2007 plant stress physiology- opportunities and challenges for the food industry

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Critical Reviews in Food Science and Nutrition, 46:749–763 (2007)
Copyright C Taylor and Francis Group, LLC
ISSN: 1040-8398
DOI: 10.1080/10408390601062211

Plant Stress Physiology:
Opportunities and Challenges for the
Food Industry

FEDERICO GOMEZ GALINDO∗ and INGEGERD SJOHOLM
´ ¨
Department of Food Engineering, Lund University, P.O. Box 124, SE-221 00, Lund, Sweden

ALLAN G. RASMUSSON and SUSANNE WIDELL
Department of Cell and Organism Biology, Lund University, S¨ lvegatan 35B, SE-223 62 Lund, Sweden
o

KARL KAACK
Department of Food Science, Danish Institute of Agricultural Sciences, Kristinebjergvej 10, DK-5792, Aarslev, Denmark

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 to become stressful. Cellular responses to stress may include
changes in cell cycle and division, cell membranes, cell wall
Because plants are confined to the place in which they grow, architecture, and metabolism (e.g. accumulation of osmotically
they have a limited capacity to avoid unfavorable conditions active substances).
in their environment, such as extremes of temperature, water From a biological point of view, industrial treatment of plant
shortage, insufficient or excessive light or mineral nutrients, tissue will mimic stress (Fig. 1) and therefore, knowledge of
wounding by herbivores, or attack by pathogenic bacteria, fungi, how the plant material will be affected in relation to time, the
viruses, and viroids. Plants have developed sophisticated molec- environment, and industrial manipulation is of fundamental im-
ular chemical strategies to defend themselves against such abi- portance for quality assurance and process optimization. We
otic and biotic stress, often combined with changes in growth here focus our attention on reviewing and analyzing possible
and development patterns (Boyer, 1982; Gaspar et al., 2002). advantages and disadvantages of the stress responses of fruits
Stress is usually defined as an external factor that exerts a dis- and vegetables during industrial processing operations. Reports
advantageous influence on the plant. This concept is closely on attempts to implement physiological and biochemical prin-
associated with stress tolerance, which is the plant’s capacity ciples in the industrial processing of fruit and vegetables are
to cope with unfavorable conditions (Taiz and Zeiger, 2002). not common in the literature, but a few recent investigations,
In both natural and agricultural conditions, environmental fac- referred to in the following sections, have laid the foundation
tors, such as air temperature, can become stressful in just a few for a fascinating area of research and technological innovation.
minutes. Soil water content may take days to weeks, whereas
other factors such as soil mineral deficiency, can take months
STRESS LEADING TO CELL DAMAGE
∗ Address Correspondence to Federico G´ mez Galindo, Department of Food
o
Engineering, Lund University, P.O. Box 124, SE-221 00, Lund, Sweden. Tel: During harvesting, transportation, washing, sorting, and
+4646 222 9814; Fax: +4646 222 9846; E-mail: federico.gomez@food.lth.se packing, fruits and vegetables are subjected to mechanical stress
749


750 F. G. GALINDO ET AL.

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.

that may lead to crushing of surface cell layers. When the fresh during harvesting, handling and storage, leading to significant
products reach the processing line for producing, for example, levels of rejection of potato harvests (Potato Marketing Board,
ready-to-use salads, they are typically peeled, sliced, diced, or 1994). The synthesis of melanin is thought to be a defence
shredded before packaging. These operations cut through cells mechanism in which the polymerized, insoluble complexes
and leave intact cells of previously internal tissues exposed. form a resistant barrier, sealing tuber tissues against the entry
These postharvest and processing operations are traumatic for and spread of pathogens. The predisposition of tubers to melanin
the cells proximal to the damage site and induce a complex se- production depends on growth and storage conditions and
ries of molecular events aimed at repairing the damage caused temperature during processing, and exhibits a wide range of ge-
to the tissue (Surjadinata and Cisneros-Zevallos, 2003). netic 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
Response to Postharvest Handling undesirable physiological changes, further reducing quality and
storability.
Mechanical stress, imposed on plant cells by a variety of phys- In spite of the many detrimental consequences of posthar-
ical stimuli during harvesting and handling of fresh horticultural vest mechanical stress on the quality of fruit and vegetables,
products, induces a wide range of cellular responses such as in- some reports have shown that slight mechanical stress during
creased respiration rate, ethylene production, and higher suscep- growth can improve the postharvest processability of lettuce,
tibility to pathogen attack (Charron and Cantliffe, 1995; Stanley, cauliflower, celery (Biddington and Dearman, 1985; P¨ ntinen
o
1991). In carrots, mechanical stress brings about a decrease in and Voipio, 1992), and baby leaf salad (Clarkson et al., 2003),
root pressure potential and water potential during the initial stor- when the stress is applied to the seedlings. Mechanical stress
age period (Mempel and Geyer, 1999; Herppich et al., 1999). during growth results in modified leaf architecture producing
Furthermore, the production of ethylene and 6-methoxymellein smaller, more compact new leaves. After industrial unit oper-
(a bitter compound) increases, whereas the levels of several ter- ations including washing, drying, and packing, baby leaves of
penes associated with the characteristic aroma of carrots de- lettuce and spinach showed an increase in shelf-life. This in-
creases (Selj˚ sen et al., 2001). The accelerated aging in cu-
a crease was associated with a reduction in the area of individual
cumbers involves the induction of cell-wall-degrading enzymes, epidermal cells and modification of the biophysical properties
leading to tissue degeneration (Miller and Kelley, 1989). of the cell wall (Clarkson et al., 2003). The mechanical stress
Potatoes are particularly susceptible to mechanical stress. manipulation of the seedlings led to the development of new
Physically stressed tuber tissue produces melanin-based adapted leaves with stiffer cell walls, so that the leaves would
pigments, leading to the blue-black discoloration of subdermal have greater protection against mechanical stress during pro-
tissues known agronomically as black-spot bruising (Johnson cessing; stress that may otherwise cause damage and browning
et al., 2003).This is a serious agronomic problem manifested of the leaves (Lopez-Galvez et al., 1996). Smaller cells have


