4. Temperature

4.1.1 Temperature Dependence of Life

For most organisms, temperature ranges of active life can be determined, as well as temperature limits beyond which life is not possible. Since metabolism requires water, active life is confined to conditions under which water is in the liquid state, and only under high pressure (e.g. in the deep sea) is life possible at temperatures around and even above 100 °C. On the other side, freezing of cellular liquids can be avoided—for example, by high concentrations of compatible solutes or antifreeze compounds, such as specialised proteins—and therefore life processes can continue even in a small temperature range below zero, albeit at low intensity. A dose–response diagram for temperature usually shows an asymmetrical curve with an optimum range of about 10 K, a steep drop towards higher temperatures and a smooth decline towards lower temperatures (Fig. 4.1). Imbalances of metabolism increase with deviation from the optimal range, thereby hampering cellular and developmental processes such as growth and reproduction. On the high-temperature side, heat damage to proteins contributes the most to such imbalances, while on the low-temperature side, membrane functions and different rates of deceleration of enzymatically catalysed reactions are the main reasons for the occurrence of imbalances. For each organism a temperature range of hardly detectable life functions can be derived. Extreme temperatures beyond this range result in lethal damage. Within the tolerable temperature range, the extent of damage does not exceed the capacity of a plant for repair at more favourable temperatures. This status of “latent life” is known as one of the five cardinal points of temperature tolerance of an organism (cold death, cold latent life, temperature optimum for active life, heat latent life and heat death) (Fig. 4.1).

4.1.2 Plants as Poikilothermic Organisms

Only very few examples of plant organs that can produce heat by alternative respiration are known. One of these is the spadix of Aracean flowers. Plants are poikilothermic organisms, which cannot maintain their temperature at a level different from that of their immediate environment (Chap. 9). However, as the exterior temperatures can vary substantially for different organs such as roots (soil temperature) or leaves (air temperature, local heating by sunrays), temperatures within one individual plant can be very different at any given time. Furthermore, environmental temperatures can change rapidly. Thus, plant cells, tissues and organs require the ability to autonomously respond to temperature fluctuations.

4.1.2.1 Temperature Dependence of Metabolism

In contrast to homeothermic organisms, which maintain a stable inner temperature, plants need to balance the biochemical reactions of their metabolism. This is not a trivial task, as the rate even of an enzyme-catalysed reaction depends on the temperature, just like any non-catalysed reaction (with the only difference being that the enzyme lowers the activation energy). The simple measure for the temperature dependency of a reaction is the so-called Q ₁₀ (the quotient of the rates (V) of a reaction at two temperatures (T) differing by 10 K):

(4.2)

An increase in the temperature speeds up the reaction; a decrease slows it down. The Q₁₀ is a direct indicator of the temperature dependence of a reaction. The Q₁₀ for enzymatic reactions lies between 1.4 and 2.5, while for biophysical processes it is between 1.03 and 1.3. Both the range of Q₁₀ values for enzymatic reactions and the difference between biochemical and biophysical reactions pose fundamental problems for plants.

(4.1)

When the rate constants, and are inserted instead of the rate (V) of the reaction, the activation energy (E _a) can be calculated:

(4.3)

where R is the universal gas constant.

Because of variations in the efficiency of catalysis, not all biochemical reactions that take place at the same time in a plant cell require the same activation energy. Therefore, a change in the temperature could easily disturb the metabolic balance in reaction chains—in particular, those involving steps with a high Q₁₀. Plants must be able to compensate for potential temperature-caused metabolic imbalances—that is, changes of pool sizes of metabolites.

The challenges arising from the much lower temperature dependence of biophysical reactions apply to photosynthesis in particular. Absorption of light energy is barely affected by temperature, while the CO ₂ fixation in the Calvin cycle is substantially slower at cooler temperatures. This leads to an excess of energy, which has to be dissipated in order to prevent injury (Sect. 4.1.2.2 and Chap. 3) (Fig. 4.2).

Since the cellular metabolism is composed of a large number of individual reactions whose reaction rates are dependent not only on the temperature but also on the equilibrium constants and sizes of the substrate pools, heat inactivation of the cellular metabolism is not as sudden as that of individual enzymes. Comparably strong inactivation does not take place at temperatures below the optimum.

4.1.2.2 Temperature and Photosynthesis

Practically any type of stress that impairs CO₂ assimilation in the Calvin cycle under high light can cause oxidative damage and photoinhibition (of photosynthesis). Low temperatures are among the most prominent of these stresses (Chap. 12). Essentially, two processes account for oxidative damage. First, excitation energy cannot be channelled efficiently into the reduction of nicotinamide adenine dinucleotide phosphate (NADP⁺), because the balance is disturbed between photosynthetic thylakoid reactions (the so-called light reactions, which are virtually not decelerated), and the biochemical CO₂ fixation reactions which, in contrast, are slowed down considerably in the cold. As a consequence, reduced NADP (NADPH) is not oxidised at a sufficient rate in the reducing phase of the Calvin cycle, leading to a shortage of the electron acceptor NADP⁺. Under such conditions, electrons are transferred to O₂ instead and thereby radicals and reactive oxygen species (ROS) are produced. Depending on the scavenging capacity of the cell, ROS can be detoxified or cause damage. In addition, they can act as signals activating pathways that target genes for ROS-detoxifying proteins such as ascorbate peroxidase, glutathione reductase or superoxide dismutase (Chap. 2, Sect. 2.​2).

ROS generation can inhibit the repair of the D1 protein, which is the primary site of photoinhibition due to excessive light intensity (Yamamoto 2016). The D1 protein is a component of photosystem II (PS II), which contains—in addition to the paired P₆₈₀ reaction centre—several other photopigments, as well as the quinones Q_A and the mobile Q_B (plastoquinone in the bound state). During unlimited electron drainage from PS II to plastoquinone the excited chlorophylls of P₆₈₀ are in the singlet state ¹P₆₈₀, which readily dissipates its energy through Chl_D1 and pheophytin_D1 to Q_A. Impaired reoxidation of Q_A ^Ɵ—e.g. because of over-reduction of plastoquinone or other components of the linear photosynthetic electron transport—results in a spin conversion of P₆₈₀ ⁺Q_A ^Ɵ to ³[P₆₈₀ ⁺Q_A ^Ɵ] (triplet state) (Chap. 2, Sect. 2.​2). This triplet state of P₆₈₀ reacts readily with molecular oxygen ³O₂, producing the extremely reactive singlet oxygen ¹O₂. (Quenching of singlet oxygen formed at a low rate by carotenoids is described in detail in Chap. 2, Sect. 2.​2.​3). At low temperatures, quenching cannot keep pace with the production of singlet oxygen. This is the second process (besides ROS production) that causes photoinhibition of PS II. A similar process has also been described for photosystem I (PS I), albeit with ROS species other than singlet oxygen. Here the equivalent to the D1 protein, PsaB, is damaged when ROS production (in particular, the hydroxyl radical) exceeds detoxification. Photoinhibition of PS I is particularly pronounced in tropical species sensitive to chilling—that is, cold temperatures above freezing (Fig. 4.3). The difference in the damage to D1 and PsaB is the time required for repair. Recovery from photoinhibition of PS II requires only hours, whereas restoration of PS I takes several days.

4.1.2.4 Freezing

A fourth temperature-related stress phenomenon—besides metabolic imbalances, changes in membrane fluidity and heat denaturation of proteins—is the freezing of water (i.e. the formation of ice crystals) within an organism (Boxes 4.1 and 4.2). This represents a severe additional threat. Thus, chilling stress (exposure to low positive temperatures) is distinguished from freezing stress (exposure to sub-zero temperatures) (Fig. 4.4). Massive injury can occur because of severe dehydration upon formation of ice crystals (Box 4.2). Ice crystals predominantly build up in the apoplast because of the lower concentration of solutes relative to the cytosol. Ice formation in the apoplast leads to a drastic drop in water availability. The concomitant decrease in the water potential to extremely negative values causes damage to membranes and in turn the cellular compartmentation. Ice, in contrast to liquid water, is hydrophobic, and ice crystals at the surface of a biomembrane cannot exert the membrane-forming forces required for the formation of a lipid bilayer. Amphiphilic lipids unite with micelles or membranes only when they are in a sufficiently hydrophilic medium—that is, stabilised by an ordered water film. If this film disappears, the stabilising effect of the hydrophobic interaction vanishes and the lipids aggregate into droplets. This is then called the “lipid hexagonal II phase” (Fig. 4.5). Thus, intracellular crystallisation of water, which inevitably brings ice into contact with biomembranes, results in membrane disintegration and collapse of the cellular compartmentation. Loss of membrane integrity is indicated by electrolyte leakage, a parameter commonly used to quantify freezing tolerance (Box 4.3). Furthermore, freezing of water in xylem vessels and subsequent thawing causes air breakage of the water column, called an embolism.

