•Tolerate temperature decline only to thepoint of water crystallization in cells (withstand lower temperatures by activating ffee2e retardation mechanisms which lower crystallization temperature and increase the ra uge of water super cooling inside the cell).

• Species that are frost-sensitive throughout the year (algae colonizing cool oceans, selected fresh water alga e, arborescent plants in the tropical and subtropical zone) as well as various plant species in a temperate climate that arefieeze-sensitivein the summer

• Able to survive extra cellular water crystallization andthe a ccomp anying dehy di a tion stress aft era period of growth at low temp eratuies

* Selected fresh water algae, intertidalzone algae, land algae, mosses in all climate zones (including tropical), biennial and perennial land plants colonizing regions with harsh winters.

•The frost resistance may increase significantly after a period of acclimatization, reaching -SCC or even -70°C, while some species are able to withstand temperature -196°C.

Fig. 5.6 Classification of the plants, subject to their range oflethal temperatures and the characteristics of mechanisms conditioning their resistance to low temperatures (adapted from Stushnoff et al. 1984)

Thermotropic phase changes are the primary cause of membrane dysfunctions that lead to irreversible damage and cell death. The above may produce reactive oxygen species and the accompanying oxidative stress. According to recent research, the phospholipid which initiates the phase transition of the cell membrane is phos-phatidylglycerol (PG). If a PG molecule contains fatty acids with a high melting point, that is, saturated fatty acids, then the phase transition of this lipid takes place relatively easily at low temperatures and this, in turn, induces the transformation of other phospholipids and galactolipids adjacent to PG (Los and Murata 2004; Wang et al. 2006). According to the second chilling injury theory, the primary cause of damage is the sudden increase in the concentration of free calcium ions in the cytosol (Minorsky 1989). Calcium ion concentrations increase as calcium channels in the plasmalemma become opened due to sudden depolarization (Lecourieux et al. 2006). In chilling-sensitive plants, calcium opens the stomata, and transpiration significantly exceeds water uptake by the roots (Liang et al. 2009) . In many sensitive species, the first indication of cold stress is striking wilting of the leaves, despite optimal water supply in the soil (Mahajan and Tuteja 2005; Solanke and Sharma 2008) . The release of calcium ions into the cytosol has many secondary effects, including induced gene expression which could result from changes in the content or distribution of cell hormones, mainly abscisic acid (ABA). This phenomenon is in particularly related to the acidification of the cytoplasm at low temperatures (and the corresponding alkal-ization of the vacuoles) which, at least in part, is actively controlled by H+-transport from the cytoplasm to the vacuole catalyzed by H+-ATPase located on the vacuolar membrane. The inactiva-tion of this enzyme has been reported to occur much earlier than other symptoms of cell injury (Yoshida et al. 1999; Lindberg et al. 2005). Chilling affects the entire internal environment of each cell and each molecule within the cells (Kartsch and Wise 2000). The rate and extent of injury is determined by temperature, its duration as well as the chilling rate. Sudden temperature drops (thermal shock) have particularly damaging consequences. The lower the temperature and the longer its effect, the greater the extent of the sustained injury (Mahajan and Tuteja 2005; Solanke and Sharma 2008). Plant structures and physiological cell processes have varied sensitivity to chilling temperatures (Fig. 5.7). Most injuries are sustained in the cell membrane which may represent a potential site of perception and/or injury (Lindberg et al. 2005). There are changes in the viscosity and liquidity of the membrane, leading to an increase in diffusion resistance and, in many cases, enzyme inactivation. The reversibility of those effects is determined by the severity of damage. Changes in chemical composition may be observed as the result of lipid degradation, the release of fatty acids and changes in the activity of metabolizing enzymes, peroxidation, disintegration of lipid-protein bonds, and higher membrane permeability. The chemical composition of the cytoplasm and differences in lipid quality in various chilling-sensitive species determine the phase transition point, that is, the point at which the membrane is transformed from a liquid-crystal state into a gel state (Solanke and Sharma 2008; Jan et al. 2009). This change in the membrane's physical state impairs its normal functioning. In most chilling-sensitive plants, the phase transition point is around 10°C. Chilling sensitivity is mostly related to a higher content of saturated fatty acid residues in lipids, while the cold-hardiness mechanism is explained by the desaturation of fatty acids which enables the plant to quickly acclimatize to low temperatures. The above is only one of the factors explaining variations in the plants' response to temperature stress (Lindberg et al. 2005; Zhang and Tian 2010). Interactions between membrane components, including lipid-lipid and lipid-protein, are also believed to play an important role. Higher sterol concentrations increase membrane rigidity. The role of membrane proteins during chilling is also a source of controversy, but there is general agreement that conformational changes in protein-lipid systems may lead to membrane disintegration and dysfunction (Los and Murata 2004; Lindberg et al. 2005). Frost-induced

