept is closely associated with stress tolerance, which is the plant’s capacity to cope with unfavorable conditions (Taiz and Zeiger, 2002). In both natural and agricultural conditions, environmental factors, such as air temperature, can become stressful in just a few minutes. Soil water content may take days to weeks, whereas other factors such as soil mineral deficiency, can take months to become stressful. Cellular responses to stress may include changes in cell cycle and division, cell membranes, cell wall architecture, and metabolism (e.g. accumulation of osmotically active substances).

From a biological point of view, industrial treatment of plant tissue will mimic stress (Fig. 1) and therefore, knowledge of how the plant material will be affected in relation to time, the environment, and industrial manipulation is of fundamental importance for quality assurance and process optimization. We here focus our attention on reviewing and analyzing possible advantages and disadvantages of the stress responses of fruits and vegetables during industrial processing operations. Reports on attempts to implement physiological and biochemical principles in the industrial processing of fruit and vegetables are not common in the literature, but a few recent investigations, referred to in the following sections, have laid the foundation for a fascinating area of research and technological innovation.

STRESS LEADING TO CELL DAMAGE

During harvesting, transportation, washing, sorting, and packing, fruits and vegetables are subjected to mechanical stress that may lead to crushing of surface cell layers. When the fresh products reach the processing line for producing, for example, ready-to-use salads, they are typically peeled, sliced, diced, or shredded before packaging. These operations cut through cells and leave intact cells of previously internal tissues exposed. These postharvest and processing operations are traumatic for the cells proximal to the damage site and induce a complex series of molecular events aimed at repairing the damage caused to the tissue (Surjadinata and Cisneros- Zevallos, 2003).

Figure 1 Schematic representation of the topics discussed in this review. Industrial treatment of plant tissue will mimic stress responses in nature, influencing the quality of fresh and processed products.

Response to Postharvest Handling

Mechanical stress, imposed on plant cells by a variety of physical stimuli during harvesting and handling of fresh horticultural products, induces a wide range of cellular responses such as increased respiration rate, ethylene production, and higher susceptibility to pathogen attack (Charron and Cantliffe, 1995; Stanley, 1991). In carrots, mechanical stress brings about a decrease in root pressure potential and water potential during the initial storage period (Mempel and Geyer, 1999; Herppich et al., 1999). Furthermore, the production of ethylene and 6-methoxymellein (a bitter compound) increases, whereas the levels of several terpenes associated with the characteristic aroma of carrots decreases (Seljasen et al., 2001). The accelerated aging in cucumbers involves the induction of cell-wall-degrading enzymes, leading to tissue degeneration (Miller and Kelley, 1989).

Potatoes are particularly susceptible to mechanical stress. Physically stressed tuber tissue produces melanin-based pigments, leading to the blue-black discoloration of subdermal tissues known agronomically as black-spot bruising (Johnson et al., 2003).This is a serious agronomic problem manifested during harvesting, handling and storage, leading to significant levels of rejection of potato harvests (Potato Marketing Board, 1994). The synthesis of melanin is thought to be a defence mechanism in which the polymerized, insoluble complexes form a resistant barrier, sealing tuber tissues against the entry and spread of pathogens. The predisposition of tubers to melanin production depends on growth and storage conditions and temperature during processing, and exhibits a wide range of genetic variation (Hoffmann and Wormanns, 2002; Johnson et al., 2003). Therefore, mechanical stress during handling (caused, e.g. by falls and collisions) induces wound responses leading to undesirable physiological changes, further reducing quality and storability.

In spite of the many detrimental consequences of postharvest mechanical stress on the quality of fruit and vegetables, some reports have shown that slight mechanical stress during growth can improve the postharvest processability of lettuce, cauliflower, celery (Biddington and Dearman, 1985; Pontinen and Voipio, 1992), and baby leaf salad (Clarkson et al., 2003), when the stress is applied to the seedlings. Mechanical stress during growth results in modified leaf architecture producing smaller, more compact new leaves. After industrial unit operations including washing, drying, and packing, baby leaves of lettuce and spinach showed an increase in shelf-life. This increase was associated with a reduction in the area of individual epidermal cells and modification of the biophysical properties of the cell wall (Clarkson et al., 2003). The mechanical stress manipulation of the seedlings led to the development of new adapted leaves with stiffer cell walls, so that the leaves would have greater protection against mechanical stress during processing; stress that may otherwise cause damage and browning of the leaves (Lopez-Galvez et al., 1996). Smaller cells have a larger relative cell wall volume and dry weight (Wurr et al., 1986; Clarkson et al., 2003).

Effects on Minimally Processed Plant Tissue

As a result of peeling, grating, or shredding, a relatively stable agricultural product with a shelf life of several weeks or months will change into one that deteriorates rapidly from a food quality perspective. Minimally processed fruit and vegetables should have storage lives of at least 4-7 days, but preferably longer, up to 21 days depending on the market (Ahvenainen, 1996; BarryRyan and O’Beirne, 1997). Deterioration is mostly the result of microbial spoilage, wound healing, biochemical changes, and loss of nutritional quality.

The quality and shelf life of minimally processed fruit and vegetables are directly influenced by the extent of wounding and the size of the wounded surface caused by the processing operation. For example, it has been demonstrated that carrots peeled by abrasion or sliced with a blunt machine blade show higher respiration rates, greater microbial contamination and microbial growth rates, higher pH values in the carrot tissue, higher rates of weight loss, and higher white tissue development than those that had been hand peeled or sliced with razor blades (Barry-Ryan and O’Beirne, 1998; 2000).