Box 4.1: The Physics of Freezing: Homogeneous and Heterogeneous Ice Nucleation

A droplet of purest water, not disturbed by any agitation, can supercool to around −40 °C before instant nucleation takes place (Lorv et al. 2014). In that case, the Brownian molecular movement of the water molecules is so slow that larger clusters of molecules form, which initiate solidification in a hexagonal crystal lattice. As the nucleation in this case originates from water itself, it is called homogeneous nucleation. The cooling energy expended to achieve crystallisation is released as the heat of crystallisation, which can be recorded on a thermogram as the freezing exotherm (Fig. 4.6). The melting process requires energy as well, referred to as the heat of fusion, and is recognised by a delay in warming during the thawing process (melting point (MP) in Fig. 4.6). When nucleation of crystallisation is initiated in other ways (e.g. by seeding with ice crystals), supercooling to a less negative temperature than for homogeneous nucleation is sufficient for crystallisation. This is called heterogeneous nucleation because elements of the environment trigger crystallisation. These elements can be particular surface structures (e.g. of an ice crystal or a cell wall), whose microstructures can force the water clusters into a lattice-like structure which, together with the sub-zero temperature, facilitates crystallisation. Thus, heterogeneous nucleation requires less supercooling and, considering the situation in a plant tissue, with the physicochemical equilibrium between the extracellular ice and the cellular solutions, the proportion of water that crystallises is smaller and the freeze desiccation is less dramatic. This is important for preventing freezing damage by sudden ice formation.

The water vapour pressure over ice is less than that over supercooled liquid water at the same sub-zero temperature (Table 4.2). In line with the lower water vapour pressure is the more negative water potential of ice compared with supercooled water. Both are directly dependent on the sub-zero temperature. The water potential of ice (Ψ_ice) can be calculated from the vapour pressures over liquid water (P _water) and over ice (P _ice) by the following equation:

(4.4)

where V _w is the mole volume of water at the particular temperature. Therefore, at sub-zero temperatures, ice is thermodynamically more stable than supercooled water.

Table 4.2

Partial pressure of water vapour (in millibars) above ice and supercooled water

°C	Vapour pressure of ice (mbar)	Vapour pressure of liquid water (mbar)
0	6.1	6.1
−5	4.0	4.2
−10	2.6	2.9
−15	1.7	1.9
−20	1.0	1.3
−30	0.38	0.51

The water vapour pressure is also lower over aqueous solutions than over pure water and depends on the concentration of the solution—more precisely on the mole fraction (M) of water in the solution (Raoult’s law):

(4.5)

(4.6)

For dilute solutions it is the concentration of a solute(s):

(4.7)

where n = number of moles in a particular volume of water and the osmotic potential (water potential of the solution, π) is:

(4.8)

It follows that the freezing/melting point of a solution is lower than that of pure water (Fig. 4.6b) and that the depression (Δ_Tm) is proportional to the concentration of the solution, whereby the molar Δ_Tm = 1.86 °C; thus the actual Δ_Tm = 1.86 × n _s (mole solute in 1 kg water (molality)).

This applies strictly to dilute (i.e. “ideal”) solutions. In concentrated solutions, interactions occur between the dissolved particles, resulting in an apparent reduction in the concentration. The factor α, by which the concentrations appears to decrease, is the activity factor.

The following considerations are important for the freeze dehydration of plant cells. Cooling of dilute solutions to sub-zero temperatures results first in freezing of pure water and an increase in the concentration of the cellular liquid. Separated by the cell wall, extracellular ice coexists with the liquid content of the cell, the proportions responding to the water potential of both compartments (within the cell, equilibrium of the water potentials of the organelles must also be assumed, otherwise considerable intracellular fluxes of water would occur between the organelles).

Box 4.2: How Much of the Tissue Water Can Freeze?

Liquid water can be distinguished from ice by nuclear magnetic resonance (NMR) spectroscopy. The frozen and the still liquid portions of the water content of a leaf, for example, can thus be determined by running the NMR scans at a series of sub-freezing temperatures. Plotting the portion of liquid water versus the temperature produces so-called freezing curves (Fig. 4.7). The lower the temperature, the more cellular water is deposited as ice in the intercellular cavities. The nearly hyperbolic freezing curve of ivy (Hedera helix) can be transformed into a straight line by plotting the residual liquid water against the reciprocal of the temperature. The line cuts the y axis at a value, K, which is the amount of water that, because of its binding to macromolecules, cannot freeze. In the example shown in Fig. 4.7, K ≈ 5%. Interestingly, at a normal winter temperature of −10 °C, about 80% of the water in an ivy leaf is already frozen outside the cells and accordingly shrinkage of the cell volumes is dramatic and can result in considerable negative turgor (Zhu and Beck 1991). The concomitant increase in the concentration of the intracellular solutes is likewise dramatic (e.g. fivefold in the presented example). A highly concentrated solution has a substantially lower freezing point than the original cellular solutions, thus counteracting intracellular ice formation.

4.1.3 Variations in Temperature Range

A classification most commonly used in microbiology sorts organisms into three categories based on their temperature optima: psychrophiles, mesophiles and thermophiles. Psychrophiles are organisms adapted to cold habitats where water frequently changes from the liquid to the crystalline state. Most of them are unicellular snow and ice algae (e.g. Haematococcus pluvialis and various diatoms) or bacteria that prevent freezing of cellular water. At the other end of the scale are the thermophiles (and extremophiles) with optimal temperatures for growth between +80 °C and above 100 °C. They comprise certain unicellular algae such as Cyanidioschyzon merolae, cyanobacteria, bacteria and archaea, most of them living in hot springs, in geysers or on the black smokers of the deep sea. Most organisms, including practically all plants—in particular, the (homoiohydric) terrestrial and the marine species—belong to the mesophiles, which thrive at day temperatures between 15 °C and 25 °C and (in the case of terrestrial species) night temperatures about 10 K lower.

However, while temperature optima are not that different between plants, the tolerable temperature range varies widely (Tables 4.3 and 4.4). Most of the variation is attributable to differences in the ability to withstand temperatures below the optimum. The temperature at which cold damage occurs largely corresponds to the geographical distribution of a species. For instance, plants from temperate zones are usually more cold tolerant than plants from tropical regions. They are able to withstand sub-zero temperatures, whereas many tropical plants already show damage when exposed to temperatures slightly above freezing (chilling) (Fig. 4.3). Palms, mangroves, and tropical ornamental plants such as the African violet (Saintpaulia ionantha) (Fig. 4.8) or the coffee tree (Fig. 4.9) have been studied intensively. They are all adapted to a temperature range of between +5 and about 50 °C. Many other crops are chilling sensitive as well (e.g. cotton, soybean, maize and rice).

The chilling sensitivity of the African violet has been used to demonstrate that the strength or quantity of stress is composed of its intensity and its duration (Fig. 4.8). Chilling damage, visible as necroses, starts at temperatures below 8 °C. Fifty per cent damage results from 6 h of exposure to +1 °C or maintenance at +5 °C for 50 h. In this case, the low rate of repair allows comparative quantification of stress damage.

The variation in the ability to tolerate temperature extremes is far less pronounced at the other end of the temperature scale. Critical temperatures causing heat damage barely vary between plants, and hardly any correlation with the climatic zone of origin is apparent (Tables 4.3 and 4.4).

Not all life processes of a plant have the same optimum temperature, because the individual developmental steps may take place in different ambient conditions. Winter cultivars of cereals, for instance, germinate at temperatures considerably lower than those required for growth (in spring and summer). Nevertheless, the rates of germination show typical temperature dependence with Q₁₀ values between 1.5 and 2.5. In that context it is also important to mention that not all organs of a higher plant are similarly cold sensitive or tolerant. Those most sensitive are the roots and meristems of the shoots, while axial tissues and mature leaves are the most tolerant parts (Fig. 4.9). Also, certain stages in the development of a plant are particularly vulnerable—namely, seed germination and fruit ripening.

The correlation between the cold/frost tolerance of a species and the average minimum temperature of the climatic zone it inhabits shows the adaptive importance of mechanisms to cope with temperatures near and especially below the freezing point. Likewise, within-species variation in frost tolerance is associated with distribution of ecotypes along temperature gradients. The frost tolerance of Arabidopsis thaliana accessions correlates well with the mean minimum temperature at the site of origin. Individuals from Sweden or Russia, for instance, survive exposure to two consecutive nights of −10 °C much better than individuals from Spain, Libya or Morocco (Zhen and Ungerer 2008, Fig. 4.10). Similarly, there are pronounced differences in freezing tolerance between winter cereals and spring cereals.