Fig. 5.7 Functional disturbance occurred in chilling-sensitive plants, subjected to stress duration (adapted from Kacperska 1998)

changes may lead to inhibited protoplast movement, excessive protoplast vacuolization, damage to the endoplasmic reticulum, drop in turgidity, and higher membrane permeability. Cytoplasmic streaming and photosynthesis, including thyla-koid functioning in chloroplasts (as demonstrated by enhanced in vivo chlorophyll fluorescence), are most susceptible to reversible disruptions. Irreversible damage, including injuries caused by stressors other than temperature, is also most likely to affect thylakoid membranes, mostly photosystem II. Chloroplast lipids undergo various metabolic changes in both chilling-sensitive and cold-hardy plants. Higher levels of galactoli-pase activity and, consequently, higher free fatty acid concentrations are noted in the chloroplasts of chilling-sensitive species (faba beans, beans, tomatoes, maize) than in cold-hardy plants (spinach, pea). Lower temperatures disrupt the maintenance of the proton gradient in thylakoid membranes conditioning ATP synthesis. Powerful radiation during or directly after chilling intensifies the relevant injuries and retards, or even disables, damage repair in both chilling-sensitive and cold-hardy plants. Long-term frost inhibits the synthesis of chlorophyll and starch (Muller et al. 2005; Liang et al. 2009; Sun et al. 2010). Other membranes (plasmalemma and tonoplast) are damaged after relatively longer exposure, as demonstrated by membrane cells' ability to plas-molyze and vital staining. Those injuries are irreversible. Other metabolic functions are marked by varied sensitivity to low temperatures which cause metabolic disorders and lead to toxin accumulation, for example, respiration efficiency may be higher or lower subject to environmental factors that accompany freezing temperatures. Chilling may also inhibit the activity of many oxidoreductive enzymes, such as catalase, leading to the accumulation of hydrogen peroxide and the production of free radicals (Suzuki and Mittler 2006; Liang et al. 2009; Sun et al. 2010). In sublethal cold stress, fruit ripening and seed germination are most severely inhibited (Kumar and Bhatla 2006).

Frost leads to the appearance of stress which is linked not directly to low temperature, but to freezing (crystallization) of water in the plant (Mahajan and Tuteja 2005) . Intracellular and extracellular crystallization produces different effects. Ice crystals are formed readily in those parts of the plant where temperature drops most rapidly and where water freezes most easily (due to high water potential), mostly vascular bundles and intracellular spaces in above-ground parts where water vapor undergoes condensation. Ice crystals spread quickly via vessels and other tissues with uniform structure. The presence of air-filled intercellular spaces as well as tissues with lignified or cutinized walls slows down crystallization. Ice formation is accelerated by ice-nucleation active bacteria of the genera Ervinia and Pseudomonas. The proteins formed on the outer bacterial cell wall react with water particles and facilitate the formation of ice crystals at temperatures just below 0°C. In the absence of ice-nucleation active bacteria on the surface of tissues and on the walls of intracellular spaces, ice formation would begin at temperatures several degrees lower due to the supercooling of water solutions.