The respiration rate of fresh vegetable slices is in most cases 3 to 5 times that of the intact organ, but aging of the sliced tissue elicits additional increase. Thus, the respiration rate of an aged slice may be 25 times that of the intact organ (Laties, 1978). Wadso et al. (2004) found that the overall metabolic activity of diced carrot, rutabaga, and potato tissue rose linearly with an increase in cut surface area per unit volume (intensity of wounding), being as much as 40% higher when the surface area was doubled. This increase in metabolic activity is the consequence of a large number of biosynthetic events taking place during wound healing (Laties, 1978).

The initial physiological steps following wounding and the generation of wound signals are not fully understood (Saltveit, 2000). Products of lipid metabolism and lipid oxidation as well as compounds such as ethylene and abscisic acid (ABA), are thought to be possible candidates for the wound signals in plant cells (Pena- Cortes and Willmitzer, 1995).

When plant tissues are wounded, the cells near the site of the wounding stress strengthen their cell walls by the secretion of additional structural components such as lignin or suberin, creating a protective layer immediately below the site of damage, to prevent dehydration and potential penetration by pathogens (Satoh et al.,1992; Kaack et al., 2002b). The synthesis of several secreted proteins, such as hydroxyproline-rich glycoproteins, and their cross- linking to the cell wall after wounding has also been observed (Showalter and Varner, 1987; Bradley et al., 1992). Suberization is a regulated process whereby the intercellular spaces in tissues become impregnated with a poly(phenolic) matrix concomitant with the deposition of a poly(aliphatic) matrix between the plasmalemma and carbohydrate cell wall (Friedman, 1997; Bernards et al., 1999). The oxidative coupling of the poly(phenolic) component of suberin is thought to be a peroxidase/H^sub 2^O^sub 2^-dependent, free-radical process. In response to wounding, and in association with suberization, plant tissues generate reactive oxygen species (ROS), including superoxide (O^sup -^^sub 2^), hydrogen peroxide (H^sub 2^O^sub 2^), and the hydroxyl radical (OH). It has been shown that H^sub 2^O^sub 2^ is essential for suberization in potato slices (Razem and Bernards, 2002). Immediately following wounding, a rapid increase in oxygen uptake is followed by an initial burst of ROS (oxidative burst) (Bolwell et al., 1995). In wounded potatoes, this burst reaches a maximum within 30 to 60 min and is followed by at least three other massive bursts at 42,63, and 100 h post-wounding. These later bursts were associated with wound healing and are probably involved in the oxidative cross-linking of suberin poly(phenols) (Razem and Bernards, 2003). The initial deposition of suberin in potato requires approximately 18 h at 18[degrees]C (Lulai and Corsini, 1998) and reaches a stage in which the suberized layer has sufficient structural integrity to be peeled off intact 3 days after wounding (Razem and Bernards, 2002).

Deposition of suberin may cause detrimental quality characteristics. For example, in the production of pre-peeled potatoes, a common industrial product in Scandinavia, hardening of the tuber surface takes place (Fig. 2a) (Kaack et al., 2002b). These potatoes are too hard for consumption, even after cooking at 98100[degrees]C for one hour. Microscopic examination shows that when hard potatoes are cooked, brick-like cells at the potato surface remain intact (Fig. 2b). It was demonstrated that potato hardening was significantly correlated to the mechanical impact of the peeler, and was increased by blows during sorting or transport (Kaack et al. 2002a). However, the hardening of potato tissue does not occur if the tubers are steamed or cooked immediately or a few hours after peeling, probably because the exposed intact cells are killed. Therefore, understanding the dynamics and time scales of the metabolic processes taking place in vegetables during industrial unit operations is of great importance in processing design and optimization.

LOW-TEMPERATURE STRESS

Among the various kinds of environmental stress affecting plants, low temperature is of particular interest to food science, since low temperature, either chilling or freezing, is one of the most widespread and effective methods of conservation.

Low-Temperature Sweetening

Physiology

During the storage of some plant tissues at temperatures lower than those for optimum growth or storage (i.e.,

Figure 2 Microstructure of the surface of wounded potato tissue, (a) Raw surface with thick suberized (s) and curly cell walls (w). (b) Surface of cooked potato showing a few brick-like cells at (u) and gelatinized starch (G) with small regions of suberin.

In parsnip roots and potato tubers the increase in levels of sucrose and other hexoses during low-temperature storage is known as “cold sweetening” (Hart et al., 1986; Shattuck et al., 1989; Wismer et al., 1995; Espen et al., 1999). The cold-induced increase in soluble sugars may play a role in osmoregulation, cryoprotection (Espen et al., 1999) and possibly also in the activation of respiratory metabolism. The genetic control and the metabolic pathways of sugar synthesis have been studied (Deiting et al., 1998). In potatoes, the mechanism of cold sweetening is complex and is mediated by many interrelated metabolic pathways, such as the induction of the enzymes required in starch degradation, alterations in the biochemical pathways of sucrose metabolism, glycolysis and mitochondrial respiration, as well as electrolyte leakage and membrane lipid peroxidation (Blenkinsop et al., 2004).

Implications for the Potato Crisp Industry

Blenkinsop et al. (2004) underlined the importance of the understanding of metabolic changes in potatoes during cold storage to ensure satisfactory chip color in the potato chip industry. Color control is complicated as the color is determined by the chemical composition of the tubers, which not only varies with season and cultivar, but changes during storage. Sugar levels and free amino acids are important in determining the chip color, which is attributed to the products of the Maillard reaction. In addition to the complex carbohydrate metabolism, storage conditions and the length of storage are also known to increase the free amino acid content and the amount of reducing sugars (Brierley et al., 1996).