Depending on the temperature climate of their habitat, plants must be adapted to and acclimate to not only the extremes of the temperature range (i.e. to seasonal variations) but also to smaller or greater diurnal temperature fluctuations. Considerable diurnal temperature oscillations are typical of deserts and the alpine and nival vegetation belts of high mountains, especially in the tropics. The air temperature during daytime may reach 20–30 °C, whereas the nocturnal temperature usually falls below the freezing point and the leaves may be stiffly frozen at the end of the night. While freezing of leaf tissue water is a process of several hours, thawing takes place within minutes once the tropical morning sun hits the leaves (Sect. 4.2.6). These plants have developed mechanisms to slow down freezing or even avoid it. They have to be frost tolerant all year round.

On a sunny day in temperate regions (e.g. in Central Europe) the surface of bare soil may become as hot as +50 °C, while the air temperature above the soil decreases rapidly with the distance from the soil surface. The gradient in the soil itself is even steeper. Reverse temperature gradients occur also during clear nights when no cloud cover mitigates radiation emission from horizontal and slightly inclined surfaces of the vegetation or from the soil. Young plants whose shoots or leaves are close to the ground are therefore exposed to a stronger temperature stress during both day and night than plants whose leaves are supported by a stem, which experience a considerably less challenging temperature microclimate (for details of the energy balance of leaves, Chap. 9).

Box 4.3: Measurement of Cold Hardiness and Damage by Cold and Freezing of Plant Tissue

The degree of cold/frost hardiness of a plant or plant tissue can be experimentally examined by subjecting the sample to a so-called freeze–thaw cycle with subsequent quantification of the induced damage. To warrant full equilibration of the sample with the applied temperature, low rates of cooling and rewarming must be applied (Wisniewski et al. 2014). A frequently used rate is 2 K/h. The protocol for a freeze–thaw cycle is shown in Fig. 4.11. For quantification of the damage, which can result from cooling as well as from rewarming, the sample is kept after rewarming at room temperature for at least 24 h to allow development of potential damage. Commonly used assays for the degree of damage and cold resistance, respectively, are biochemical activity tests such as the triphenyl-tetrazolium test for dehydrogenase activity or the electrical conductivity test for membrane leakage (Fig. 4.12). Controls are undamaged as well as completely killed (by dipping in liquid nitrogen) samples. Cold hardiness is commonly expressed as the LT₅₀—the temperature at which 50% damage (e.g. ion leakage of 50%) becomes apparent.

4.1.4 Strategies to Cope with Temperature Fluctuations and Temperature Extremes

Seasonally varying temperature extremes can be avoided through various escape strategies (Chap. 2). Annual plants can endure unfavourable conditions as dry seeds that are far less frost or heat sensitive. A new life cycle then starts with germination only after a cold winter or a hot, dry summer, depending on the habitat. Alternatives for perennial plants are the shedding of cold-sensitive organs such as the leaves or the overwintering only of the subterranean plant parts (rhizomes, bulbs) (Chap. 12). Tropical alpine plants have developed mechanisms of short-term freezing avoidance, as the daily periods of sub-zero temperatures are usually shorter than 12 h (Sect. 4.2.6).

Besides such escape strategies, every plant needs to be able to adjust metabolic fluxes and membrane fluidity to fluctuating temperatures and to protect cellular structures during short periods of temperature extremes. In the absence of homeostatic temperature regulation, most of the mechanisms involved operate at the cellular level and protect the tissues directly exposed to an unfavourable temperature.

Acclimative changes in heat tolerance (= acquired thermotolerance), albeit not nearly as pronounced as cold hardening, are also well established. The underlying mechanisms are only partly specific to the stressor heat (Sect. 4.3); rather, they provide general stress tolerance. This is important, as heat stress (a) occurs in a temperature range of high metabolic activity and (b) is almost always accompanied by or associated with other stresses such as drought, high light intensity and generation of ROS.

4.2.1 Adjustment of Membrane Fluidity

A very important aspect of cold stress physiology is the increase of membrane fluidity with decreasing temperatures (Sect. 4.1.2.3). Cold/frost hardening and de-hardening are therefore strongly associated with changes in the chemical composition of the cellular membranes to maintain the fluidity required for proper functioning. Acclimation to a particular temperature involves modification of the lipid composition with respect to the proportions of saturated and unsaturated fatty acids—that is, changes in the desaturation index (double-bond index (DBI)):

(4.10)

At lower growth temperatures, membrane fluidity is maintained by a higher proportion of unsaturated fatty acids. The respective acclimative responses (Fig. 4.14) have been documented for many different plant species.

Membrane sterols play a special role, due to an amphipathic structure that differs from that of the glycerolipids. Since their hydrophobic moiety is much larger relative to the polar heads than in glycerolipids, they are able to diffuse rapidly horizontally as well as vertically (the so-called flip-flop mechanism). They serve as buffers for membrane fluidity. At low temperatures, they increase membrane fluidity by disrupting gelling of phospholipid domains and thus preventing the formation of semi-crystalline patches. At high temperatures, they decelerate the motion of the fatty acid tails and thus stiffen the membrane (Buchanan et al. 2015).

Unsaturated fatty acids originate from saturated ones by the oxygen-dependent action of desaturases. Although desaturation is an oxidative process, it requires, in addition, two electrons from electron donors such as ferredoxin (in plastids) or reduced cytochrome b₅ (in the endoplasmic reticulum (ER); for more details, see plant physiology and plant biochemistry textbooks). Each fatty acid desaturase introduces a double bond at a specific position—for example, at the Δ6, Δ9 or Δ12 position. In the ER of plants and in cyanobacteria, desaturation is commonly catalysed by acyl-lipid desaturases, which introduce unsaturated bonds into fatty acids that are in a lipid-bound form. Various types of membrane-bound and soluble desaturases are also present in the plastids of plant cells. The importance of desaturases for cold acclimation is demonstrated by the inability of desaturase mutants to survive at low non-freezing temperatures. In contrast, overexpression of the ω-3 fatty acid desaturases FAD3 (localised in the ER) and FAD7 (localised in the chloroplasts), which catalyse the conversion of linoleic acid (18:2) to linolenic acid (18:3) in tomato, increased the chilling tolerance of the plants (Domínguez et al. 2010) but also affected the photosynthetic performance in a different way, depending on the intracellular localisation of the respective desaturase (Fig. 4.15).

Adjustment of the lipid composition of membranes takes time, as degradation, synthesis and trafficking of lipids are required. Temperature changes during the course of a day/night cycle are probably too rapid, as changes in membrane composition usually take several days. Still, during a day/night cycle associated with a corresponding diurnal temperature cycle, the plasma membranes of Arabidopsis seedlings showed slightly lower fluidity during the cold phase. Whether this was due to a reorientation of the membrane domains (van Meer et al. 2008) or to small changes in lipid desaturation was not clear. The fluidity buffer function exerted by the membrane sterols may play an important role in compensating for diurnal fluidity fluctuations (Martinière et al. 2011).

4.2.2 Prevention of Photoinhibition

Cold hardening has to include not only modifications of the lipid/protein composition of the biomembranes but also adjustments of other metabolic processes. The foremost adjustment is the reduction in the light-harvesting capacity of the chloroplasts. The decelerated metabolism in the cold can lead to dramatically increased rates of ROS formation, since light absorption by the photosynthetic pigments takes place irrespective of the temperature, while the biochemical utilisation of the absorbed energy cannot keep pace with the pigment excitation. Thus, in cold-hardened plants the chlorophyll content is lower and the photosynthetic apparatus is partially degraded (Fig. 4.16). Conversely, xanthophyll concentrations are higher, so the capacity for non-photochemical quenching is higher.

The limitation of photosynthesis does not necessarily result in a shortage of plant reserve material, as respiration is also markedly restricted at lower temperatures (Hansen and Beck 1994). Concomitantly with the slowdown in photosynthesis, frost hardening of evergreens is associated with a change in carbohydrate metabolism in favour of the production and accumulation of oligosaccharides instead of starch (Hansen et al. 1997).

4.2.3 Cryoprotective Proteins

In the course of cold acclimation, the proteome of plant cells undergoes pronounced changes. Many proteins increase in abundance upon exposure to low temperature. Collectively they are often referred to as cold-responsive proteins (COR proteins). Among them are isoforms of housekeeping enzymes with a lower temperature optimum, as well as enzymes involved in the biosynthesis of cryoprotectants (Sect. 4.2.4.1). The integrity and function of biomembranes have to be protected against damage resulting from freeze dehydration and the concomitant increase in the solute concentrations of the intracellular fluids. Such protection has been associated with the synthesis of dehydrins (DHNs) and dehydrin-like proteins (Late Embryogenesis Abundant (LEA) proteins and Responsive to ABA (RAB) proteins; for more details, Chap. 6). These proteins are all extremely hydrophilic glycine-rich proteins of a wide range of molecular weights. They lack enzymatic activity, are heat and acid stable and possess a high proportion of a random coil secondary structure, which binds water intramolecularly (Hanin et al. 2011). Interaction with biomembranes is enabled by a hydrophobic domain while the hydrophilic domain(s) face the cytosol. Attachment to the biomembranes displaces low molecular weight solutes, such as ions, from the membrane, thus avoiding changes in the membrane potential and disintegration or inactivation of ion pumps and transporters.