If tissue is supercooled rapidly (e.g., faster than 5 K min-1) and the cells have high water potential, or if cell water had been first deeply supercooled, ice may be formed in the protoplast. The above invariably leads to cytoplasm destruction and cell death (Fitter and Hay 2002; Rajashekar 2000; Jan et al. 2009; Janska et al. 2010). Water freezing in intracellular spaces is a less dangerous phenomenon. In nature, where temperature decline is generally slow (1-5 K min-1), crystallization usually takes place outside the protoplast in intra-cellular spaces and between the cell wall and the protoplast (partly due to the extracellular fluid having a higher freezing point, i.e., lower solute concentration, than intracellular fluid). The above leads to extracellular crystallization. Vapor pressure decreases in the spaces above ice, and a water potential gradient is created between the unfrozen interior of the cell and the extracellular environment. Water moves along this gradient into extracellular spaces where it is crystallized (Fitter and Hay 2002; Jan et al. 2009; Janska et al. 2010). Cells are dehydrated (secondary stress) and they contract due to desiccation. The lower the surrounding temperature, the longer it takes for an equilibrium to be reached between the water potential above ice and inside cells, and the greater the effect of cell dehydration (Solanke and Sharma 2008).

Multiple forms of membrane damage can occur as a consequence of freeze-induced cellular dehydration including expansion-induced-lysis, lamellar-to-hexagonal-II chase transitions, and fracture jump lesions. The above leads to cell contraction and the associated changes in reactions between the plasmalemma and the cell wall, partial loss of plasmalemma due to exocy-tosis and endocytosis, changes in the structure of the plasmalemma and other cell membranes, and the creation of protein-deprived lipid areas in the membrane. The greatest damage is done to the plasmalemma. Dehydration also increases the concentration of solutions in the cytosol and the cell sap, leading to higher salinity (Mahajan and Tuteja 2005; Solanke and Sharma 2008; Jan et al. 2009). Conformational changes in proteins found in the plasmalemma and other membranes lead to changes in the activity of various membrane enzymes, including ATPases responsible for the movement of protons and other ions through membranes (Lindberg et al. 2005). Some ions, accumulated in cells by ion transporters (e.g., potassium ions), are diffused after thawing into intracellular spaces together with water, for example, in leaf tissue. Certain proteins, such as the thylakoid coupling factor, become dissociated in the process. The effect of chill injury on life processes is often visible when plants resume their normal growth after freezing temperatures subside. Even partial degradation of thylakoid membranes inhibits photosynthesis, and the process may be reversible. PS II activity may be partially or completely inhibited, and the balance between the light-dependent phase and CO2 assimilation may be upset. There is a rise in photorespiration intensity (Alam et al. 2005). Changes in the mitochondria and the respiration process are not as profound. In strongly dehydrated cells, the membrane undergoes lyotropic phase transitions, and hexagonal arrangements are formed in lipid bilayers of a single membrane or two layers of two adjoining membranes (e.g., plasmalemma and endoplas-mic reticulum). The membranes' primary structure is not always restored after thawing, and water is diffused into the extracellular environment together with ions through membrane channels. Cell dehydration caused by extracellular crystallization increases the concentrations of salt and organic acids in the protoplast which, in turn, may lead to protein denaturation and enzyme inactivation (Mahajan and Tuteja 2005; Solanke and Sharma 2008) . Few enzymes remain active at below zero temperatures, but some of them are activated, such as phospholi-pase D which catalyzes the hydrolysis of phos-pholipids (Ruelland et al. 2002) . The degradation of membrane lipids begins during freezing and after thawing, releasing unsaturated fatty acids which are peroxidized. Chlorophyll may be also be photooxidized in green tissues exposed to light (Sung et al. 2003). Chill injuries may occur not only during freezing, but also during the thawing of tissue. Plant survival is also determined by post-thawing environmental factors -rapid temperature growth and high light intensity may disturb metabolic pathways in cells and cause additional damage. During rapid melting of ice, the cell is rehydrated, and it quickly increases its volume. The above leads to tension and cracks in cell structures, mostly in the cytoplasm which is the site of primary cell injury. The above changes have less damaging consequences for dormant plants. In a temperate climate, winter frost is not a typical stressor for plants, but freezing temperatures could be a source of stress if they occurred in the spring or summer (Muller et al. 2005) .