In April 2002, the National Food Administration of Sweden and the University of Stockholm announced the presence of acrylamide, a possible carcinogenic, in foods processed at high temperatures (Roseh and Hellenas, 2002; Tareke et al., 2002). As a result of high- temperature frying, potato-based food products, such as potato crisps, contain higher levels of acrylamide than other baked or fried products (Chuda et al., 2003). Changes in the levels of reducing sugars and amino acids in potato tubers during storage were investigated in relation to the presence of acrylamide in the crisps. Chuda et al. (2003) found that crisps made from tubers stored at 2[degrees]C contained ten times more acrylamide than those made from tubers stored at 20[degrees]C. In the crisps, the acrylamide level did not depend on the levels of total amino acids or aspargine, but on the availability of reducing sugars in the raw potato.

Generally, the tubers used to manufacture potato crisps are not stored at such low temperatures as 2[degrees]C, as they will produce dark-colored crisps that are unacceptable to the consumer due to their appearance and bitter taste (Roe et al., 1990). Therefore, potatoes are generally stored at around 10[degrees]C in order to maintain low levels of sugars during long-term storage (Blenkinsop et al., 2004). However, at this storage temperature potatoes will sprout, and the application of chemicals to inhibit sprouting may be necessary. According to Blenkinsop et al. (2004), there has been great interest during recent years in developing potato cultivars (through traditional breeding and selection methods and/or through the use of genetic engineering) that are more resistant to low- temperature sweetening, and which have an acceptable color when processed directly after low-temperature storage (e.g. 4[degrees]C), thus avoiding the application of sprout inhibitors. As stated above, levels of formation of acrylamide during frying should be another criterion for the development of such cultivars.

Chilling Injury

During growth and postharvest handling, chilling injury, defined as damage to susceptible plant species during exposure to low temperatures above the freezing point, leads to losses in yield and growth potential of crop plants and to reduced quality of detached, edible tissues (Purvis and Shewfelt, 1993). Fruits of many species, especially those of tropical and subtropical origin suffer chilling injury upon exposure to non-freezing temperatures below 12[degrees]C (Lafuente et al., 1991; Jaitrong et al., 2004).

Causes of Tissue Damage

A common response of sensitive plant cells to low temperatures is the disruption of membrane integrity (Purvis and Shewfelt, 1993). In chilling-sensitive plants, the lipids in the bilayer have a high percentage of saturated fatty acid chains, and membranes with this composition tend to solidify into a semi-crystalline state at a temperature well above 0[degrees]C (Parkin et al., 1989). Low temperature also affects membrane proteins and enzymes. Protein- protein and protein-lipid interactions may be weakened by a decrease in the relative strength of hydrophobic bonding, leading to subunit dissociation and/or polypeptide unfolding (Stanley, 1991).

It has been shown that some of the effects of low-temperature stress are mediated by reactive oxygen species (Aroca et al., 2003). The production of ROS is a phenomenon common to chilling and other stress conditions (e.g., cell damage), as indicated in the previous section. Under prolonged oxidative stress conditions, ROS cause lipid peroxidation, DNA damage, and protein oxidative inactivation (Prasad, 1996). The activities of certain enzymes involved in keeping ROS at low levels, including superoxide dismutase and catalase, decrease. The consequence of this is a reduction in defence against free radicals and repair mechanisms. During exposure to stress the balance between degradation and repair will be shifted towards greater degradation of susceptible tissues (Purvis and Shewfelt, 1993).

Minimizing Chilling Injury After Harvest

Species that are sensitive to chilling can show appreciable variation in their response to low temperatures. Also, temperatures that are considered “cold” vary between species (e.g. pineapple and carrot). Resistance to chilling injury often increases if plants are first hardened (acclimated) by exposure to cool but non-injurious temperatures. Chilling damage thus can be minimized if exposure is slow and gradual. Membrane lipids from chilling-resistant plants often have a greater proportion of unsaturated fatty acids than those from chilling-sensitive plants, and during acclimation to low temperatures the activity of lipid desaturase enzymes increases and the proportion of unsaturated lipids rises (Stanley, 1991; Palta et al., 1993). This modification lowers the temperature at which the membrane lipids begin a gradual phase change from fluid to semi- crystalline, and allows membranes to remain fluid at lower temperatures (Vandenbussche et al., 1999). For example, Marangoni et al. (1990) stored mature green commercial tomatoes at 12[degrees]C for 4 d followed by storage at 8[degrees]C for 4 d, and then chilling at 5[degrees]C for 15 d. The properties of these tomatoes were compared with those directly chilled for 15 d at 5[degrees]C. The gradual acclimation program decreased the severity of chilling injury, as reflected in a more intense red color and a harder fruit, compared with what was observed in directly chilled tomatoes. Gradually cold-treated tomatoes showed an increase in the proportion of unsaturated fatty acids in their membranes, indicating that acclimation had taken place. The described chilling response will also prepare plant tissues for potential freezing. A direct response to chilling is a decrease in cellular respiration. However, in many species acclimation results in the restoration of respiration (Atkin and Tjoelker, 2003), which may lead to increased respiratory losses during storage. Therefore, it is important to use procedures for thermal acclimation that avoid respiratory reactivation.

Other methods of reducing or avoiding chilling injury have been described in the literature. They are based on the physiological response to another stress that protects the tissue against chilling injury (cross-tolerance). These procedures will be described in more detail in following sections.

Freezing Injury

Freezing injury occurs at temperatures below the freezing point of water. Several plants, however, are able to induce tolerance to freezing, following a period of acclimation at cold, but non- freezing, temperatures (Smallwood and Bowles, 2002).