PLANT STRESS PHYSIOLOGY 751

a larger relative cell wall volume and dry weight (Wurr et al., man, 1997; Bernards et al., 1999). The oxidative coupling of
1986; Clarkson et al., 2003). the poly(phenolic) component of suberin is thought to be a
peroxidase/H2 O2 -dependent, free-radical process. In response
to wounding, and in association with suberization, plant tis-
Effects on Minimally Processed Plant Tissue sues generate reactive oxygen species (ROS), including super-
oxide (O− ), hydrogen peroxide (H2 O2 ), and the hydroxyl radical
2
As a result of peeling, grating, or shredding, a relatively stable (OH. ). It has been shown that H2 O2 is essential for suberization
agricultural product with a shelf life of several weeks or months in potato slices (Razem and Bernards, 2002). Immediately fol-
will change into one that deteriorates rapidly from a food quality lowing wounding, a rapid increase in oxygen uptake is followed
perspective. Minimally processed fruit and vegetables should by an initial burst of ROS (oxidative burst) (Bolwell et al., 1995).
have storage lives of at least 4–7 days, but preferably longer, up In wounded potatoes, this burst reaches a maximum within 30
to 21 days depending on the market (Ahvenainen, 1996; Barry- to 60 min and is followed by at least three other massive bursts
Ryan and O’Beirne, 1997). Deterioration is mostly the result of at 42, 63, and 100 h post-wounding. These later bursts were as-
microbial spoilage, wound healing, biochemical changes, and sociated with wound healing and are probably involved in the
loss of nutritional quality. oxidative cross-linking of suberin poly(phenols) (Razem and
The quality and shelf life of minimally processed fruit and Bernards, 2003). The initial deposition of suberin in potato re-
vegetables are directly influenced by the extent of wounding quires approximately 18 h at 18◦ C (Lulai and Corsini, 1998)
and the size of the wounded surface caused by the processing and reaches a stage in which the suberized layer has sufficient
operation. For example, it has been demonstrated that carrots structural integrity to be peeled off intact 3 days after wounding
peeled by abrasion or sliced with a blunt machine blade show (Razem and Bernards, 2002).
higher respiration rates, greater microbial contamination and Deposition of suberin may cause detrimental quality charac-
microbial growth rates, higher pH values in the carrot tissue, teristics. For example, in the production of pre-peeled potatoes,
higher rates of weight loss, and higher white tissue development a common industrial product in Scandinavia, hardening of the
than those that had been hand peeled or sliced with razor blades tuber surface takes place (Fig. 2a) (Kaack et al., 2002b). These
(Barry-Ryan and O’Beirne, 1998; 2000). potatoes are too hard for consumption, even after cooking at 98–
The respiration rate of fresh vegetable slices is in most cases 100◦ C for one hour. Microscopic examination shows that when
3 to 5 times that of the intact organ, but aging of the sliced tissue hard potatoes are cooked, brick-like cells at the potato surface
elicits additional increase. Thus, the respiration rate of an aged remain intact (Fig. 2b). It was demonstrated that potato harden-
slice may be 25 times that of the intact organ (Laties, 1978). ing was significantly correlated to the mechanical impact of the
Wads¨ et al. (2004) found that the overall metabolic activity
o peeler, and was increased by blows during sorting or transport
of diced carrot, rutabaga, and potato tissue rose linearly with an (Kaack et al. 2002a). However, the hardening of potato tissue
increase in cut surface area per unit volume (intensity of wound- does not occur if the tubers are steamed or cooked immediately
ing), being as much as 40% higher when the surface area was or a few hours after peeling, probably because the exposed in-
doubled. This increase in metabolic activity is the consequence tact cells are killed. Therefore, understanding the dynamics and
of a large number of biosynthetic events taking place during time scales of the metabolic processes taking place in vegeta-
wound healing (Laties, 1978). bles during industrial unit operations is of great importance in
The initial physiological steps following wounding and the processing design and optimization.
generation of wound signals are not fully understood (Saltveit,
2000). Products of lipid metabolism and lipid oxidation as well LOW-TEMPERATURE STRESS
as compounds such as ethylene and abscisic acid (ABA), are
thought to be possible candidates for the wound signals in plant Among the various kinds of environmental stress affecting
cells (Pe˜ a-Cortes and Willmitzer, 1995).
n plants, low temperature is of particular interest to food science,
When plant tissues are wounded, the cells near the site of the since low temperature, either chilling or freezing, is one of the
wounding stress strengthen their cell walls by the secretion of ad- most widespread and effective methods of conservation.
ditional structural components such as lignin or suberin, creating
a protective layer immediately below the site of damage, to pre-
Low-Temperature Sweetening
vent dehydration and potential penetration by pathogens (Satoh
et al.,1992; Kaack et al., 2002b). The synthesis of several se-
Physiology
creted proteins, such as hydroxyproline-rich glycoproteins, and
their cross-linking to the cell wall after wounding has also been During the storage of some plant tissues at temperatures lower
observed (Showalter and Varner, 1987; Bradley et al., 1992). than those for optimum growth or storage (i.e.,<9–10◦ C for
Suberization is a regulated process whereby the intercellular potatoes), the inverse hydrolysis of polysaccharides to disac-
spaces in tissues become impregnated with a poly(phenolic) ma- charides, and finally to monosaccharides takes place. This usu-
trix concomitant with the deposition of a poly(aliphatic) matrix ally occurs early during the storage period (Rutherford, 1981;
between the plasmalemma and carbohydrate cell wall (Fried- Blenkinsop et al., 2004).