2.2 Resistance to low Temperature, Acclimatization

Resistance is related to frost tolerance, that is, the ability of the organism to survive low temperatures without damage. In regions characterized by seasonal climate change, plants' resistance to freezing fluctuates periodically - it is the lowest during intensive elongation growth in the spring, and it rises significantly in the fall when growth is arrested by the direct effect of low temperature or the combined effect of shorter daytime and temperature drop (Li et al. 2005a, b). Frost resistance is usually achieved by preventing ice formation in the symplast. An important mechanism preventing or delaying symplastic ice formation is frost plasmolysis. Poorly hydrated plants which are acclimatized to water stress usually show increased cold resistance, for example, plants which are extremely tolerant to drying out, for example, embryos of ripened seeds, can be conserved alive at -200°C without damage (Jan et al. 2009) . Species-specific cold resistance is a genetically programmed trait that can be modified by both endogenous and exogenous factors. For a vast number of species, frost tolerance is not a static feature, but it is closely correlated with season, it fluctuates in various growing periods, and it is not identical for all organs (Rorat et al. 2006; Hekneby et al. 2006). The above-ground parts of wheat seedlings were acclimatized even to -20°C, but the roots' sensitivity to frost did not change. Acclimatization can be accelerated by hardening the plants, that is, exposing them to increasingly lower temperatures on successive days, initially above zero, followed by insignificantly below zero (Li et al. 2005a; Zhang and Tian 2010). This process is continued for several weeks. Plants are characterized by the greatest frost resistance 1-3 weeks from the beginning of exposure to freezing temperatures. The period of deacclimatization, that is, dehardening, is much shorter, and it usually lasts several days. The higher the ambient temperature, the faster the deacclimatization process. After dehardening, repeated exposure to frost can severely damage many plants (Li et al. 2004; Burbulis et al. 2008).

Plant organs are also marked by varied sensitivity to frost (Li et al. 2005a; Rorat et al. 2006) . Roots are most susceptible to the damaging effects of freezing temperatures, shoots are less sensitive, while tree trunks and older branches are characterized by the highest frost resistance (Muller et al. 2005; Kato-Noguchi 2007). Snow cover minimizes the temperature drop in the soil, and it protects crops from freezing. The cold sensitivity of flowers is determined by the given species' phenological growth stages (Thakur et al. 2009; Ohnishi et al. 2010).

ABA stimulates and speeds-up plant hardening. According to Weiser (1970), acclimatization, and perhaps also hardening, is determined by modifications in gene expression. In this case,

ABA can enhance cold resistance if it is able to induce the expression of the respective genes (Gusta et al. 2005). Gibberellins and auxins deliver an opposite effect. Substances that retard gibberellin synthesis accelerate hardening. Intensive nitrogen fertilization generally delays dormancy and increases susceptibility to freezing. Heavy potassium fertilization has the opposite effect by increasing the frost resistance of both herbaceous and arborescent plants. The concentrations of sugar and other osmoprotectors that protect the cell from dehydration increases in the cytosol and vacuoles (Liang et al. 2009).

Fluid supercooling inside the cell is yet another factor that increases the plants' cold resistance by delaying crystallization in the cell. The presence of substances dissolved in the vac-uolar sap lowers crystallization temperature. In small, weakly vacuolated cells, water may undergo deep supercooling. In large and hydrated parenchymal cells and xylem vessels, the supercooled state is very unstable, and it rarely lasts longer than several hours. Supercooling provides temporary protection against freezing caused by, for example, strong ground frost. In tissues comprising small, densely packed and weakly vacuolated cells whose walls prevent ice crystals from spreading, a supercooled state may persist until the temperature drops below a threshold value. The accumulation of nonpolar lipids on the surface of the plasmalemma also prevents ice penetration from the apoplast to the cell interior. In herbaceous plants, the supercooling of water is observed at -1 to -15°C, and in arborescent plants at -30°C, and even -50°C. Such a high degree of supercooling is observed only in some living tissues, such as core paren-chymal cells, meristematic tissue, leaf bud scales, and flower buds. When ambient temperature drops below the critical supercooling point, this meta-stable state is rapidly disrupted, and ice is formed inside the cells, ultimately leading to their death. In some extremely frost-resistant tree species, the protoplasm is able to vitrify. Vitrification is stimulated by a high concentration of sucrose and other sugars. In this relatively stable condition, it is possible to cool cells almost to absolute zero without destruction

(Rajashekar 2000; Hopkins 2006; Jan et al.