Physiology

The primary manifestation of cell damage by freezing is observed in the plasma membrane (Steponkus, 1984; Palta, 1990). The water potential of ice is lower than that of liquid water. Extracellular ice crystals grow by drawing water from cells, thus dehydrating them, until the water potential of the ice and that of water in the cell are equal. The water potential of ice decreases as the temperature decreases, so the extent of cellular dehydration increases with decreasing temperature, to a limit set by vitrification (glassy state) (Pearce, 2001). This deterioration is observed as wilting or softening of plant parts.

Plants that survive winter either prevent the crystallization of ice within their tissues (freeze avoidance) or can withstand ice crystallization in the apoplast (freeze tolerance) (Smallwood and Bowles, 2002). Freeze avoidance involves supercooling and hence prevention of the incursion of ice into the apoplast. Without ice nucleation, pure water can be supercooled to a certain point below 0[degrees]C. However, this supercooling is only a practical strategy at the whole plant level when exposure to subzero temperatures is relatively brief (George et al., 1982; Smallwood and Bowles, 2002). Some specialized cell types and organs use supercooling as a strategy to overwinter, such as the xylem ray parenchyma cells of many trees, which supercool to around 0 -40[degrees]C (George and Burke, 1977). Given the widespread presence of nucleators in the environment, the most common frost survival strategy is cold acclimation (freeze tolerance) and this is achieved through several changes in cell biochemistry regulated at the gene expression level (Danyluk et al., 1998).

The accumulation of osmotically active substances, such as simple sugars, organic acids, proline, and glycinebetaine, is a protective mechanism induced by cold stress (as previously described for cold sweetening). In many plants, sugars act as cryoprotectants which increase the freezing resistance through direct and/or indirect effects (Graham and Patterson, 1982; Chang and Reed, 2000). The hydrophilic nature of sugars is well-suited to replace water and stabilize the cell membrane through hydrogen bonding between hydroxyl groups on the sugar and polar residues in phospholipids, preventing dehydration effects in membranes (Danyluk etal., 1998). Accumulation of osmotically active substances leads to a decrease in the chemical potential of water. It has been suggested that this mechanism is involved in regulating the induction of cold-induced gene expression (Fu et al., 2000) and in the higher resistance of cold-acclimated plants to fungal infection (Tronsmo, 1986).

Many cold-induced proteins accumulate in the tissues during cold acclimation (reviewed by Thomashow, 1999). These can account for up to 0.9% of the total soluble proteins in winter wheat after 21 days’ of cold acclimation (Houde et al., 1995), and have been found to be accumulated in many organelles, including the endoplasmic reticulum (Ukaji et al., 2001) and mitochondria (Zykova et al., 2002). The functions of these proteins are not fully understood and have been the subject of intense research during recent years. It has been speculated that they could have a detergent-like activity, coating hydrophobic surfaces and thus preventing the coagulation of macromolecules (Smallwood and Bowles, 2002). Examples of proteins that accumulate with cold acclimation include the cryoprotective proteins of spinach, rye, and other cereal antifreeze proteins (AFPs) (Thomashow, 1999). A factor common to all these proteins is that they are predominantly located in the apoplast and are therefore more likely to come into contact with the outer surface of the plasma membrane.

AFPs are expressed in a number of plant species, such as winter rye, winter barley, winter canola, white oak, and carrots, in response to low temperature (Urrutia et al., 1992; Duman and Olsen, 1993; Feeney and Yeh, 1993; Griffith and Antikainen, 1996; Smallwood et al., 1999). Their accumulation and activity have been found to be strongly correlated with winter survival and it has been suggested that they be used as a biological marker for crop improvement (Griffith et al., 1992; Chun et al., 1998).

Antifreeze proteins interact with ice crystals by adsorption onto non-basal planes of ice at the ice-water interface thus modifying their growth. At high AFP concentrations (fiM), minimal crystal growth occurs, forming very small, stable hexagonal bipyramids. Physical damage caused by ice can occur during warming, as well as during freezing, by a process known as recrystallization (Knight and Duman, 1986; Breton et al., 2000).

Recrystallization of ice occurs when small ice crystals condense into larger ones. This can happen very quickly at temperatures just below the melting point of a frozen solution. In nature, prolonged exposure to subzero temperatures and temperature fluctuations may promote recrystallization of frozen tissues, especially those in which cells are densely packed, and allow ice access to locations from which it is usually excluded. AFPs adsorbed onto the surfaces of ice act as potent inhibitors of recrystallization, even at very low concentrations (e.g. 1 /ig/ml) (Worrall et al., 1998; Smallwood et al., 1999). Given that AFPs are also found in plant tissues where ice is allowed to crystallize in the apoplast (which includes the xylem, cell walls, and intercellular spaces), it has been speculated that inhibition of ice recrystallization may be the physiologically relevant aspect of the activity of AFPs (Smallwood and Bowles, 2002).