In unstressed plants, only low concentrations of dehydrins are present. Stress by drought, salt and cold leads to rapid up-regulation of gene expression and steady accumulation of dehydrins in all compartments of the cell. As for the majority of the COR proteins, formation of dehydrins can be triggered by abscisic acid (ABA) or can be activated independently of ABA. The promoter of the COR gene, RD29A (also known as COR78 or LTI178) contains cis-acting elements for ABA-independent signalling (cold response, osmotic stress) as well as for ABA-dependent (osmotic) signalling (Chaps. 2 and 6). Enhanced frost tolerance of A. thaliana was achieved by overexpression of RcDHN5 from Rhododendron catawbiense (Peng et al. 2008) and was attributed to maintenance of enzyme activity upon water deficiency. Nonetheless, the question as to whether particular dehydrins directly contribute to chilling and freezing tolerance remains unanswered.

Another type of proteins has been described as Cold Shock Domain Proteins (CSPs), suggesting cryoprotective properties (Sasaki et al. 2007). The typical Cold Shock Domain (CSD) contains a five-stranded β-barrel sheet with two consensus motifs, which bind to single-stranded DNA/RNA (Sasaki et al. 2013). The CSDs are found in bacteria, as well as in eukaryotes, and are considered RNA chaperones. Upon cold treatment, RNA forms unfavourable secondary structures that could interfere with RNA functions such as transcription and translation. CSPs are able to resolve these structures and restore RNA functionality. Although they are associated with cold adaptation, CSPs regulate many biological processes such as growth and flowering, thus exhibiting pleiotropic effects (Sasaki and Imai 2012).

4.2.4 Control of Ice Formation

To survive sub-freezing temperatures, organisms have developed a variety of mechanisms to maintain at least a minimum portion of their cellular liquids unfrozen. Variations in frost tolerance between acclimated and non-acclimated plants are at least partly explained by differences in the onset temperature of detrimental intracellular ice formation. Figure 4.17 shows the example of rye.

Whether crystallisation takes place inside or outside the cells depends on the cooling rate. When tissue is cooled slowly, water can move or leak out of the cells and crystallise outside the cell membranes in the extracellular spaces. This is a reversible process following the equilibrium between the water potentials of ice and of the cellular solutions. The water potential of ice is more negative than that of supercooled pure water (Box 4.1). The lower the sub-zero temperature is, the more negative the water potential of ice is and the more cellular water crystallises in the intercellular space. At a high cooling rate, there is not sufficient time for water export, and freezing takes place intracellularly, thereby killing the cells. In that case, ice crystals can grow through plasmodesmata and pits from cell to cell and rapidly expand to wider areas of the tissue.

For crystallisation, the liquid accumulating in the apoplastic space requires a nucleation trigger. Ice spreading from nearby xylem vessels could play such a role. In frost-sensitive dicots, ice spreads from only one nucleation event rapidly through the entire plant, while in graminoids, because of the polystele, each grass leaf requires separate nucleation. Thus, the shoot structure and vascular system are important for the spreading of ice and freezing damage (Hacker and Neuner 2007, 2008). Otherwise, so-called ice nucleation–active bacteria (INA bacteria; Box 4.4) in the substomatal cavity or on the surface of a leaf could trigger nucleation. With potato and cauliflower, it has been shown that even water droplets on a leaf or a flower bud surface can catalyse ice formation inside the plant, and it takes only a few minutes for the entire plant to be frozen (Fuller and Wisniewski 1998). Ice nucleation activity has recently been reported in plant cell walls of bark tissue from blueberry stems, with outstanding activity (ice nucleation at −1.0 °C). This activity showed seasonal changes, peaking in November shortly before the onset of frost (Kishimoto et al. 2014). The mechanism underlying this INA principle is not yet known.

Box 4.4: Ice Nucleation Proteins

The so-called ice nucleation–active bacteria (INA bacteria) produce cylindrical surface proteins, which facilitate heterogeneous nucleation (ice nucleation proteins (INPs)). These bacteria are commonly gram-negative, epiphytic and pathogenic (Lorv et al. 2014). An example is Pseudomonas syringae. The effect of the “decoration” of the outer membrane with INPs is shown in Table 4.6 in comparison with other bacteria, which do not produce these proteins.

Table 4.6

Ice nucleation activity of bacterial cultures (Maki et al. 1974)

Species	Ice nucleation temperature (°C)
	T 1	T 90
Pseudomonas syringae C9	−2.9	−3.5
Pseudomonas syringae wt	−3.2	−3.9
Pseudomonas aeruginosa	−7.5	−17.8
Staphylococcus epidermidis	−6.9	−19.5
Escherichia coli	−8.3	−17.1
Proteus mirabilis	−8.0	−19.4
Bacillus subtilis	−10.6	−18.0
Pure culture medium	−9.2	−17.0

Thirty droplets (each 0.01 mL) of test material were placed on a controlled surface, and the temperature was slowly lowered from room temperature to −25 °C. The temperature at which the first crystal formed (T₁) and the temperature at which 90% of the droplets were frozen (T₉₀) were recorded

The INP from P. syringae consists of a particular octapeptide in up to more than 100-fold repetition (Fig. 4.18). The N terminus is anchored with transmembrane spans to a glycosylphosphatidylinositol patch of the outer membrane of the bacterium (Turner et al. 1991; Sarhan 2011).

Deletion mutants show that a 68-fold repetition of the motif is sufficient to effectively trigger ice seeding. A change in the octapeptide sequence abolishes ice nucleation activity (Green and Warren 1985). A model of the protein predicts a largely planar molecule with a molecular mass around 150 kDa, serving as a template for forcing water into an ice lattice. This quasi-crystalline fixation of the water molecules minimises the degree of supercooling that is required to “quiet” the water clusters (Kajava and Lindow 1993) (Table 4.7).

Table 4.7

Analysis of bacterial ice nucleators (INPs) from Pseudomonas syringae. (Hew and Yang 1992)

Nucleation temperature (°C)	Estimated molecular mass (kDa)	Number of INPs required for nucleation
−12 to −13	150	1
−3	870	60
−2	19,800	132
−1	83,700	558

As mentioned above, ice seeding activity has been observed with plant cell walls from blueberry stems and also with cell walls from rye leaves (Brush et al. 1994). Apart from a general chemical composition (proteins, carbohydrates and phospholipids (Gusta et al. 2004)), the mode of function and mechanism of plant ice nucleators are not yet known.

The effects of INA bacteria are double edged. In frost-tolerant plants they trigger “high-temperature” ice seeding, thus avoiding strong supercooling and potential intracellular ice formation. On the other hand, they cause frost damage to many crops, such as maize, strawberry and citrus fruits, which are not freezing resistant but can tolerate supercooling to some extent. Attempts have been made to dilute out the INA bacterial infestation of crop plants by replacement with antagonists (Table 4.8). These are obtained by plating out wash solutions from leaves and cooling them to −5 or −9 °C. Antagonists of INA bacteria should be present wherever ice formation does not take place. These organisms are mostly natural mutants of the original bacterial strains.

Table 4.8

Antagonists (mutants) of ice nucleation–active (INA) bacteria inhibiting noxious ice formation during moderate frost. (After Lindow (1982))

Bacterial culture	Damage (% change in leaf colour after 1 week)
Control (Pseudomonas syringae)	95
Mutant A 510	12
Mutant A 509	18
Mutant A 507	27
Mutant A 506	33
Mutant A 508	51

Pear leaves were inoculated with the respective bacterial culture 3 weeks prior to frost to give the bacteria time to establish themselves. Frost damage was quantified by assessing necrosis

INA bacteria have found several technical and biotechnical applications: artificial snow making, cloud seeding and rain making in warmer regions, and use as part of a fusion protein of the N-terminus with a protein of interest (e.g. an enzyme (Gao et al. 2014)) or binding of cancer cells by an antibody or antibody mimetic protein displayed on the surface of engineered Escherichia coli cells (Zahnd et al. 2007).

4.2.4.1 Osmotic Adjustments

For the sake of survival during freezing, ice formation should take place outside the cell walls in the intercellular spaces and should be suppressed intracellularly by furnishing of the cells with compatible solutes, which allow them to supercool, thereby preserving their cell plasma in the liquid state. Both strategies entail benefits and stresses or risks. Extracellular freezing of cellular water results in cellular desiccation proportional to the frost temperature. At the same time, intracellular solutions concentrate and may exert ionic stress on biomembranes and protein complexes. Accumulation of compatible solutes counteracts problems caused by freeze desiccation, but this mechanism carries a high risk as ice formation by overstretching the capacity for supercooling leads to instantaneous death, as there is no time for water export. For both mechanisms, stress increases with the strength of the frost. Possible damage is gradual upon extracellular freezing but is sudden and absolutely detrimental upon intracellular ice formation.