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 Implications for the Potato Crisp Industry
sucrose and other hexoses during low-temperature storage is
known as “cold sweetening” (Hart et al., 1986; Shattuck et al., Blenkinsop et al. (2004) underlined the importance of the un-
1989; Wismer et al., 1995; Espen et al., 1999). The cold-induced derstanding of metabolic changes in potatoes during cold storage
increase in soluble sugars may play a role in osmoregulation, to ensure satisfactory chip color in the potato chip industry. Color
cryoprotection (Espen et al., 1999) and possibly also in the control is complicated as the color is determined by the chemical
activation of respiratory metabolism. The genetic control and composition of the tubers, which not only varies with season and
the metabolic pathways of sugar synthesis have been studied cultivar, but changes during storage. Sugar levels and free amino
(Deiting et al., 1998). In potatoes, the mechanism of cold sweet- acids are important in determining the chip color, which is at-
ening is complex and is mediated by many interrelated metabolic tributed to the products of the Maillard reaction. In addition to the
pathways, such as the induction of the enzymes required in complex carbohydrate metabolism, storage conditions and the
starch degradation, alterations in the biochemical pathways of length of storage are also known to increase the free amino acid
sucrose metabolism, glycolysis and mitochondrial respiration, content and the amount of reducing sugars (Brierley et al., 1996).
as well as electrolyte leakage and membrane lipid peroxidation In April 2002, the National Food Administration of Sweden
(Blenkinsop et al., 2004). and the University of Stockholm announced the presence of