Application of Cold Acclimation in the Frozen-Vegetable Industry

When cold-stressed, starch-rich vegetables (e.g. potatoes) are frozen industrially, the effects of cold sweetening during the storage period could be detrimental to the quality of the product after cooking by the consumer at home (e.g., excessive brown color after frying, as discussed earlier). However, if vegetables accumulating mostly sucrose in their cytoplasm (e.g. carrots) and antifreeze proteins in their cell walls during growth in the field in late autumn are to be frozen, industry may take advantage of cold- induced stress responses to optimize the quality of the frozen product. The potential application of the acquisition of freezing tolerance by cold-acclimation of carrot taproots in the frozen- carrot industry has been discussed by Gomez and Sjoholm (2004). The authors illustrated the enhancement of the tolerance to freezing by the metabolic response to low-temperature stress by freezing both acclimated and nonacclimated carrot slices at a very slow freezing rate (-5[degrees]C ambient temperature over-night). Figure 3a shows a piece of non-acclimated carrot tissue that has been extensively damaged by freezing. In remarkable contrast, Fig. 3b shows much more intact tissue of the cold-acclimated samples frozen under the same conditions. Although vegetables such as carrots are usually frozen quickly to produce small ice crystals, these ice crystals may grow larger over time through recrystallization. Recrystallization occurs when temperature gradients form within the product during freezing or thawing, or when the temperature fluctuates during storage or transportation (Griffith and Ewart, 1995; Breton et al., 2000). Recrystallization in frozen foods can result in membrane damage, thus reduced water holding capacity (high drip loss), and associated loss of nutrients (Fletcher et al., 1999; Breton et al., 2000). AFP is abundant in the acclimated carrot tap root apoplast (0.5 mg of pure protein can be isolated from 1 kg of fully acclimated carrot taproots Smallwood et al., 1999), and may be a key factor in inhibiting recrystallization and preserving the quality of the frozen product. Figure 3 Conventional scanning electron micrographs showing the parenchyma of frozen carrots. Carrot slices from non- acclimated (a) and cold-acclimated (b) field-grown carrot taproots were covered with plastic film and frozen at -5[degrees]C overnight. The samples were freeze-dried, fractured, and gold-sputtered. They were examined in a JEOL SEM 840-A microscope, operated at 15 kV and a working distance of 15 mm. The images show a remarkable contrast in the degree of tissue damage caused by the freezing treatment between the acclimated and the non-acclimated carrots.

The potential benefits of cold acclimation of frozen carrots can, however be eradicated by the common practice of blanching before freezing. Heat will damage the cells, destroying the protective system nature has created against frost damage. Above approximately 50[degrees]C, the functionality of the cell membrane is irreversibly damaged (De Belie et al., 2000). Denaturation of proteins such as AFPs in the cell walls would also compromise the cold acclimation effect, as these proteins must be folded correctly in order to be active (M. Griffith, pers. comm.). The optimization of blanching to minimize tissue damage is thus very important if the frozen-food industry is to be able to take advantage of cold acclimation to protect tissue cells (Gomez, 2004).

HEAT STRESS AND HEAT SHOCK

Most tissues of higher plants are unable to survive extended exposure to high temperatures. Non-growing cells and dehydrated tissue can tolerate much higher temperatures than hydrated, growing cells. Actively growing tissues rarely survive temperatures above 45[degrees]C, but dry seeds can endure 120[degrees]C and pollen grains of some species can remain viable after exposure to 70[degrees]C (Taiz and Zeiger, 2002).

Storage of some legumes under tropical conditions (3040[degrees]C; >75% humidity) renders them susceptible to a hardening phenomenon, causing nutritional losses and inflicting economic losses on farmers and poor urban dwellers in developing countries (Aguilera and Ballivian, 1987; Martin-Cabrejas and Esteban, 1995). This is an irreversible phenomenon known as the hard- to-cook (HTC) defect. Beans with this defect are characterized by extended cooking times to achieve cotyledon softening, are less palatable to the consumer and are of lower nutritional value (Reyes- Moreno and Paredes-Lopez, 1993).

In many crops, as further discussed in this section, periodic, brief exposure to sublethal heat stress often induces tolerance to otherwise lethal temperatures, a phenomenon known as induced thermotolerance (Viswanathan and Khanna-Chopra, 1996). Thermotolerance in crops is determined by a variety of factors such as photoperiod, light intensity and water availability (Ann et al., 2004).

Causes of cell damage

Exposure of plants to temperatures above their optimal growth temperature can disrupt many essential metabolic processes, including photosynthesis and respiration, the former being more sensitive. Activation of lipid peroxidation is one of the earliest and least stress-specific plasmalemmal responses caused by any stress agent, including heat shock. Lipid peroxidation can result in various structural and functional disturbances in the cell (Veselov et al., 2002). Furthermore, excessive fluidity of membrane lipids at high temperatures (above 50[degrees]C) is correlated with loss of functional cell compartmentalisation which considerably enhances the permeability of membranes and, in consequence, the passive flux of solutes (Kluge et al., 1991), leakage of electrolytes, and reduction of turgor pressure (De Belie et al., 2000; Gonzatez-Martinez, 2003). It has been hypothesized that in beans susceptible to the HTC defect, the effects of temperature on cell membrane are accompanied by lignification of the cell wall, pectic de-esterification in the middle lamella and breakdown of phytic acid, inhibiting chelation of divalent cations, which renders pectates in the middle lamella unsusceptible to softening during cooking (Aguilera and Ballivian, 1987, Reyes-Moreno and Paredes-Lopez, 1993).

High-temperature injury is also associated with lipid phase transitions and/or changes in transmembrane protein conformation (Hansen et al., 1994). Heat stress causes many cell proteins (enzymes or structural proteins) to become unfolded or misfolded. Such misfolded proteins can aggregate and precipitate.

Plant Strategies for Heat Tolerance

Metabolic acclimation associated with heat tolerance mechanisms includes an increase in the degree of saturation of fatty acids in membrane lipids, which makes the membranes less fluid, the synthesis of enzymes and isoenzymes with broad thermal kinetic windows, the synthesis of protective enzymes such as glutathione reductase, peroxidase, and catalase (Viswanathan and Khanna-Chopra, 1996), and the production of heat shock proteins (HSPs).

In response to sudden rises in temperature (5 to 10[degrees]C), plants produce a unique set of proteins, the HSPs. Most HSPs function as molecular chaperones, that is, they bind to unfolded or denatured proteins, prevent aggregation and induce correct refolding, facilitating correct cell function at elevated, stressful temperatures. Some HSPs assist in polypeptide transport across membranes into cellular compartments (Miroshnichenko et al., 2005).