Accordingly, frost hardening of plants is regularly associated with the accumulation of compatible solutes (also termed osmolytes or cryoprotectants) in the cells, predominantly in the vacuoles which, however, have to equilibrate their osmotic potential with the cytoplasm and other cellular organelles. These low molecular weight solutes are carbohydrates, amino acids (e.g. proline), polyamines and many more. Among the carbohydrates that accumulate during frost hardening, sucrose and its galactosides raffinose and stachyose are prominent. Also, polyols, as the reduced forms of monosaccharides, are frequently found. For winter and summer cereals, for instance, a strong correlation between freezing tolerance and the sugar accumulation rate was determined.

Compatible solutes, in addition to their colligative effects, are known also as soft ROS detoxifiers (e.g. mannitol) (Tarczynski et al. 1993). Upon extracellular freezing of cell water, these non-charged but weakly polar (like water) compounds “dilute” the ionic charge at the membrane surfaces (which results from the concentration of cellular ionic compounds) and thus stabilise the bilayer structure.

Their colligative (physicochemical) effect on the freezing point of the cellular liquids comes into play when freeze dehydration progresses. For example, the cell sap of frost-hardened spruce needles is only 1.5 osmolar, corresponding to a freezing point depression of 2.8 K. This is physiologically insignificant because ice nucleation requires stronger supercooling than −2.8 °C. Upon extracellular freezing of cellular water the concentration of the cryoprotectants increases significantly, and with a 10% remaining liquid volume, the freezing point depression attains 28.5 K, which could prevent freezing of the residual liquid water.

An example of the cold-hardening effect of cryoprotectants is the branched-chain polyol hamamelitol and its galactoside clusianose, which accumulate under natural or artificial cold stress in the leaves of the alpine primrose Primula clusiana (Sellmair et al. 1968, 1969) (Fig. 4.19). Both compounds are not degradable by the plant itself and finally reach the soil when leaves decay.

Besides the formation of cryoprotectants, the release of water from the cell into the apoplast can further reduce the probability of intracellular freezing, albeit at the cost of potentially stronger ionic stress for the cell. Membranes of frost-hardened cells allow water to exit from the cell into the apoplast more easily than frost-sensitive membranes do. This has been demonstrated with protoplasts isolated from frost-sensitive and frost-hardened rye leaves (Fig. 4.17). The mechanisms behind this facilitated efflux are not known. The simplest explanation assumes cold inhibition of ion pumps and H⁺-ATPases, leaking of low molecular weight solutes and passive water efflux, following the solutes.

4.2.4.2 Antifreeze Proteins

In particular, unicellular organisms such as bacteria are endangered by intracellular crystallisation of water. Three strategies have been recognised in frost-tolerant organisms including prokaryotes, lower and higher plants, and special groups of animals—for example, insects or inhabitants of the Arctic and Antarctic Oceans. Apart from the accumulation of compatible solutes and the activation of their biosynthetic machinery, three functional types of proteins can be differentiated in relation to the formation of ice crystals at the cellular level: proteins that promote ice formation (ice nucleation proteins (INPs)), proteins that inhibit ice nucleation (anti-nucleating proteins (ANPs)) and proteins that control the growth and recrystallisation of ice crystals (the classical antifreeze proteins (AFPs)). ANPs and AFPs are often considered one group of proteins because there is some evidence that AFPs might also bind to nucleation-promoting structures (Griffith and Yaish 2004). Up to now, INPs have been known only from prokaryotes—mainly gram-negative bacteria (Box 4.4)—while ANPs and AFPs are also known from plants.

AFPs have been detected in all types of organisms—for example, vertebrates and invertebrates, plants, fungi and prokaryotes (Table 4.9). Their presence can be monitored microscopically by examining the shape of the ice crystals. These are round and flat in water or diluted solutions. Upon binding of AFPs to their prism faces, developing ice crystals acquire a hexagonal structure (Fig. 4.20). AFPs have been extracted from the leaves of frost-hardened plants, but barely from non-acclimated frost-sensitive leaves. For instance, one of the two flowering plants found naturally in Antarctica, Deschampsia antarctica, secretes AFPs into the apoplast (Bravo and Griffith 2005). Thus, the ability to produce AFPs appears to be part of the cold-hardening process at least in some plant species.

Table 4.9

Lowering of the freezing point by antifreeze proteins, as measured by thermal hysteresis

Organism	Tissue	Lowering of freezing point, thermal hysteresis (°C)
Bony fish	Muscle	0.7–1.5
Insects	Digestive tract, muscle	3–6
Bacteria	–	0.3–0.35
Fungi	Fruiting body	0.3–0.35
Mosses	Whole plant	0.3–0.68
Horsetails (Equisetum hyemale)	Rhizome	0.2
Ferns	Shoot	0.25
Winter wheat	Leaves	0.2
Carrot	Root	0.4
Carrot	Shoot	0.15
Poplar	Twig	0.22

AFPs are also known as thermal hysteresis proteins (THPs) because they lower the freezing point but not the melting point (thermal hysteresis activity). Thus, the freeze–thaw characteristic appears as a hysteresis curve (Griffith and Yaish 2004) which can be monitored in a temperature-controlled microscope. AFP interaction with tiny ice crystals suppresses their growth, at least at moderate freezing temperatures (ice recrystallisation inhibition (IRI)); considerably deeper supercooling is required for the crystallisation of that portion of water, which corresponds to the equilibrium between the water potentials of ice and the supercooled cellular solutions (Box 4.1). As they are efficient at very low concentrations (µM), it is clear that AFPs act in a non-colligative (non-physicochemical) manner. AFPs adsorb to the surface of ice crystals by forces that are not yet fully clear; hydrogen bonds, hydrophobic interactions or a particular structure of the hydration layer of the AFPs have been discussed (Lorv et al. 2014). One common character of all AFPs is a flat, hydrophobic ice-binding surface (Smolin and Daggett (2008). Another common feature are amino acid tandem repeats (X-Gly-Thr-Gly-Asn-Asp-X-U-X-U-Gly-Gly-X-U-X-Gly-X-U-X, in which X = hydrophilic and U = hydrophobic residue), which are aligned on one side forming that surface (Fig. 4.20a). Adsorption inhibits both further growth of the ice crystal and recrystallisation into harmful shapes, such as long needles. Probably the IRI activity is the more important since the actual effect on the freezing point depression (Table 4.9) is small. In recrystallisation assays with rye leaf extracts, AFPs present in cold-acclimated plants have been demonstrated to inhibit the growth of ice crystals (Fig. 4.20). AFPs are a mixture of small to medium-sized proteins, which can bind to both planes of an ice crystal, covering most of its surfaces. The binding is irreversible until the ice melts.

The efficacy of AFPs in lowering the freezing point differs greatly between organisms. Plant AFPs, although not very effective in lowering the freezing temperature (less than 1 K; Table 4.9) are very efficient in inhibiting recrystallisation of ice nuclei. Because of multiple ice-binding domains of individual plant AFPs or AFP oligomers, the same protein can bind simultaneously to different planes of the ice crystals (Griffith and Yaish 2004). Intracellular AFPs, when binding to ice nucleators, can prevent intracellular ice formation. Furthermore, they can be secreted into the apoplast, where they interfere with growth and recrystallisation of extracellular ice. Thus, they have a dual task.

Amino acid sequences of plant antifreeze proteins do not establish them as a distinct protein family. Instead, AFPs are highly variable and display similarities to a wide range of proteins. Some AFPs—in particular, those from cereals—are highly homologous to pathogenesis-related (PR) proteins produced upon pathogen attack. They include chitinases, β-1,3-glucanases, polygalacturonase inhibitors, and osmotin- and thaumatin-like enzymes (Griffith and Yaish 2004; Gupta and Deswal 2014)—that is, enzymes that degrade fungal cell walls and inhibit fungal enzymes. In cold-acclimated winter rye, AFPs exhibit both antifungal and antifreeze activity. In contrast, PR proteins induced by pathogens at room temperature in non-acclimated plants lack antifreeze activity. The dual function of the PR-AFPs has been interpreted with respect to resistance against low-temperature pathogens such as snow mould. The excreted PR-AFPs form hetero-oligomers that inhibit growth of such fungal pathogens (Hiilovaara-Teijo et al. 1999).

4.2.5 Signalling Networks Involved in Cold Acclimation

Drought, high salinity and freezing stress all pose osmotic challenges for plants. Accordingly, there is considerable overlap in the molecular responses of cells to these different stress conditions (Chap. 2). When one is judging by the hundreds to thousands of changes elicited in the A. thaliana transcriptome, however, there are also highly complex responses specific to cold acclimation.