acrylamide, a possible carcinogenic, in foods processed at It has been shown that some of the effects of low-temperature
high temperatures (Ros´ n and Hellen¨ s, 2002; Tareke et al.,
e a stress are mediated by reactive oxygen species (Aroca et al.,
2002). As a result of high-temperature frying, potato-based 2003). The production of ROS is a phenomenon common to
food products, such as potato crisps, contain higher levels of chilling and other stress conditions (e.g., cell damage), as indi-
acrylamide than other baked or fried products (Chuda et al., cated in the previous section. Under prolonged oxidative stress
2003). Changes in the levels of reducing sugars and amino acids conditions, ROS cause lipid peroxidation, DNA damage, and
in potato tubers during storage were investigated in relation to protein oxidative inactivation (Prasad, 1996). The activities of
the presence of acrylamide in the crisps. Chuda et al. (2003) certain enzymes involved in keeping ROS at low levels, in-
found that crisps made from tubers stored at 2◦ C contained ten cluding superoxide dismutase and catalase, decrease. The con-
times more acrylamide than those made from tubers stored at sequence of this is a reduction in defence against free radi-
20◦ C. In the crisps, the acrylamide level did not depend on the cals and repair mechanisms. During exposure to stress the bal-
levels of total amino acids or aspargine, but on the availability ance between degradation and repair will be shifted towards
of reducing sugars in the raw potato. greater degradation of susceptible tissues (Purvis and Shewfelt,
Generally, the tubers used to manufacture potato crisps are 1993).
not stored at such low temperatures as 2◦ 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, Minimizing Chilling Injury After Harvest
potatoes are generally stored at around 10◦ C in order to main- Species that are sensitive to chilling can show appreciable
tain low levels of sugars during long-term storage (Blenkinsop variation in their response to low temperatures. Also, tem-
et al., 2004). However, at this storage temperature potatoes will peratures that are considered “cold” vary between species
sprout, and the application of chemicals to inhibit sprouting may (e.g. pineapple and carrot). Resistance to chilling injury often
be necessary. According to Blenkinsop et al. (2004), there has increases if plants are first hardened (acclimated) by exposure to
been great interest during recent years in developing potato culti- cool but non-injurious temperatures. Chilling damage thus can
vars (through traditional breeding and selection methods and/or be minimized if exposure is slow and gradual. Membrane lipids
through the use of genetic engineering) that are more resistant from chilling-resistant plants often have a greater proportion of
to low-temperature sweetening, and which have an acceptable unsaturated fatty acids than those from chilling-sensitive plants,
color when processed directly after low-temperature storage and during acclimation to low temperatures the activity of lipid
(e.g. 4◦ C), thus avoiding the application of sprout inhibitors. desaturase enzymes increases and the proportion of unsaturated
As stated above, levels of formation of acrylamide during frying lipids rises (Stanley, 1991; Palta et al., 1993). This modification
should be another criterion for the development of such cultivars. lowers the temperature at which the membrane lipids begin
a gradual phase change from fluid to semi-crystalline, and
Chilling Injury allows membranes to remain fluid at lower temperatures
(Vandenbussche et al., 1999). For example, Marangoni et al.
During growth and postharvest handling, chilling injury, de- (1990) stored mature green commercial tomatoes at 12◦ C for 4
fined as damage to susceptible plant species during exposure d followed by storage at 8◦ C for 4 d, and then chilling at 5◦ C
to low temperatures above the freezing point, leads to losses in for 15 d. The properties of these tomatoes were compared with
yield and growth potential of crop plants and to reduced quality those directly chilled for 15 d at 5◦ C. The gradual acclimation
of detached, edible tissues (Purvis and Shewfelt, 1993). Fruits of program decreased the severity of chilling injury, as reflected
many species, especially those of tropical and subtropical origin in a more intense red color and a harder fruit, compared with
suffer chilling injury upon exposure to non-freezing tempera- what was observed in directly chilled tomatoes. Gradually
tures below 12◦ C (Lafuente et al., 1991; Jaitrong et al., 2004). 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
Causes of Tissue Damage
will also prepare plant tissues for potential freezing.
A common response of sensitive plant cells to low tem- A direct response to chilling is a decrease in cellular res-
peratures is the disruption of membrane integrity (Purvis and piration. However, in many species acclimation results in the
Shewfelt, 1993). In chilling-sensitive plants, the lipids in the restoration of respiration (Atkin and Tjoelker, 2003), which may
bilayer have a high percentage of saturated fatty acid chains, lead to increased respiratory losses during storage. Therefore,
and membranes with this composition tend to solidify into a it is important to use procedures for thermal acclimation that
semi-crystalline state at a temperature well above 0◦ C (Parkin avoid respiratory reactivation.
et al., 1989). Low temperature also affects membrane proteins Other methods of reducing or avoiding chilling injury have
and enzymes. Protein-protein and protein-lipid interactions may been described in the literature. They are based on the physio-
be weakened by a decrease in the relative strength of hydropho- logical response to another stress that protects the tissue against
bic bonding, leading to subunit dissociation and/or polypeptide chilling injury (cross-tolerance). These procedures will be de-
unfolding (Stanley, 1991). scribed in more detail in following sections.