Plants and most other organisms produce HSPs that have different functions in response to increases in temperature: HSP100, HSP90, HSP70, HSP60, and small HSPs (smHSPs, 15-30 kDa) (Vierling, 1991). HSP expression has been characterized in a variety of higher plants, including tomato (Banzet et al., 1998), maize (Cooper and Ho, 1983), soybean (Hsieh et al., 1992), carrot (Malik et al., 1999), pea (DeRocher et al., 1991), sugarcane (Hoisydai and Harrington, 1989), apple (Bowenet al., 2002), and potato (Ann et al., 2004). Some smHSPs are known to play an important role in the protection of biomembranes and organelles (Viswanathan and Khanna-Chopra, 1996). Synthesizing a number of smHSPs at elevated temperatures is one of the unique features of the heat-shock response of plants (Ahn et al., 2004). Cells that have been induced to synthesize HSPs show improved thermal tolerance and can withstand exposure to temperatures that are otherwise lethal (Malik et al., 1999; Bowen et al., 2002). It has been shown that a smHSP in tomato (VIS1) plays a role in facilitating fruit ripening, senescence, and seed dispersal by protecting the cellular machinery against thermal denaturation during the daily cycles of daytime rise in temperature. VIS 1 acts as a chaperone by binding reversibly to enzymes, including cell wall polymer-modifying enzymes, and protecting them from thermal denaturation (Ramakrishna et al., 2003).

In the food industry, heat treatment (generally 500 – 70[degrees]C) has been used for the past 40 years to improve the texture of vegetables prior to high-temperature processing. The firming effect of low-temperature blanching has been studied in a number of vegetables (Bartolome et al., 1972; Lee et al., 1979; StolleSmits et al., 2000). Evidence indicates that the firming effect is due to the temperature activation of pectin methylesterase (PME, EC 3.1.1.11). The resulting reduction in the degree of methylesterification of the pectins in the cell wall and middle lamella allows the more calcium cross-linking between calcium molecules, increasing firmness (Pilnik and Voragen, 1991). The mechanism governing temperature activation of PME is not well- understood. It has been speculated that, at elevated temperatures, a change in the PME enzyme or its environment may occur such that the enzyme is converted into a more active form. Loss of membrane integrity and leakage between cellular compartments at temperatures >40[degrees]C may contribute to this activation (Anthon and Barrett, 2006). However, to our knowledge, no previous study has associated PME activation with induction of signal cascades at the genetic level and/or metabolic transformations strictly associated with the concept of “stress response” that we have been using throughout this review. It appears that mild blanching treatment, for example, at 70[degrees]C for 30 min (Lee et al., 1979), is used by the industry as a direct way of regulating the activity of the enzyme. This treatment may mimic a true stress response that may occur at lower temperatures (around 40[degrees]C) for a longer time, for example, when harvested material lies in the sun for hours before processing. A more detailed study of the time and temperature dependence of PME activation and the molecular mechanisms regulating it would be of interest.

Cross-Tolerance and its Application in Postharvest Handling and Minimal Processing

In general, stress responses involve changes in the proteome and metabolome with increased expression of proteins and compatible solutes. Cross-talk between stress signalling pathways may result in co-expression of stress responses (Joyce et al., 2003). Thus, cells previously exposed to one kind of stress may gain protection against another kind (cross-tolerance). For example, some of the HSPs are not unique to high-temperature stress and can be induced by other forms of stress such as drought (Alamillo et al., 1995; Wehmeyer and Vierling, 2000), wounding, low temperature, and salinity (Wang et al., 2001). Symptoms of chilling injury are reduced after heat pretreatment, and this reduction is correlated with persistence of several HSPs in fruit tissue (Sabehat et al., 1996). Tomato and avocado fruits, in which heat shock was induced (48 h at 38[degrees]C), accumulated HSPs and were protected from injury by subsequent chilling at 2[degrees]C (Lurie, 1998). Reduced chilling injury of cucumber cotyledons and cultured apple cells after exposure to 37 or 42[degrees]C has also been reported (Lafuente et al., 1991; Wang et al., 2001). Heat treatment at 38[degrees]C for 8 h applied to evening-harvested sweet basil reduced its sensitivity to chilling. This reduction may have involved the antioxidative system of ROS protection, as suggested by the increased reductive potential in the leaves, as well as the induction of superoxide dismutase and catalase activity following heat treatment. Elevated activity remained through subsequent cold storage below 12[degrees]C (Faure-Mlynski et al., 2004). Heat shock treatment has been used to reduce decay and chilling injury, and to enhance host resistance to pathogens in fruits. Treatment by dipping in water at 52- 53[degrees]C for 2 min or 62[degrees]C for 20 s promoted the accumulation of HSPs and prolinerich proteins in the skin of grapefruit. Heat application has been shown to markedly reduce decay and the sensitivity of citrus fruit to chilling injury without any deleterious effects on fruit quality (Ben-Yehoshua, 2003). Several types of machines for hot water treatment are already in operation in many countries in packing houses for citrus (Ben-Yehoshua, 2003) and other fruits, such as bell peppers, com cobs, lychees, mangos, melons, nectarines, and peaches (Fallik et al., 1999).

When cells are subjected to a stressful, but non-lethal temperature, the synthesis of HSPs increases dramatically, white the continuous translation of other proteins is lowered or ceases (Vierling, 1991). This effect has been seen in studies on the wounding stress response of carrots and lettuce. In the case of carrot slices, exposure to 40[degrees]C for 1 h caused the cessation of the synthesis and secretion of extensin proteins, a typical response to wounding stress (Brodl and Ho, 1992). Maximum accumulation of HSPs was seen in the carrot slices one hour after a temperature increase from 28[degrees]C to 40[degrees]C. The synthesis of HSPs diminished sharply after 3 h of continuous incubation at40[degrees]C and the carrots resumed the secretion of extensin proteins during that period of time. Upon recovery from 40[degrees]C, the carrot slices resumed the secretion of extensin and other cell wall proteins (Brodl and Ho, 1992). This study demonstrates that high-temperature stress reduces the response to wounding and nicely illustrates the fact that plant tissues follow a certain temporal order and hierarchy in their response to multiple stimuli. The heat-stressed, wounded tissue has basically redirected its resources towards the response to more severe stress.