A large number of COR genes belong to the CBF (for C-REPEAT BINDING FACTOR) regulon—that is, they are activated under the control of CBF transcription factors (also termed DRE-Binding proteins (DREB)) (Yamaguchi-Shinozaki and Shinozaki 2006). The promoters of these COR genes, which encode many of the proteins discussed above (i.e. dehydrins, antifreeze proteins, ROS-detoxifying enzymes, fatty acid desaturases, lipid transfer proteins, sugar and proline transporters, osmolyte producing enzymes) share a 5-bp consensus sequence (CCGAC), the so-called Dehydration Responsive Element (DRE), which is known also as Low Temperature Responsive Element (LTRE) or C-Repeat (CRT). Binding of CBFs rapidly activates transcription of COR genes.

The CBF regulon is present in many if not all higher plants (Box 4.5). Its central role in cold acclimation has been demonstrated by overexpression studies. Constitutive strong transcription of CBF genes results in the formation of COR proteins and a permanent cold-acclimated state even in the absence of low-temperature exposure. Such plants can survive transfer to freezing temperatures even without prior acclimation (Jaglo-Ottesen et al. 1998) (Fig. 4.21). CBF overexpression leads, on the other hand, to a strong reduction in the growth rate because CBFs stimulate the production of DELLA proteins, which are central negative regulators of growth (Fig. 4.21b). This is one of many examples for the active dampening of growth as an integral part of stress acclimation responses (Chap. 2).

Activation of cold acclimation involves a cascade of regulatory steps that have been unravelled in A. thaliana (Fig. 4.22). CBF genes are rapidly up-regulated upon exposure to low non-freezing temperatures. Their expression is controlled by transcription factors that are themselves predominantly controlled at the posttranslational level. The transcription factor ICE1 (Inducer of CBF Expression 1) triggers the transcription of CBF genes by binding to cis elements in the CBF promoters. The stability of ICE1 is controlled by ubiquitination. The ubiquitin E3 ligase HOS1 targets ICE1 for proteasomal degradation, which prevents the expression of CBF genes in the absence of cold. HOS1-antagonistic regulation of ICE1 stability is mediated by SUMO-Ligase-mediated (SIZ1) sumoylation—that is, the addition of SUMO proteins, which protect ICE1 from degradation (Knight and Knight 2012).

Events upstream from ICE1 are less well defined. The primary sensor of cold temperature is still unknown (Sect. 4.4). What is clearly established is a rapid increase in cytosolic Ca ²⁺ levels upon transfer of plants to cold conditions, which activates proteins that inhibit ICE1 degradation and activate ICE1 sumoylation. In addition, Ca²⁺ influences CBF expression via a calmodulin-binding transcription factor (CAMTA). The same regulatory steps leading to enhanced ICE1 activity are triggered by a cold-responsive Ca²⁺-independent MAP kinase phosphorylation cascade.

Box 4.5: The Role of the CBF Regulon in Cold Tolerance and Cold Acclimation

The CBF regulon is present even in chilling-sensitive species such as tomato (Chap. 2 and Sect. 4.2.1), and tomato CBF genes can trigger cold acclimation when expressed in Arabidopsis thaliana. Thus, the responses activated by CBFs appear to vary between species that are able to cold acclimate (e.g. Arabidopsis) and those that cannot (e.g. tomato). In addition, however, differences in CBF expression can be linked to natural variations in freezing tolerance (Fig. 4.23). The higher sensitivity to freezing temperatures of A. thaliana accessions from North Africa and Southern Europe (Fig. 4.23) correlates with lower CBF expression and concomitantly lower COR gene expression relative to accessions from Northern and Eastern Europe. Comparative sequencing of CBF genes from these accessions indicated relaxed selection on these genes in accessions from habitats that have a much lower incidence of frost events (Zhen and Ungerer 2008). However, not for all CBF genes expression can be directly linked with cold tolerance or cold acclimation (Fig. 4.23e).

4.2.6 Freezing Avoidance and Freezing Tolerance in Tropical High Mountain Plants

Cold hardening and de-hardening is observed only in plants of regions with seasons characterised by temperature changes. Tropical plants are not known to harden, which corresponds to virtually invariable high temperatures throughout the year. Nevertheless, plants of the tropical high mountains (e.g. the Andes, the East African and Hawaiian volcanoes, and the high mountains of New Guinea) are frost hardy. Furthermore, their frost hardiness is maintained throughout the entire year because they are subjected to the so-called Frostwechsel climate, described as summer every day and winter every night (Hedberg 1964) (Fig. 4.24). The regularly occurring frosts are only moderate (temperatures below −15 °C are rare and occur mostly in topographic depressions) and may last for a maximum of 12 h, and the life forms of the vegetation are adapted to cope with this recurrent temperature stress (Hedberg 1964; Beck 1994a). Adaptation to this type of climate requires mechanisms of freezing avoidance in addition to acclimative reactions. The plants are frost tolerant throughout the whole year but are not tolerant of an uninterrupted frost period.

Several types of response to sub-freezing temperatures can be recognised in the five common functional plant types of the tropical high alpine flora (Chap. 19): grass tussocks, sclerophyllous shrubs, acaulescent rosette plants, cushion plants and the spectacular giant rosette plants. All rely on the principle of daily changes between the nocturnal frost and the “summer” temperatures during the day, caused by the intense tropical sunshine. Mechanisms to overcome the different primary and secondary abiotic stress factors (climate, high light intensities and ultraviolet radiation, seasonal shortage of water during the dry season, low partial pressure of CO₂ and oxygen) imposed on the tropical alpine plants by their environment have been discussed in some detail, but the molecular level has hardly been touched upon (Beck 2011). Ecophysiological data show that separate investigation of the impact of one abiotic stressor on these plants cannot sufficiently explain the real situation, and a more holistic consideration of their performance is required, as claimed by Gusta and Wisniewski (2013), for the understanding of plant cold hardiness. The example of the “giant rosette plant” life form (Fig. 4.25) shows that more than one “strategy” may be adopted by a plant individual—namely, freezing tolerance as well as freezing avoidance by permanent insulation (Fig. 4.25) or nocturnal insulation, thermal buffering by liquids, or supercooling. Each of these “strategies” may be considered to meet a special physiological requirement. While the stems, inflorescences and roots follow the “strategy” of freezing avoidance, the leaves of the African Senecio and Lobelia are freezing tolerant and are stiffly frozen in the morning before sunrise. Extracellular freezing of cellular water has been documented, concomitant with freeze cytorrhysis of mesophyll cells or intravacuolar emergence of gas bubbles, which disappear upon thawing in the hypodermis (Beck 1994a). In nature the mesophyll cells produce sucrose as a cryoprotectant, and only a minor accumulation of starch can be detected in the chloroplasts. While the mature leaves of the giant rosette freeze every night, the young leaves in the central leaf bud, despite being freezing resistant as well, are protected from freezing through the formation of a so-called night bud (Hedberg 1964) by the outer rosette leaves. Since extracellular crystallisation of cell water results in a loss of cell turgor, growth of the young leaves would be interrupted every night. The night bud prevents nocturnal sub-zero temperatures and concomitant freezing of the meristematic tissue, thus enabling uninterrupted growth. A question still to be solved is how propagation of ice from the frozen leaves to the other parts of the plants is prevented. Fast spreading of ice has been observed by infrared differential thermal analysis in dicotyledonous alpine plants, starting from the xylem vessels and spreading into the rest of the plants (Hacker and Neuner 2007, 2008). In the case of Senecio, ice formation is confined to the leaves and cannot be triggered from the xylem, because of the existence of a kind of “thermal ice barrier” (Hacker et al. 2011). Interestingly, the leaves of the corresponding giant rosette plants of the neotropical Andes (Espeletia spp.) are reported to supercool to about 8–12 °C below the ambient temperature and to be killed by intracellular ice formation (Goldstein et al. 1984, 1985).

4.3.1 Heat Stress Avoidance

The timing of key developmental steps such as germination and sexual reproduction can help plants reduce the probability of heat exposure and damage (Chap. 2). Besides that, not many different mechanisms of heat stress avoidance have been demonstrated. At the morphological level, positioning of the leaves in a tree crown or canopy and the leaf angle may reduce heating by sunrays. Segmentation of large leaf areas into narrower parts can have a similar effect. Transpiration cooling (Chap. 10) appears to be an effective mechanism. However, heat frequently coincides with low water availability, and then the necessary closure of stomata greatly reduces the cooling potential. More details about heat avoidance are presented in Chap. 9. Generally, however, because of the extreme fluctuations in temperature that can occur daily or seasonally and at the micro-scale (Sect. 4.1), it is in most habitats virtually impossible for terrestrial plants, as sessile organisms, to avoid heat stress altogether. Accordingly, all plants are capable of mounting a response to heat stress (Vierling 1991).

4.3.2 Acquired Thermotolerance

It is well documented that exposure to either a continuously applied non-lethal heat stress or single heat shocks results in the acquisition of thermotolerance or, in other words, heat hardening. These types of heat exposure elicit cellular processes that enhance the ability of plants to survive a later and more severe heat stress. Acquired thermotolerance therefore represents a classic acclimation. After just 15 min of exposure to heat, plants start to acclimatise, as indicated by a steady rise in the maximum tolerated temperatures (Kaplan et al. 2004). Fig. 4.28 shows the differences in survival rates of A. thaliana seedlings after heat stress of 45 °C, depending on the temperature exposure prior to the heat stress.