Freezing Injury wheat after 21 days’ of cold acclimation (Houde et al., 1995),
and have been found to be accumulated in many organelles,
Freezing injury occurs at temperatures below the freezing including the endoplasmic reticulum (Ukaji et al., 2001) and
point of water. Several plants, however, are able to induce mitochondria (Zykova et al., 2002). The functions of these
tolerance to freezing, following a period of acclimation at cold, proteins are not fully understood and have been the subject of
but non-freezing, temperatures (Smallwood and Bowles, 2002). 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
Physiology (Smallwood and Bowles, 2002). Examples of proteins that
The primary manifestation of cell damage by freezing is ob- accumulate with cold acclimation include the cryoprotective
served in the plasma membrane (Steponkus, 1984; Palta, 1990). proteins of spinach, rye, and other cereal antifreeze proteins
The water potential of ice is lower than that of liquid water. Ex- (AFPs) (Thomashow, 1999). A factor common to all these
tracellular ice crystals grow by drawing water from cells, thus proteins is that they are predominantly located in the apoplast
dehydrating them, until the water potential of the ice and that of and are therefore more likely to come into contact with the
water in the cell are equal. The water potential of ice decreases as outer surface of the plasma membrane.
the temperature decreases, so the extent of cellular dehydration AFPs are expressed in a number of plant species, such as
increases with decreasing temperature, to a limit set by vitrifica- winter rye, winter barley, winter canola, white oak, and carrots,
tion (glassy state) (Pearce, 2001). This deterioration is observed in response to low temperature (Urrutia et al., 1992; Duman and
as wilting or softening of plant parts. Olsen, 1993; Feeney and Yeh, 1993; Griffith and Antikainen,
Plants that survive winter either prevent the crystallization 1996; Smallwood et al., 1999). Their accumulation and activity
of ice within their tissues (freeze avoidance) or can withstand have been found to be strongly correlated with winter survival
ice crystallization in the apoplast (freeze tolerance) (Smallwood and it has been suggested that they be used as a biological marker
and Bowles, 2002). Freeze avoidance involves supercooling and for crop improvement (Griffith et al., 1992; Chun et al., 1998).
hence prevention of the incursion of ice into the apoplast. With- Antifreeze proteins interact with ice crystals by adsorption
out ice nucleation, pure water can be supercooled to a certain onto non-basal planes of ice at the ice-water interface thus mod-
point below 0◦ C. However, this supercooling is only a practical ifying their growth. At high AFP concentrations (µM), mini-
strategy at the whole plant level when exposure to subzero tem- mal crystal growth occurs, forming very small, stable hexago-
peratures is relatively brief (George et al., 1982; Smallwood and nal bipyramids. Physical damage caused by ice can occur during
Bowles, 2002). Some specialized cell types and organs use su- warming, as well as during freezing, by a process known as re-
percooling as a strategy to overwinter, such as the xylem ray crystallization (Knight and Duman, 1986; Breton et al., 2000).
parenchyma cells of many trees, which supercool to around Recrystallization of ice occurs when small ice crystals con-
-40◦ C (George and Burke, 1977). Given the widespread pres- dense into larger ones. This can happen very quickly at tem-
ence of nucleators in the environment, the most common frost peratures just below the melting point of a frozen solution. In
survival strategy is cold acclimation (freeze tolerance) and this is nature, prolonged exposure to subzero temperatures and tem-
achieved through several changes in cell biochemistry regulated perature fluctuations may promote recrystallization of frozen
at the gene expression level (Danyluk et al., 1998). tissues, especially those in which cells are densely packed, and
The accumulation of osmotically active substances, such as allow ice access to locations from which it is usually excluded.
simple sugars, organic acids, proline, and glycinebetaine, is a AFPs adsorbed onto the surfaces of ice act as potent inhibitors of
protective mechanism induced by cold stress (as previously de- recrystallization, even at very low concentrations (e.g. 1 µg/ml)
scribed for cold sweetening). In many plants, sugars act as cry- (Worrall et al., 1998; Smallwood et al., 1999). Given that AFPs
oprotectants which increase the freezing resistance through di- are also found in plant tissues where ice is allowed to crystal-
rect and/or indirect effects (Graham and Patterson, 1982; Chang lize in the apoplast (which includes the xylem, cell walls, and
and Reed, 2000). The hydrophilic nature of sugars is well-suited intercellular spaces), it has been speculated that inhibition of ice
to replace water and stabilize the cell membrane through hydro- recrystallization may be the physiologically relevant aspect of
gen bonding between hydroxyl groups on the sugar and po- the activity of AFPs (Smallwood and Bowles, 2002).
lar residues in phospholipids, preventing dehydration effects in
membranes (Danyluk et al., 1998). Accumulation of osmotically
Application of Cold Acclimation in the
active substances leads to a decrease in the chemical potential of
Frozen-Vegetable Industry
water. It has been suggested that this mechanism is involved in
regulating the induction of cold-induced gene expression (Fu When cold-stressed, starch-rich vegetables (e.g. potatoes)
et al., 2000) and in the higher resistance of cold-acclimated are frozen industrially, the effects of cold sweetening during
plants to fungal infection (Tronsmo, 1986). the storage period could be detrimental to the quality of the
Many cold-induced proteins accumulate in the tissues during product after cooking by the consumer at home (e.g., exces-
cold acclimation (reviewed by Thomashow, 1999). These can sive brown color after frying, as discussed earlier). However,
account for up to 0.9% of the total soluble proteins in winter if vegetables accumulating mostly sucrose in their cytoplasm



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◦ 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.

(e.g. carrots) and antifreeze proteins in their cell walls during by G´ mez and Sj¨ holm (2004). The authors illustrated the en-
o o
growth in the field in late autumn are to be frozen, industry hancement of the tolerance to freezing by the metabolic response
may take advantage of cold-induced stress responses to opti- to low-temperature stress by freezing both acclimated and non-
mize the quality of the frozen product. The potential application acclimated carrot slices at a very slow freezing rate (−5◦ C
of the acquisition of freezing tolerance by cold-acclimation of ambient temperature over-night). Figure 3a shows a piece of
carrot taproots in the frozen-carrot industry has been discussed non-acclimated carrot tissue that has been extensively damaged