This principle has been applied in the minimal processing of vegetables to prevent browning of wounded lettuce leaf tissue. Wound- induced browning has been significantly reduced in iceberg lettuce by the application of short thermal stress (Loaiza-Velarde et al., 1997). A heat shock of 45[degrees]C for 90 s effectively prevents the synthesis of phenylalanine ammonia-lyase (PAL), whose increased activity leads to the accumulation of phenolic compounds (e.g., chlorogenic acid, dicaffeoyl tartaric acid, and isochlorogenic acid) and tissue browning (Salveit, 2000). Inhibition of PAL synthesis appears to result from the redirection of protein synthesis away from wound-induced proteins to the synthesis of HSPs. The effect of heat shock (45[degrees]C for 90 s followed by rapid cooling to 0[degrees]C) either 4 h before wounding or 2 h after wounding was so persistent that the fresh-cut lettuce did not show any browning after 15 days in air at 5[degrees]C (Salveit, 2000). However, it has also been shown that this treatment was not successful in tissues with constitutively or induced high levels of phenolic compounds. The heat shock acts only on the synthesis of PAL and not on the activity of other enzymes involved in tissue browning (Salveit, 2000).

DROUGHT STRESS AND DESICCATION TOLERANCE

Water deficit can be defined as any water content of a tissue or cell that is below the highest water content exhibited in the most hydrated state (Taiz and Zeiger, 2002). Lack of water has several detrimental effects on plants, including modification of the cell wall crystallinity, clumping of microfibrils, denaturation of proteins, loss of cell turgor and membrane fluidity, and oxidative damage by reactive oxygen species (Aguilera et al., 2003; Prothon et al., 2003).

Strategies for Desiccation Tolerance

Some plant tissues can acquire desiccation tolerance, defined as the ability to function while dehydrated, or desiccation postponement, defined as the ability to maintain tissue hydration (Davies, 2004). Desiccation tolerance involves a co-ordinated set of mechanisms that help certain tissues to survive dehydration. These mechanisms include stomatal closure (Davies et al., 2002), osmotic adjustment, removal of reactive oxygen species, and the accumulation of late embryogenesis-abundant (LEA) proteins (Oliver et al., 2001).

Plants can continue to take up water only when their water potential is below that of the water source. Osmotic adjustment, in which cells accumulate osmotically active solutes (also known as compatible solutes or osmolytes and including sugars, organic acids, glycine betaine, sorbitol, proline, amino acids, polyols, quaternary amines, and ions), is a process in which the water potential of the tissue can be decreased without an accompanying decrease in turgor (see Gomez et al., 2004 for definitions of plant water relations). The change in tissue water potential results simply from changes in the osmotic potential (Fan et al., 1994; Zhang et al., 1999). In radish tubers the total concentration of free sugars increases with soil water deficit (Herppich et al., 2001a). During storage, carrots can increase their concentration of glucose and fructose from their sucrose stores (Herppich et al., 2001b,c). These monosaccharides contribute twice as much to osmotic pressure per unit weight as disaccharides.

Cellular electron transport chains are impaired upon dehydration and may generate increasing amounts of reactive oxygen species (Hoekstra, 2002). Free radical attack on phospholipids, DNA and proteins is one of the molecular mechanisms of damage leading to death in desiccation-sensitive cells upon drying (Oliver et al., 2001). Protection against ROS is thought to play a role in desiccation tolerance. Therefore, free radical scavenging systems are important components among the mechanisms governing desiccation tolerance. Over-expression of some enzymes, such as manganese superoxide dismutase and glutathione S-transferase/glutathione peroxidase, has been associated with an enhanced tolerance to water deficit in transgenic tobacco plants and cotton cells (Serrano and Montesinos, 2003). Moreover, desiccation-tolerant organisms (seeds) can reduce and adapt their metabolic activities early during drying to decrease the generation of ROS (Leprince et al., 1994).

Stress often induces the accumulation of proteins, as has been described for AFPs in the case of low-temperature stress and HSPs in heat stress. In the case of drought stress, a large group of genes code for hydrophilic LEA proteins, which are suspected to play a rote in the acquisition of desiccation tolerance (Blackman et al., 1995). Although the function of LEA proteins is not well- understood, they accumulate in vegetative tissues during episodes of drought. Their protective role may be associated with their ability to retain water and to prevent crystallization of cellular proteins during desiccation (Serrano and Montesinos, 2003). Oliver et al. (2001), summarize the possible protective roles of LEA proteins. At high hydration levels, LEA proteins might play a role in sequestering ions and preventing of the damaging effects of free radical reactions. In the dried state, LEA proteins may act together with carbohydrates in the formation of a tight hydrogen-bound network, providing stability to macromolecular and cellular structures in the cytoplasm. This network would inhibit the fusion of cellular membranes, denaturation of cytoplasmic proteins, and the detrimental effects of free radical reactions.

Drought typically leads to the accumulation of ABA. Numerous genes are induced by both drought and ABA accumulation during the stress episode (Liu et al., 2005). Exogenous application of ABA has been shown to induce desiccation tolerance in somatic alfalfa embryos. Heat shock pretreatment, at 38[degrees]C for as little as 10 min, induced a degree of desiccation tolerance in the somatic embryos which was equivalent to ABA application and was therefore shown to be a viable alternative to exogenous ABA treatment. The drying rate did not influence the survival of the heat-stressed embryos (Senaratna et al., 1989).