Membranes are restructured under exposure to cold before the temperature drops below zero. In these conditions, the water potential is gradually lowered with a simultaneous drop in the osmotic potential due to the accumulation of carbohydrates in vacuoles. ABA is accumulated, and it induces the synthesis of specific proteins. The next stage brings intensified changes in the cell membrane - degradation of phosphatidylcholine and phosphoinositol, accompanied by a continued increase in ABA levels and protein synthesis modifications (Gusta et al. 2005; Lindberg et al. 2005). Cryoprotectants, substances that directly protect the membrane from damage, are also synthesized at this stage. Rigid membranes are less likely to be deformed during frost-induced dehydration, and they protect cells against freezing more effectively. This parameter is largely dependent on the sterol content of cells (Hopkins 2006; Janska et al. 2010).

In addition to membrane unsaturation, it appears that lipid asymmetry in the membrane also contributes to the physical structure of the membrane at low temperature (Gomes et al. 2000) .

The mechanism protecting chloroplast membranes enables the plant to begin photosynthesis as soon as ambient temperature increases.

The cold resistance of plants is also determined by the following mechanisms:

1. Thermal insulation which delays and minimizes heat loss, for example, shoot apices are often covered with dense foliage (rosette plant habit) or they winter under a layer of leaves or litter (geophytes). Frost tender organs are often rejected before the onset of very low temperatures (deciduous plants shed leaves in the fall). In high mountainous regions of tropical zones, the leaves of large rosette plants close above the tip at night to protect the interior from freezing (Hopkins 2006) .

2. Water freezing in intertissue spaces, for example, between the seed coat and the embryo or between bud scales, where extensive areas are covered with ice.

3. Cell structures are protected against excessive dehydration with an accompanying increase in the effectiveness of barriers that prevent ice crystals from propagating from the apoplast inside the cell. The following mechanisms are involved:

(a) Osmotic pressure increases to keep water inside the cell, and the water potential decreases due to the accumulation of osmotically active compounds (simple sugars and oligosaccharides, polyols, low-molecular-weight nitrogen compounds, such as selected amino acids) in vacuoles and hydrophilic proteins in the cytoplasm (Rorat 2006; Liang et al. 2009). The share of highly polar lipids in the membrane structure increases, such as phosphatidylcholine and phosphatidyle-thanolamine in the plasmalemma and cytoplasmic membranes or digalactosyl-diacylglycerol in chloroplast membranes, which increases matrix interactions inside the cell.

(b) The membrane is enriched with more stable lipids containing polyunsaturated fatty acid residues, selected sterols, and cryopro-tectants are accumulated in the cytoplasm to protect cell structures against strong dehydration (Lindberg et al. 2005; Zhang and Tian 2010). These substances stabilize membrane structure and prevent conforma-tional protein changes. They counteract the accumulation of salt ions and selected organic acids in the cell, and they protect proteins against denaturation. Small proteins, whose synthesis is enhanced or induced under exposure to low temperatures, play a protective role. Some of them show significant homology to proteins synthesized in response to water stress, for example, to dehydrin (Rorat et al. 2006). The cell wall plays an important role in protecting the cell against the adverse consequences of dehydration, and it is the main barrier to ice penetration.

In addition to mechanisms responsible for resistance to the primary consequences of frost, cold-resistant plants develop acclimatization mechanisms that enable them to avoid secondary thermal stress at below zero temperatures, such as photoinhibition, draught, oxygen deficiency (under ice cover), or mechanical effects of ice load (Alcázar et al. 2011).

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