by freezing. In remarkable contrast, Fig. 3b shows much more Causes of cell damage
intact tissue of the cold-acclimated samples frozen under the
same conditions. Although vegetables such as carrots are usually Exposure of plants to temperatures above their optimal
frozen quickly to produce small ice crystals, these ice crystals growth temperature can disrupt many essential metabolic pro-
may grow larger over time through recrystallization. Recrys- cesses, including photosynthesis and respiration, the former be-
tallization occurs when temperature gradients form within the ing more sensitive. Activation of lipid peroxidation is one of the
product during freezing or thawing, or when the temperature earliest and least stress-specific plasmalemmal responses caused
fluctuates during storage or transportation (Griffith and Ewart, by any stress agent, including heat shock. Lipid peroxidation
1995; Breton et al., 2000). Recrystallization in frozen foods can can result in various structural and functional disturbances in
result in membrane damage, thus reduced water holding capac- the cell (Veselov et al., 2002). Furthermore, excessive fluidity
ity (high drip loss), and associated loss of nutrients (Fletcher of membrane lipids at high temperatures (above 50◦ C) is cor-
et al., 1999; Breton et al., 2000). AFP is abundant in the accli- related with loss of functional cell compartmentalisation which
mated carrot tap root apoplast (0.5 mg of pure protein can be considerably enhances the permeability of membranes and, in
isolated from 1 kg of fully acclimated carrot taproots Smallwood consequence, the passive flux of solutes (Kluge et al., 1991),
et al., 1999), and may be a key factor in inhibiting recrystalliza- leakage of electrolytes, and reduction of turgor pressure (De
tion and preserving the quality of the frozen product. Belie et al., 2000; Gonz´ lez-Martńez, 2003). It has been hypoth-
a ı
The potential benefits of cold acclimation of frozen carrots esized that in beans susceptible to the HTC defect, the effects of
can, however be eradicated by the common practice of blanching temperature on cell membrane are accompanied by lignification
before freezing. Heat will damage the cells, destroying the pro- of the cell wall, pectic de-esterification in the middle lamella
tective system nature has created against frost damage. Above and breakdown of phytic acid, inhibiting chelation of divalent
approximately 50◦ C, the functionality of the cell membrane is cations, which renders pectates in the middle lamella unsuscep-
irreversibly damaged (De Belie et al., 2000). Denaturation of tible to softening during cooking (Aguilera and Ballivian, 1987,
proteins such as AFPs in the cell walls would also compromise Reyes-Moreno and Paredes-L´ pez, 1993).
o
the cold acclimation effect, as these proteins must be folded High-temperature injury is also associated with lipid phase
correctly in order to be active (M. Griffith, pers. comm.). The transitions and/or changes in transmembrane protein conforma-
optimization of blanching to minimize tissue damage is thus very tion (Hansen et al., 1994). Heat stress causes many cell proteins
important if the frozen-food industry is to be able to take advan- (enzymes or structural proteins) to become unfolded or mis-
tage of cold acclimation to protect tissue cells (G´ mez, 2004).
o folded. Such misfolded proteins can aggregate and precipitate.

HEAT STRESS AND HEAT SHOCK
Plant Strategies for Heat Tolerance
Most tissues of higher plants are unable to survive extended
exposure to high temperatures. Non-growing cells and dehy- Metabolic acclimation associated with heat tolerance mech-
drated tissue can tolerate much higher temperatures than hy- anisms includes an increase in the degree of saturation of fatty
drated, growing cells. Actively growing tissues rarely survive acids in membrane lipids, which makes the membranes less fluid,
temperatures above 45◦ C, but dry seeds can endure 120◦ C and the synthesis of enzymes and isoenzymes with broad thermal
pollen grains of some species can remain viable after exposure kinetic windows, the synthesis of protective enzymes such as
to 70◦ C (Taiz and Zeiger, 2002). glutathione reductase, peroxidase, and catalase (Viswanathan
Storage of some legumes under tropical conditions (30– and Khanna-Chopra, 1996), and the production of heat shock
40◦ C; >75% humidity) renders them susceptible to a hardening proteins (HSPs).
phenomenon, causing nutritional losses and inflicting economic In response to sudden rises in temperature (5 to 10◦ C), plants
losses on farmers and poor urban dwellers in developing produce a unique set of proteins, the HSPs. Most HSPs func-
countries (Aguilera and Ballivian, 1987; Martin-Cabrejas and tion as molecular chaperones, that is, they bind to unfolded or
Esteban, 1995). This is an irreversible phenomenon known denatured proteins, prevent aggregation and induce correct re-
as the hard-to-cook (HTC) defect. Beans with this defect are folding, facilitating correct cell function at elevated, stressful
characterized by extended cooking times to achieve cotyledon temperatures. Some HSPs assist in polypeptide transport across
softening, are less palatable to the consumer and are of lower membranes into cellular compartments (Miroshnichenko et al.,
nutritional value (Reyes-Moreno and Paredes-L´ pez, 1993).
o 2005).
In many crops, as further discussed in this section, peri- Plants and most other organisms produce HSPs that have
odic, brief exposure to sublethal heat stress often induces tol- different functions in response to increases in temperature:
erance to otherwise lethal temperatures, a phenomenon known HSP100, HSP90, HSP70, HSP60, and small HSPs (smHSPs,
as induced thermotolerance (Viswanathan and Khanna-Chopra, 15–30 kDa) (Vierling, 1991). HSP expression has been charac-
1996). Thermotolerance in crops is determined by a variety of terized in a variety of higher plants, including tomato (Banzet et
factors such as photoperiod, light intensity and water availability al., 1998), maize (Cooper and Ho, 1983), soybean (Hsieh et al.,
(Ahn et al., 2004). 1992), carrot (Malik et al., 1999), pea (DeRocher et al., 1991),