Application to Food Dehydration

The quality of air-dehydrated plant products is often very low, with shrunken, shrivelled, darkened tissue, and poor rehydration ability (Nijhuis and Torringa, 1996). Over the years, techniques such as freeze-drying and vacuum drying have been developed in attempts to improve the characteristics of the dried material. However, much remains to be done if we are to attain high quality characteristics of semi-prepared fruit and vegetables (Prothon, 2002) regarding color, texture, and the appearance of freshness of rehydrated products (Serrano and Montesinos, 2003). Processes should be optimized so as to prevent tissue damage. The quality of the product will be associated with the preservation of the structure and the function of biological membranes and proteins during desiccation, storage, and rehydration (Serrano and Montesinos, 2003).

To the best of our knowledge, no application of the principles of desiccation tolerance described above to food dehydration has been reported. Few studies have pointed out that a better understanding of the mechanisms by which desiccation-tolerant organisms survive desiccation may lead to the development of new methods of preserving foods and biological materials. Further research is therefore needed on the following topics:

* The use of glass-forming carbohydrates. Aguilera and Karel (1995) and Aguilera (2003) highlighted the importance of searching for carbohydrates, e.g. trehalose (which appears to be the preferred sugar synthesized before dehydration of yeasts, fungi and bacteria) or sucrose, raffinose, stachyose and verbascose (with an analogous role in seeds of higher plants) to preserve food materials as low- moisture glasses. Survival under desiccation has been suggested to be associated with vitrification of the cytoplasm (Aguilera and Karel, 1995). In the glassy state, the molecular mobility and biochemical activity are restricted in the cytoplasm, and possibly in key organelles, but normal activities resume upon rehydration (Williams and Leopold, 1989). * The application of stress pretreatment aimed at inducing certain levels of desiccation tolerance (cross-tolerance). Heat stress has been suggested as an effective method of achieving this aim (Senaratna et al., 1989; Tunnacliffe et al., 2001). The effect of cold stress could also be investigated. Plant tissue acclimated to cold implies protection of cell membranes against dehydration (in this case the intended protection is against freezing-induced dehydration) and an increase in the strength of cell wall (which may help to prevent tissue collapse (Prothon et al., 2003)).

* The combination of the above, alone or together with different processing technologies. The combination of osmotic and microwave dehydration or calcium pretreatment prior to microwave-assisted dehydration (Prothon et al., 2001; Ahrne et al., 2003) could be applied after stress pre-treatment or application of glass-forming carbohydrates.

* Genetic engineering it can be used to generate transgenic plants suitable for growth under drought conditions and destined for the production of dried foods (Aguilera et al., 2003; Serrano and Montesinos, 2003). The question here is whether transgenic products will be accepted by consumers.

FUTURE PERSPECTIVES

The carbon and oxygen metabolism of plant cells is connected to several processes of which we have limited understanding. These include ROS metabolism and signalling, cell survival, stress resistance, and redox homeostasis. Industrial practices involved, for example, in the production of minimally processed fruit and vegetables are likely to influence these metabolic processes and, therefore, research is needed to gain knowledge on novel (i.e. not found in nature) tissue stress conditions during processing operations in the food industry.

For example, packaging in a modified atmosphere may affect the metabolism of the product, influencing storage and quality properties. Jacobsson (2004) demonstrated that different levels of O2 and CO2 in the package affect the metabolism of broccoli, resulting in changes in the aroma, which, in many cases, were not noticeable until after the broccoli was cooked. Plant stress responses often involve changes in secondary metabolism. It can not be excluded that factors affecting primary metabolism, such as the exposure to different atmospheres inside the package, can affect secondary metabolic pathways involved in the production of aroma compounds.

Recent publications on novel processing techniques such as the application of high hydrostatic pressure or pulsed electric fields (Dornenburg and Knorr, 1998; Ye et al., 2004) have suggested the possibility of using these techniques to stress the cells and stimulate secondary metabolism and thus the biosynthesis of desirable health-promoting metabolites.

External application of substances used in the food industry to prevent enzymatic browning, such as citric acid and L-cysteine (Laurila et al., 1998), is another example of novel stress conditions occurring due to industrial practices. Cysteine induced severe down-regulation of the cytochrome pathway followed by the induction of alternative oxidase (AOX) expression in tobacco cells (Vanlerberghe et al., 2002). In cells with suppressed AOX expression, the application of cysteine even induced programmed cell death. At neutral pH, citrate has been shown to increase AOX expression in cell suspensions potentially increasing respiratory catabolism (Vanlerberghe and Ordog, 2002). However, the consequences of cell metabolism resulting from the application of cysteine and citrate are not well-understood. Citric acid application would cause acidic stress to the cells by lowering the apoplastic pH below normal levels, and a decrease in pH has been shown to increase gene expression of alternative respiratory pathways, including AOX (Escobar et al., 2006). According to Lambers et al. (1998) the tissue copes with excess H+ uptake at low pH by increasing active H+ pumping by plasma membrane ATPases, increasing the demand for respiratory energy. Therefore, changes in metabolic activity due to the application of anti-browning substances in the food industry must be understood at the gene expression level with regard to the consequences on rates of oxygen consumption, browning inhibition, sugar metabolism, and cell wall changes during wound-induced reactions in fresh-cut fruit and vegetables.

Investigations of the genetic control of metabolism during industrial processing of fresh fruit and vegetables, through techniques such as transcriptomics and metabolic profiling, will provide knowledge on the consequences of industrial practices essential for quality assurance and optimization.

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