Similarly, heat exposure is less damaging to the photosynthetic capacity of fir needles when they have been exposed to a previous non-lethal heat stress (Fig. 4.29).

The most important mechanism underlying acquired thermotolerance is the massive production of heat shock proteins (HSPs) (Sect. 4.3.3.1) (Vierling 1991). Regardless of the optimal growing temperature, this response is highly conserved not only among plants but also among prokaryotes, animals and fungi. According to recent genome-wide analyses in A. thaliana, however, not only the expression level of HSP genes changes. Up to 2% of all genes are affected in their activity (Kotak et al. 2007). For many of them the contribution to thermotolerance is functionally not understood. What is evident is an up-regulation of anti-oxidative defences to counteract the increased production of ROS under heat stress. Documented is also the accumulation of so-called chemical chaperones, which can increase the heat resistance of cells and shift the onset of the heat stress response to higher temperatures. These chemical chaperones are compatible solutes such as glycerol, proline and betaines (Chap. 6). They stabilise the folding and structure of mature proteins, and they can buffer the cellular redox potential. Another critical response is the modulation of membrane lipids towards a higher saturation level of fatty acids. Some of the reactions may initiate signalling cascades that result in the induction of HSPs. For instance, the dehydration-responsive transcription factor DREB2A (Chap. 6) regulates the heat shock factor Hsf3A (Sect. 4.3.3.2 and Box 4.7). ABA concentrations rise in heat-stressed tissues.

4.3.3 The Heat Shock Response

Heat stress causes the unfolding (denaturation) of proteins (Sect. 4.3.3) (Qu et al. 2013). In eukaryotic cells this triggers a reaction sequence termed the unfolded protein response (UPR). Stress-induced accumulation of unfolded proteins activates transcription factors (heat shock transcription factors (HSFs)), which then bind to specific cis elements (heat shock elements (HSEs)) in the promoters of genes encoding heat shock proteins (HSPs).

4.3.3.1 Heat Shock Proteins

HSPs are named after their enhanced production upon heat treatment. However, they are essential for prokaryotic as well as eukaryotic cells, both in the absence of stress and for heat stress survival. HSPs act as chaperones required for correct folding of (unglycosylated) nascent proteins upon release from the ribosome or after stress-induced misfolding, unfolding or aggregation of unfolded proteins (Box 4.6). This essentiality is the reason why the contribution of HSPs to heat stress survival—while not in doubt—cannot be accurately assessed genetically. Loss-of-function mutants are not viable.

HSPs are constitutively expressed and involved in protein and membrane stabilisation, in protein assembly into functional complexes, in intracellular transport of protein precursors to their target organelles and as part of cellular signalling processes. In plants they occur in plastids, mitochondria, peroxisomes, the nucleus, the cytosol and the ER. However, not all chaperones occur in all of these plant cell organelles. Prokaryotic HSPs such as HSP60 are known only from mitochondria and chloroplasts—plant organelles of prokaryotic origin. HSPs are classified by their molecular weight (Wang et al. 2004) and only in the bacterial (Escherichia coli) system do they have specific designations (in brackets). The most important classes are HSP100 (Clp), HSP90, HSP70 (DnaK), HSP60 (GroEL, the so-called GroE chaperonins) and the small HSPs (sHSPs): the HSP40 (DnaJ), HSP10 (GroES) and HSP23 (GroE) families. HSPs can cooperate with co-chaperones (e.g. HSP70 with HSP40 and HSP23).

Chaperones, particularly of the HSP70 type, are also involved in the removal of irreversibly denatured proteins. After a strong heat stress, some unfolded proteins cannot be repaired anymore. They are irreversibly denatured and tend to form—or have already formed—aggregates via their exposed hydrophobic domains. Such proteins have to be removed from the cell. They are tagged for degradation in the 26S proteasome by the attachment of several units of the small protein ubiquitin (poly-ubiquitination). Targeting to the proteasome where the adenosine triphosphate (ATP)-consuming degradation takes place is then dependent on HSP activity.

Box 4.6: Chaperones and Chaperonins Repair Misfolded Proteins

The HSP70 cycle (Fig. 4.30): The heat shock protein 70 (Hsp70) (DnaK) proteins contain three functional domains; the N-terminal domain has ATPase activity. Hydrolysis of adenosine triphosphate (ATP), as well as the exchange of ATP for adenosine diphosphate (ADP), changes the conformation of the other domains. The C-terminal domain functions as a lid for the interior domain, which binds the misfolded protein(s). The lid is open whenever ATP is bound to the N-terminal domain. Misfolded proteins bind weakly to the substrate-binding domain by hydrophobic interaction (1). Binding of a co-chaperone (J-domain protein) activates ATP hydrolysis, whereupon the lid closes and refolding of the protein can take place in the groove of the substrate-binding domain (2). The J-domain protein dissociates from the Hsp70–ADP complex. By interaction with a nucleotide exchange factor (NEF), ADP is replaced by ATP and the lid opens (3) to release the completely (4) or partially repaired protein which, in the latter case, can undergo another HSP cycle (5). Under normal conditions, the HSP70 cycle is also operative in preventing misfolding of hydrophobic stretches of a nascent protein protruding from the ribosome.

The prokaryotic chaperonin GroEL/GroES cycle operates in the plastids and mitochondria of a plant cell in a mechanism that resembles that of the HSP70 system but also shows some differences, as the GroEL forms a back-to-back double chamber, with each chamber consisting of seven GroEL subunits, which all show ATPase activity. Both chambers work alternately. GroES is the lid protein and DnaJ replaces the J-domain protein. One GroEl cycle consumes seven ATPs. Step 3, as the rate-limiting step, requires about 10 s.

4.3.3.2 Heat Stress Transcription Factors

Transcriptional activation of HSP genes is dependent on heat stress transcription factors (Hsfs). Hsfs have a modular structure (Box 4.7) which, irrespective of variations in size, is conserved in the entire eukaryotic kingdom (Scharf et al. 2012). The mode of interaction with the promoters is equally conserved. Plant genomes are particularly rich in Hsfs: in A. thaliana, 21 Hsf genes are known; in soybean there are 52 such genes. According to similarities and differences in their sequences, plant Hsfs have been classified into HsfA, HsfB and HsfC, each class consisting of a number of members (e.g. HsfA1–A8). Further grouping is indicated by lower-case letters (e.g. HsfB4a–h). In comparison with plants the diversity of Hsfs in other organisms is smaller.

Hsfs mediate the acute heat shock response, as well as the acquisition of thermotolerance. Thus, responses of different magnitude can be controlled by Hsfs. This is exemplified by representatives of the A1, A2 and B1 groups in tomato (Fig. 4.31). Under normal conditions (Fig. 4.31a), monomeric HsfA1 is inactive because of its association with HSP70 and HSP90, while HsfB1 is targeted to the 26S proteasome for degradation. Acute heat stress and concomitant denaturation of proteins results in the dissociation of the chaperone–Hsf complexes and association of the HSPs with the misfolded or unfolded proteins. The liberated Hsfs (particularly HsfA1 as a master regulator) oligomerise (trimerise), and HsfA1 and HsfB1 bind to the heat stress elements in the promoters of heat stress–activated genes, which usually harbour more than one HSE. The HsfA1–HsfB1 complex further recruits a histone acetyltransferase (HAC1-CBP in Fig. 4.31) as a co-regulator, and this ternary complex enhances the expression of the HSPs (70, 90 and 17) and, in particular, that of Hsfs B1 and A2, which play a crucial role in the acquisition of thermotolerance. HsfA2 is expressed only in stressed plants and accumulates to high levels upon long-term heat stress. Loss-of-function and overexpression experiments have revealed HsfA2 as one of the key elements in the plant stress response.

HsfA2 is involved in protection against heat, ROS, salt stress and even anoxia. It hetero-oligomerises with HsfA1, forming a kind of superactivator complex (a so-called enhancosome) for the expression of HS genes (Fig. 4.31c). Apparently, the combination of the HsfA2 activator domains (AHA in Fig. 4.32) with the HAC1-CBP results in strongly enhanced activation of the transcription machinery. Hetero-oligomerisation with HsfA1a is also necessary for the import of HsfA2 into the nucleus in order to overcome the strong C-terminal nuclear export signal (NES) of this Hsf.