sugar cane (Hoisydai and Harrington, 1989), apple (Bowen et al., 2003). Thus, cells previously exposed to one kind of stress may
2002), and potato (Ahn et al., 2004). Some smHSPs are known gain protection against another kind (cross-tolerance). For exam-
to play an important role in the protection of biomembranes and ple, some of the HSPs are not unique to high-temperature stress
organelles (Viswanathan and Khanna-Chopra, 1996). Synthe- and can be induced by other forms of stress such as drought
sizing a number of smHSPs at elevated temperatures is one of (Alamillo et al., 1995; Wehmeyer and Vierling, 2000), wound-
the unique features of the heat-shock response of plants (Ahn ing, low temperature, and salinity (Wang et al., 2001). Symptoms
et al., 2004). Cells that have been induced to synthesize HSPs of chilling injury are reduced after heat pretreatment, and this
show improved thermal tolerance and can withstand exposure to reduction is correlated with persistence of several HSPs in fruit
temperatures that are otherwise lethal (Malik et al., 1999; Bowen tissue (Sabehat et al., 1996). Tomato and avocado fruits, in which
et al., 2002). It has been shown that a smHSP in tomato (VIS1) heat shock was induced (48 h at 38◦ C), accumulated HSPs and
plays a role in facilitating fruit ripening, senescence, and seed were protected from injury by subsequent chilling at 2◦ C (Lurie,
dispersal by protecting the cellular machinery against thermal 1998). Reduced chilling injury of cucumber cotyledons and cul-
denaturation during the daily cycles of daytime rise in tempera- tured apple cells after exposure to 37 or 42◦ C has also been re-
ture. VIS1 acts as a chaperone by binding reversibly to enzymes, ported (Lafuente et al., 1991; Wang et al., 2001). Heat treatment
including cell wall polymer-modifying enzymes, and protecting at 38◦ C for 8 h applied to evening-harvested sweet basil reduced
them from thermal denaturation (Ramakrishna et al., 2003). its sensitivity to chilling. This reduction may have involved the
In the food industry, heat treatment (generally 50–70◦ C) has antioxidative system of ROS protection, as suggested by the
been used for the past 40 years to improve the texture of veg- increased reductive potential in the leaves, as well as the induc-
etables prior to high-temperature processing. The firming ef- tion of superoxide dismutase and catalase activity following heat
fect of low-temperature blanching has been studied in a number treatment. Elevated activity remained through subsequent cold
of vegetables (Bartolome et al., 1972; Lee et al., 1979; Stolle- storage below 12◦ C (Faure-Mlynski et al., 2004).
Smits et al., 2000). Evidence indicates that the firming effect Heat shock treatment has been used to reduce decay and
is due to the temperature activation of pectin methylesterase chilling injury, and to enhance host resistance to pathogens in
(PME, EC 3.1.1.11). The resulting reduction in the degree of fruits. Treatment by dipping in water at 52–53◦ C for 2 min or
methylesterification of the pectins in the cell wall and middle 62◦ C for 20 s promoted the accumulation of HSPs and proline-
lamella allows the more calcium cross-linking between calcium rich proteins in the skin of grapefruit. Heat application has been
molecules, increasing firmness (Pilnik and Voragen, 1991). The shown to markedly reduce decay and the sensitivity of citrus fruit
mechanism governing temperature activation of PME is not to chilling injury without any deleterious effects on fruit quality
well-understood. It has been speculated that, at elevated tem- (Ben-Yehoshua, 2003). Several types of machines for hot water
peratures, a change in the PME enzyme or its environment may treatment are already in operation in many countries in packing
occur such that the enzyme is converted into a more active form. houses for citrus (Ben-Yehoshua, 2003) and other fruits, such
Loss of membrane integrity and leakage between cellular com- as bell peppers, corn cobs, lychees, mangos, melons, nectarines,
partments at temperatures >40◦ C may contribute to this activa- and peaches (Fallik et al., 1999).
tion (Anthon and Barrett, 2006). However, to our knowledge, no When cells are subjected to a stressful, but non-lethal tem-
previous study has associated PME activation with induction of perature, the synthesis of HSPs increases dramatically, while
signal cascades at the genetic level and/or metabolic transfor- the continuous translation of other proteins is lowered or ceases
mations strictly associated with the concept of “stress response” (Vierling, 1991). This effect has been seen in studies on the
that we have been using throughout this review. It appears that wounding stress response of carrots and lettuce. In the case of
mild blanching treatment, for example, at 70◦ C for 30 min (Lee carrot slices, exposure to 40◦ C for 1 h caused the cessation of the
et al., 1979), is used by the industry as a direct way of regulat- synthesis and secretion of extensin proteins, a typical response to
ing the activity of the enzyme. This treatment may mimic a true wounding stress (Brodl and Ho, 1992). Maximum accumulation
stress response that may occur at lower temperatures (around of HSPs was seen in the carrot slices one hour after a temperature
40◦ C) for a longer time, for example, when harvested material increase from 28◦ C to 40◦ C. The synthesis of HSPs diminished
lies in the sun for hours before processing. A more detailed study sharply after 3 h of continuous incubation at 40◦ C and the carrots
of the time and temperature dependence of PME activation and resumed the secretion of extensin proteins during that period of
the molecular mechanisms regulating it would be of interest. time. Upon recovery from 40◦ 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 re-
Cross-Tolerance and its Application in Postharvest Handling duces the response to wounding and nicely illustrates the fact
and Minimal Processing that plant tissues follow a certain temporal order and hierarchy
in their response to multiple stimuli. The heat-stressed, wounded
In general, stress responses involve changes in the proteome tissue has basically redirected its resources towards the response
and metabolome with increased expression of proteins and com- to more severe stress.
patible solutes. Cross-talk between stress signalling pathways This principle has been applied in the minimal processing of
may result in co-expression of stress responses (Joyce et al., vegetables to prevent browning of wounded lettuce leaf tissue.