Upon abatement (Fig. 4.31d) of the heat stress, the heat stress response is switched off. HsfA2 is inactivated by association with small HSPs (e.g. HSP17-CII and HSP70). This complex accumulates in the so-called heat stress granula (HSGs; Sect. 4.3.3.3). Release of HsfA2 from these granula requires another small heat stress protein, HSP17C-1, which is important for long-term heat stress. Prolonged heat stress results in high levels of heat stress proteins and of HsfA2. This is the state of acquired thermotolerance. Fading of the heat stress releases HSPs because of the decreasing level of misfolded proteins. An increase in the concentrations of free HSPs leads to monomerisation and thus inactivation of the Hsfs because of their reassociation with the HSPs. HsfA1 binds again to HSPs 70 and 90, and HsfB1 binds accordingly to HSP90. This complex can elicit degradation of HsfB1. Furthermore, it can interact with transcription factors of housekeeping genes and reactivate their expression. HSP90 interacts with HSGs, whereupon HsfA2 is released and degraded.

The interaction between heat shock factors and heat shock proteins exerts effective control over the up-regulation of HSP synthesis. The presence of more denatured proteins than under non-stressed conditions triggers the interaction of Hsfs with HSP gene promoters, and a decline in the number of denatured proteins gradually tunes out the extra HSP synthesis. Nonetheless, other factors are known to contribute to the activation of the heat shock/heat stress response (Kotak et al. 2007; Saidi et al. 2011). These include ROS and the plant hormones ABA, ethylene and salicylic acid. A change in the ROS homeostasis resulting from ROS production and detoxification appears to be a signal. Several mechanisms of ROS sensing have been proposed—for example, unidentified receptor proteins and redox-sensitive transcription factors (Chap. 2), including heat shock transcription factors (Mittler et al. 2004). However, neither ROS signalling under heat stress nor the roles of signalling molecules are understood nearly as well as the Hsf-dependent activation of HSP synthesis triggered by protein denaturation.

A second mode of sensing protein denaturation is described for the ER. One of the sensors is an ER membrane–associated transcription factor for chaperones. It is a basic leucine zipper protein, bZIP28 (Srivastava et al. 2012) (Fig. 4.33), with a single-pass transmembrane domain (TMD), a cytosolic N-terminus containing the bZIP element and a C-terminus protruding into the ER lumen. The RRIL site (Arg-Arg-Ile-Leu) on the luminal part serves the processing by specific proteases in the Golgi vesicles. bZIP28 is arrested in the ER by association of its C-terminal domain with the major luminal ER chaperone, the “binding immunoglobulin protein” (BIP). Upon stress-induced accumulation of misfolded proteins in the ER, BIP is competed away from the C-terminus of bZIP28 and associates with the accumulating misfolded proteins. Dissociation of the bZIP28–BIP complex enables the transcription factor to leave the ER and enter the Golgi. Processing by specific proteases in the Golgi removes the C-terminal part, liberating the cytosolic part of the bZIP28, which can now enter the nucleus as a transcription factor. For binding to the heat shock elements in the promoter of the heat shock genes, oligomerisation of the transcription factor is necessary.

Box 4.7: The Structure of Heat Stress Transcriptions Factors

In all investigated heat stress transcription factors (Hsfs), the DNA-binding domain is close to the N-terminus (Fig. 4.32). The hydrophobic core of that domain of about 100 amino acids specifies the selectivity of the interaction with the repetitive heat shock elements (HSEs) on the promoters of the heat shock genes. Since usually more than two HSEs are required for a proper heat stress response, oligomerisation of the Hsfs is also necessary for promoter activation.

Downstream from the DNA-binding domain is the Hsf oligomerisation domain (OD). Depending on the structure of this domain, homo-oligomerisation—as well as hetero-oligomerisation—is possible. The classification of HsfA, B or C results from sequence differences of the OD, which is therefore also termed the HR-A/B region. Clusters of basic amino acids downstream from the OD represent the nuclear localisation signal (NLS), which mediates import into the nucleus. For acclimation of the Hsf concentration in the nucleus, import must be balanced by export, and a nuclear export signal (NES) has also been identified in many plant Hsfs. Depending on the rates of import and export into and out of the nucleus, the intracellular distribution of the free Hsfs changes dynamically between the nucleus and the cytoplasm.

Activator elements (AHA motifs)—consisting of aromatic, large hydrophobic and acidic amino acids—are typical of the members of the HsfA class, except for the HsfA8 types. The activator elements can interact with the transcription apparatus and thereby activate it. Similar activator motifs are known from other transcription factors (Baniwal et al. 2004). Hsfs of the B class contain a characteristic tetrapeptide (LFGV, Leu-Phe-Gly-Val) in their C-terminal domains which, by comparison with other transcription factors, is interpreted as a repressor-binding motif. However, the corresponding transcription repressor is not yet known.

Specific roles of the plant heat stress transcription factors: The high diversity and multitude of the principally similar plant Hsfs suggest specific functional roles of these proteins. For instance, in tomato, HsfA1a is a master regulator of the heat stress response and recovery, interacting with Hsfs A2 and B1 in a functional triad upon extended heat stress (Fig. 4.31c). In this triad, HsfA2 functions as an enhancer of heat stress–induced thermotolerance. HsfB1 acts as a synergistic co-activator of HsfA1, whereas in the presence of a co-repressor, class B Hsfs can attenuate the activation of the HS genes. HsfA3 is involved in drought stress signalling, and HsfA9 controls HSP gene expression during seed development (Scharf et al. 2012).

4.3.3.3 Cell Biological Aspects of the Heat Stress Response

Organisms can acquire thermotolerance by maintaining a high level of HSPs while the normal “housekeeping metabolism” continues. During an acute heat shock, in contrast, the cell redirects its activity to almost exclusive synthesis of HSPs. Several mechanisms contribute to that rearrangement of the cellular processes: the activation of the heat stress transcription factors and the transcription of the unique HSP genes, the inhibition of the processing of the pre-messenger RNAs (pre-mRNAs) of the housekeeping genes combined with preferential translation of the heat stress gene mRNAs, and the conservation of housekeeping pre-mRNA in the nucleus or the surrounding cytosol in the form of heat stress granula (Fig. 4.34). Since housekeeping mRNAs are not functional under acute heat stress, they would be predestined for rapid degradation by RNAses. By association with small heat stress proteins and HSP70, as well as accumulated HsfA2 protein, these mRNAs are protected from degradation and stored in the multi-chaperone granula of 40 nm diameter. This might promote a return to normal cell physiology after the heat shock or upon development of the acquired thermotolerance. Dissociation of these heat stress granula requires ATP-dependent interaction with further HSPs.

4.4.1 Sensing of Extreme Temperatures

Biological membranes are strongly affected in their biophysical properties by cold or heat. For this reason and because the respective mechanisms have been revealed in bacteria, yeast and animal cells, changes in membrane fluidity or (alternatively, at least in the case of cold) disturbance and destabilisation of the microfilaments—as cytoskeletal elements are associated with the biomembranes (Orvar et al. 2000; Knight and Knight 2012)—have long been hypothesised to play a primary sensory role in temperature perception by plant cells (Saidi et al. 2011). In these cells, the list of messengers used by signalling pathways includes Ca²⁺, lipids, pH, and cyclic guanosine monophosphate (cGMP). However, no other messenger has been found that responds to more stimuli than cytosolic free Ca ²⁺ (Sanders et al. 1999). Such Ca²⁺ signals have been well documented in many plant species as the earliest measurable events (within seconds) in response to both cold (Knight et al. 1996) and heat (Gong et al. 1998) (Fig. 4.37). It is assumed that temperature-dependent membrane or cytoskeleton changes are translated into transient increases in cytosolic Ca²⁺ concentrations, mediated by Ca²⁺ channels in the plasma and the tonoplast membranes.

Evidence exists for distinct Ca signatures (Chap. 2)—that is, specific kinetics of the transient [Ca²⁺] increases that carry information and enable specificity of the downstream events (Plieth et al. 1999). In mammalian systems, different classes of Ca²⁺-permeable channels have been identified as essential sensors for cold or heat, respectively. However, no Ca²⁺ channels orthologous to the ones identified outside the plant kingdom are present in plants, according to genome analyses.

4.4.2 Sensing of Ambient Temperature Changes

Changes in temperature within the sub-extreme range, which act as important environmental cues for the modulation of developmental processes (Fig. 4.35), are apparently sensed by mechanisms that are different from the ones involved in sensing a shift to potentially stressful temperatures. A component of the ambient temperature sensing is dependent on histone H2A.Z, which can be dynamically replaced by the canonical histone 2A in the nucleosome, and vice versa (Kumar and Wigge 2010). H2A.Z-containing nucleosomes wrap DNA more tightly, which renders the DNA less accessible to RNA polymerases and in turn mRNA synthesis. Upon a shift to higher temperatures, H2A.Z is evicted from nucleosomes. This then enables key regulators such as PIF4 (Fig. 4.36) to bind to regulatory sequences and initiate the transcription of genes that activate developmental transitions. In the case of flowering, this is the florigen-encoding gene, FT (Kumar et al. 2012) (Fig. 4.38). However, while these components are clearly important in mediating temperature-controlled changes, they do not represent the primary thermosensory mechanisms. These remain to be uncovered (Wigge 2013).