Wound-induced browning has been significantly reduced in ice- 2001a). During storage, carrots can increase their concentration
berg lettuce by the application of short thermal stress (Loaiza- of glucose and fructose from their sucrose stores (Herppich
Velarde et al., 1997). A heat shock of 45◦ C for 90 s effectively et al., 2001b,c). These monosaccharides contribute twice as
prevents the synthesis of phenylalanine ammonia-lyase (PAL), much to osmotic pressure per unit weight as disaccharides.
whose increased activity leads to the accumulation of phenolic Cellular electron transport chains are impaired upon dehy-
compounds (e.g., chlorogenic acid, dicaffeoyl tartaric acid, and dration and may generate increasing amounts of reactive oxygen
isochlorogenic acid) and tissue browning (Salveit, 2000). Inhi- species (Hoekstra, 2002). Free radical attack on phospholipids,
bition of PAL synthesis appears to result from the redirection of DNA and proteins is one of the molecular mechanisms of
protein synthesis away from wound-induced proteins to the syn- damage leading to death in desiccation-sensitive cells upon
thesis of HSPs. The effect of heat shock (45◦ C for 90 s followed drying (Oliver et al., 2001). Protection against ROS is thought
by rapid cooling to 0◦ C) either 4 h before wounding or 2 h af- to play a role in desiccation tolerance. Therefore, free radical
ter wounding was so persistent that the fresh-cut lettuce did not scavenging systems are important components among the
show any browning after 15 days in air at 5◦ C (Salveit, 2000). mechanisms governing desiccation tolerance. Over-expression
However, it has also been shown that this treatment was not suc- of some enzymes, such as manganese superoxide dismutase
cessful in tissues with constitutively or induced high levels of and glutathione S-transferase/glutathione peroxidase, has been
phenolic compounds. The heat shock acts only on the synthesis associated with an enhanced tolerance to water deficit in trans-
of PAL and not on the activity of other enzymes involved in genic tobacco plants and cotton cells (Serrano and Montesinos,
tissue browning (Salveit, 2000). 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).
DROUGHT STRESS AND DESICCATION TOLERANCE Stress often induces the accumulation of proteins, as has been
described for AFPs in the case of low-temperature stress and
Water deficit can be defined as any water content of a tissue HSPs in heat stress. In the case of drought stress, a large group
or cell that is below the highest water content exhibited in the of genes code for hydrophilic LEA proteins, which are sus-
most hydrated state (Taiz and Zeiger, 2002). Lack of water has pected to play a role in the acquisition of desiccation tolerance
several detrimental effects on plants, including modification of (Blackman et al., 1995). Although the function of LEA proteins
the cell wall crystallinity, clumping of microfibrils, denaturation is not well-understood, they accumulate in vegetative tissues
of proteins, loss of cell turgor and membrane fluidity, and oxida- during episodes of drought. Their protective role may be
tive damage by reactive oxygen species (Aguilera et al., 2003; associated with their ability to retain water and to prevent
Prothon et al., 2003). 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
Strategies for Desiccation Tolerance levels, LEA proteins might play a role in sequestering ions and
preventing of the damaging effects of free radical reactions.
Some plant tissues can acquire desiccation tolerance, defined In the dried state, LEA proteins may act together with carbo-
as the ability to function while dehydrated, or desiccation post- hydrates in the formation of a tight hydrogen-bound network,
ponement, defined as the ability to maintain tissue hydration providing stability to macromolecular and cellular structures
(Davies, 2004). Desiccation tolerance involves a co-ordinated in the cytoplasm. This network would inhibit the fusion of
set of mechanisms that help certain tissues to survive dehydra- cellular membranes, denaturation of cytoplasmic proteins, and
tion. These mechanisms include stomatal closure (Davies et al., the detrimental effects of free radical reactions.
2002), osmotic adjustment, removal of reactive oxygen species, Drought typically leads to the accumulation of ABA. Numer-
and the accumulation of late embryogenesis-abundant (LEA) ous genes are induced by both drought and ABA accumulation
proteins (Oliver et al., 2001). during the stress episode (Liu et al., 2005). Exogenous applica-
Plants can continue to take up water only when their water tion of ABA has been shown to induce desiccation tolerance in
potential is below that of the water source. Osmotic adjust- somatic alfalfa embryos. Heat shock pretreatment, at 38◦ C for
ment, in which cells accumulate osmotically active solutes (also as little as 10 min, induced a degree of desiccation tolerance in
known as compatible solutes or osmolytes and including sugars, the somatic embryos which was equivalent to ABA application
organic acids, glycine betaine, sorbitol, proline, amino acids, and was therefore shown to be a viable alternative to exogenous
polyols, quaternary amines, and ions), is a process in which the ABA treatment. The drying rate did not influence the survival
water potential of the tissue can be decreased without an accom- of the heat-stressed embryos (Senaratna et al., 1989).
panying decrease in turgor (see G´ mez et al., 2004 for definitions
o
of plant water relations). The change in tissue water potential Application to Food Dehydration
results simply from changes in the osmotic potential (Fan et al.,
1994; Zhang et al., 1999). In radish tubers the total concentration The quality of air-dehydrated plant products is often very low,
of free sugars increases with soil water deficit (Herppich et al., with shrunken, shrivelled, darkened tissue, and poor rehydration

2007 plant stress physiology- opportunities and challenges for the food industry

2007 plant stress physiology- opportunities and challenges for the food industry

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2007 plant stress physiology- opportunities and challenges for the food industry